CN121013904A - Clinical-grade organoids - Google Patents

Abstract

Disclosed herein are improved methods of expanding cell populations and methods of producing organoids from pluripotent stem cells, as well as compositions and uses thereof, including compositions of liquid components that are complementary to amino acids that can be used to perform these methods, and kits including components for performing these methods and/or products of using these methods. These methods can be performed without the need for heterogeneous and ambiguous basement membrane matrices for cell culture, which enables Good Manufacturing Practice (GMP) -compliant methods for producing organoids for various uses such as clinical screening and treatment, including methods for treating liver-related diseases or disorders and methods for screening candidate compounds or compositions for treating liver-related diseases or disorders.

Description

Clinical grade organoids

Statement regarding federally sponsored research and development

The present invention was completed with government support under UH3 DK119982-02 and DP2 DK128799-01 awarded by the national institutes of health (National Institutes of Health). The government has certain rights in the invention.

Cross Reference to Related Applications

The present application is based on the priority rights of U.S. patent application No. 63/493,269, entitled "CLINICAL-GRADE ORGANOIDS," filed on page 2023, month 3, and 30, as claimed in 35 u.s.c. ≡119 (e), which is incorporated by reference in its entirety.

Technical Field

Aspects of the present disclosure relate generally to organoid compositions and methods of preparing organoid compositions. These methods can be performed without the use of a heterogeneous basement membrane matrix during organoid differentiation and culture. Also disclosed herein are methods of cryopreserving organoids for long term storage and subsequent use.

Background

The culture of pluripotent stem cells and related downstream cell types (including organoid compositions) typically involves the use of a basement membrane matrix (also referred to as extracellular matrix) that provides a biological niche comprising extracellular proteins and growth factors that support cell growth. A popular basement membrane matrix for biological research is the extracellular matrix produced by murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. However, this basement membrane matrix is incompatible with human clinical purposes because it consists of xenogenic mouse components, is ambiguous (i.e., there is variability from batch to batch), and may be pathogenic.

Thus, there is a need to be able to produce organoid compositions and other compounds that are not derived from heterologous sources that are involved in organoid production without the use of a basement membrane matrix.

Disclosure of Invention

Embodiments of the present disclosure include methods for expanding metaforegut cells and/or metaforegut endoderm cells comprising a) dissociating a monolayer of foregut endoderm cells into metaforegut cells and/or metaforegut endoderm cells, b) seeding the metaforegut cells and/or metaforegut endoderm cells onto a tissue culture surface, and c) culturing the metaforegut cells and/or metaforegut endoderm cells with a TGF-b pathway inhibitor, a FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator.

In some embodiments, enzymatic and/or mechanical dissociation may be used to dissociate the monolayer of foregut endoderm cells into posterior foregut cells and/or posterior foregut endoderm cells. In some embodiments, the posterior foregut cells and/or the posterior foregut endoderm cells can be seeded onto the tissue container surface at a cell density equal to or about 1 x 10 ⁵, 2x 10 ⁵, 3 x 10 ⁵, 4 x 10 ⁵, 5x 10 ⁵, 6 x 10 ⁵, 7 x 10 ⁵, 8 x 10 ⁵, 9 x 10 ⁵, 1 x 10 ⁶, 2x 10 ⁶, 3 x 10 ⁶, 4 x 10 ⁶, or 5x 10 ⁶ cells/cm ² of the surface area of the tissue culture surface, or at any cell density having a range defined by any two of the cell densities described above.

In some embodiments, wherein the tissue culture surface is coated with a basement membrane matrix or a component thereof. In some embodiments, the basement membrane matrix or component thereof does not comprise a non-human animal component that renders the basement membrane matrix or component thereof xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-swart (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel ^®、Cultrex^® or Geltrex ^®. In some embodiments, the basement membrane matrix or component thereof comprises human fibronectin, collagen IV, entactin, basement membrane glycans, fibrin, and/or hydrogels.

In some embodiments, the metaintestinal cells and/or metaintestinal endoderm cells may be cultured until three-dimensional (3D) spheroids can spontaneously form, optionally wherein the spheroids comprise a structure having a single lumen, and/or wherein the spheroids are free of hematopoietic tissue and acquired immune cells. In some embodiments, the metaintestinal cells and/or metaintestinal endoderm cells can be cultured for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days.

In some embodiments, the TGF-b pathway inhibitor may be selected from A83-01, repSox, LY365947, and SB431542, optionally A83-01. In some embodiments, the TGF-b pathway inhibitor may be provided at a concentration equal to or about 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the TGF-b pathway inhibitor is provided at a concentration equal to or about 500 nM. In some embodiments, the FGF pathway activator may be selected from FGF1、FGF2、FGF3、FGF4、FGF4、FGF5、FGF6、FGF7、FGF8、FGF8、FGF9、FGF10、FGF11、FGF12、FGF13、FGF14、FGF15、FGF16、FGF17、FGF18、FGF19、FGF20、FGF21、FGF22 and FGF23, optionally FGF2. In some embodiments, the FGF pathway activator can be provided at a concentration equal to or about 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, or 10ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the FGF pathway activator is provided at a concentration equal to or about 5 ng/mL. In some embodiments, the Wnt pathway activator may be selected from Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11、Wnt16、BML 284、IQ-1、WAY 262611、CHIR99021、CHIR 98014、AZD2858、BIO、AR-A014418、SB 216763、SB 415286、 aloxin (aloisine), indirubin (indirubin), altretglon (alsterpaullone), kenarone (kenpaullone), lithium chloride, TDZD 8, and TWS119, optionally CHIR99021. In some embodiments, the Wnt pathway activator may be provided at a concentration equal to or about 1 μΜ, 1.5 μΜ,2 μΜ, 2.5 μΜ,3 μΜ, 3.5 μΜ, 4 μΜ, 4.5 μΜ,5 μΜ, 5.5 μΜ,6 μΜ, 6.5 μΜ,7 μΜ,7.5 μΜ, or 8 μΜ, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the Wnt pathway activator is provided at a concentration equal to or about 3 μΜ. In some embodiments, the VEGF pathway activator may be selected from the group consisting of VEGF or GS4012, optionally VEGF. In some embodiments, the VEGF pathway activator may be provided at a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the VEGF pathway activator is provided at a concentration equal to or about 10 ng/mL.

In some embodiments, the metaintestinal cells and/or the metaintestinal endoderm cells of step c) may be cultured in a medium further comprising EGF, or in a medium not comprising EGF. In some embodiments, EGF may be provided at a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein EGF is provided at a concentration equal to or about 20 ng/mL. In some embodiments, the metaintestinal cells and/or metaintestinal endoderm cells of step c) may be cultured in a medium that also includes ascorbic acid, or in a medium that does not include ascorbic acid. In some embodiments, the ascorbic acid may be provided at a concentration equal to or about 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, or 100 μg/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ascorbic acid is provided at a concentration equal to or about 50 μg/mL.

In some embodiments, the metaintestinal cells and/or metaintestinal endoderm cells of step c) may be cultured in a medium further comprising a ROCK inhibitor, or in a medium not comprising said ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632. In some embodiments, the ROCK inhibitor may be provided at a concentration equal to or about 1 μΜ,2 μΜ,3 μΜ, 4 μΜ,5 μΜ, 6 μΜ, 7 μΜ,8 μΜ, 9 μΜ,10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ,15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, or 20 μΜ, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ROCK inhibitor is provided at a concentration equal to or about 10 μΜ.

In some embodiments, the cells of step c) may be passaged one or more times. In some embodiments, the cells of step c) may be passaged until the metaforegut cells and/or metaforegut endoderm cells do not spontaneously form spheroids. In some embodiments, the cells of step c) may be passaged no more than 3 times.

In some embodiments, the metaintestinal cells and/or metaforeintestinal endoderm cells may be cultured, and the metaforeintestinal cells and/or metaforeintestinal endoderm cells may differentiate into liver organoids. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells may be cultured until a three-dimensional (3D) spheroid spontaneously forms, optionally wherein the spheroid comprises a structure having a single lumen, and/or wherein the spheroid is free of hematopoietic tissue and acquired immune cells, and the metaforegut cells and/or metaforegut endoderm cells may be collected from the spheroid, optionally further comprising dissociating the spheroid into individual metaforegut cells and/or metaforegut endoderm cells and/or a pellet of metaforegut endoderm cells prior to the differentiating step. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are collected from the monolayer of foregut endoderm cells prior to the differentiating step by dissociating the monolayer of foregut endoderm cells into individual metaforegut cells and/or metaforegut endoderm cells and/or clumps of metaforegut cells and/or metaforegut endoderm cells.

Additional embodiments of the present disclosure include methods of differentiating metaforegut cells and/or metaforegut endoderm cells into liver organoids comprising i) contacting the metaforegut cells and/or metaforegut endoderm cells, optionally in the form of spheroids, optionally in the form of individual cells or clusters of cells dissociated from the spheroids, and/or optionally cells aggregated in a microwell or other device described herein, optionally wherein the spheroids comprise a structure having a single lumen, and/or wherein the spheroids are free of hematopoietic tissue and acquired immune cells, with a culture medium, and ii) contacting the cells of step i) for a period of time such that the metaforegut cells and/or metaforegut endoderm cells differentiate into liver organoids, optionally wherein the culture medium is a liver cell culture medium.

In some embodiments, the medium is supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid. In some embodiments, the cMET tyrosine kinase receptor agonist may be selected from Hepatocyte Growth Factor (HGF), PG-001, fugonidone (fosgonimeton), ter Lei Walai phenanthrene (terevalefim), recombinant InlB321 protein, and an agonistic c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokine may be selected from the group consisting of IL-6, oncostatin M (OSM), leukemia Inhibitory Factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokine (CLC). In some embodiments, the corticosteroid may be selected from the group consisting of dexamethasone, beclomethasone, betamethasone, flucortisone, halometasone, and mometasone. In some embodiments, the medium may be supplemented with HGF, OSM, and dexamethasone. In some embodiments, the medium may be supplemented with dexamethasone.

In some embodiments, the metaforegut cells and/or metaforegut endoderm cells may comprise metaforegut cells and/or metaforegut endoderm cells produced by the foregoing methods. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells can be in the form of spheroids or single metaforegut cells and/or metaforegut endoderm cells, and/or in the form of a pellet derived from dissociated spheroids of metaforegut cells and/or metaforegut endoderm cells, optionally wherein the spheroids comprise a structure having a single lumen, and/or wherein the spheroids are free of hematopoietic tissue and acquired immune cells.

In some embodiments, the retinoic acid pathway activator may be selected from retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally retinoic acid. In some embodiments, the retinoic acid pathway activator may be provided at a concentration of 1.0µM、1.1µM、1.2µM、1.3µM、1.4µM、1.5µM、1.6µM、1.7µM、1.8µM、1.9µM、2.0µM、2.1µM、2.2µM、2.3µM、2.4µM、2.5µM、2.6µM、2.7µM、2.8µM、2.9µM or 3.0 μm, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the retinoic acid pathway activator may be provided at a concentration equal to or about 2.0 μm. In some embodiments, HGF may be provided at a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein HGF is provided at a concentration equal to or about 10 ng/mL. In some embodiments, the OSM is provided at a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the OSM is provided at a concentration equal to or about 20 ng/mL. In some embodiments, dexamethasone may be provided at a concentration equal to or about 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, or 200nM, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein dexamethasone is provided at a concentration equal to or about 100 nM. In some embodiments, the cells of step i) and/or step ii) are not contacted with EGF.

In some embodiments, the cells of step ii) may be cultured in a growth medium supplemented with non-essential amino acids, essential amino acids and glycine. In some embodiments, the post-supplementation growth medium comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% nonessential amino acids by total volume, or nonessential amino acids within a range defined by any two of the foregoing values, optionally wherein the post-supplementation growth medium comprises about 4% -10%, 6% -12%, 10% -16%, 12% -15%, 13% -19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% nonessential amino acids by total volume. In some embodiments, the post-supplementation growth medium comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% essential amino acids by total volume, or within a range defined by any two of the foregoing values, optionally wherein the post-supplementation growth medium is about 4% -10%, 6% -12%, 10% -16%, 12% -15%, 13% -19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% essential amino acids by total volume. In some embodiments, the supplemental glycine may be provided at a concentration equal to or about 5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL、21mg/mL、22mg/mL、23mg/mL、24mg/mL、25mg/mL、26mg/mL、27mg/mL、28mg/mL、29mg/mL、30mg/mL、31mg/mL、32mg/mL、33mg/mL、34mg/mL or 35mg/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the supplemental glycine is provided at a concentration equal to or about 18mg/mL-22mg/mL or 20 mg/mL.

In some embodiments, the cells of step ii) may be further contacted with a low/first concentration of bilirubin, wherein the liver organoid formed is a mature liver organoid. In some embodiments, the low/first concentration of bilirubin may be a human fetal physiological concentration of bilirubin. In some embodiments, the low concentration/first concentration of bilirubin may be, may be less than, or may be less than about ：a) 0.1mg/L、0.2mg/L、0.3mg/L、0.4mg/L、0.5mg/L、0.6mg/L、0.7mg/L、0.8mg/L、0.9mg/L、1mg/L、1.25mg/L、1.5mg/L、1.75mg/L、2.0mg/L、2.25mg/L、2.5mg/L、2.75mg/L or 3.0mg/L, or a range defined by any two of the foregoing concentrations (e.g., 0.1mg/L to 3mg/L, 0.5mg/L to 2.0mg/L, 0.5mg/L to 1.5mg/L, 0.3mg/L to 2.5mg/L, or 0.5mg/L to 1.75 mg/L), or b) 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, or 1mg/L, or a range defined by any two of the foregoing concentrations (e.g., 0.1mg/L to 1mg/L, 0.1mg/L to 2.5mg/L, or 0.5mg/L to 1.75 mg/L), or b) 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.5mg/L, 0.7mg/L, or 0.8 mg/L.

In some embodiments, the mature liver organoid may exhibit a luminal protrusion resembling a bile duct, and/or have a single lumen and generally spherically shaped structure, and/or wherein the mature liver organoid is free of hematopoietic tissue and acquired immune cells. In some embodiments, the mature liver organoid may express reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to a liver organoid not contacted with the low/first dose of bilirubin. In some embodiments, the mature liver organoid may express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid not contacted with the low/first dose of bilirubin. In some embodiments, the mature liver organoid can express CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof. In some embodiments, mature liver organoids may exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids not contacted with low/first doses of bilirubin.

In some embodiments, the cells of step ii) may be further contacted with a high/second concentration of bilirubin, wherein the liver organoid formed is a hyperbilirubinemia liver organoid. In some embodiments, the high/second concentration of bilirubin may be, may be about, may be greater than, or may be greater than about ：a) 2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 2mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L), or b) 4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 4mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L). In some embodiments, hyperbilirubinemia liver organoids may express elevated levels of UGT1A1 or NRF2, or both, relative to liver organoids not treated with high/second concentrations of bilirubin.

In some embodiments, the liver organoids may include a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA encoding a functional GULO protein or both, wherein the liver organoid functions are capable of synthesizing ascorbic acid. In some embodiments, the functional GULO protein may be murine GULO (mGULO). In some embodiments, optionally using a tetracycline-inducible system, genes encoding functional GULO proteins may be conditionally expressed. In some embodiments, liver organoids can be engineered with gene engineering encoding functional GULO proteins using CRISPR. In some embodiments, the gene encoding the functional GULO protein or mRNA, or both, may be introduced into the liver organoid by transfection. In some embodiments, a liver organoid comprising functional GULO protein may express increased levels of NRF2 relative to a liver organoid that does not comprise functional GULO protein. In some embodiments, a liver organoid comprising functional GULO protein may express reduced levels of IL1B, IL or TNFa or any combination thereof, optionally when cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid, relative to a liver organoid that does not comprise functional GULO protein. In some embodiments, the liver organoids comprising functional GULO protein exhibit reduced caspase-3 activity relative to liver organoids that do not include functional GULO protein, optionally when cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid. In some embodiments, liver organoids comprising functional GULO protein may express increased levels of ALB relative to liver organoids not comprising functional GULO protein. In some embodiments, a liver organoid comprising functional GULO protein may resemble periportal liver tissue and express periportal liver markers. In some embodiments, the periportal liver marker may include FAH, ALB, PAH, CPS a1, HGD, or any combination thereof. In some embodiments, a liver organoid comprising functional GULO protein may exhibit increased CYP3A4 and CYP1A2 activity relative to a liver organoid that does not comprise functional GULO protein. In some embodiments, liver organoids comprising functional GULO protein may exhibit increased bilirubin conjugation activity relative to liver organoids not comprising functional GULO protein. In some embodiments, liver organoids comprising functional GULO protein may exhibit increased viability in culture relative to liver organoids not comprising functional GULO protein. In some embodiments, the liver organoid has been differentiated from a pluripotent stem cell comprising a functional GULO protein and/or a gene or mRNA encoding a functional GULO protein or both, whereby the pluripotent stem cell is capable of synthesizing ascorbic acid. In some embodiments, the liver organoid comprises an inactive UGT1A1 gene, wherein the liver organoid can be used as a model of Crigler-Najjar syndrome.

In some embodiments, posterior foregut cells and/or posterior foregut endoderm cells may be aggregated in a microwell or other device (e.g., aggresell) prior to step i), wherein aggregating the posterior foregut cells and/or posterior foregut endoderm cells may result in a liver organoid of more uniform size. In some embodiments, the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or a component thereof, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or a component thereof that is heterologous to the human, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or a component thereof isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the cells of step i) and/or step ii) are not contacted with Matrigel ^®、Cultrex^® or Geltrex ^®. In some embodiments, the cells of step i) and/or step ii) and/or liver organoids formed therefrom may be cultured in a static bioreactor or a non-static bioreactor, optionally a rotating bioreactor, optionally a 3D rotating bioreactor. In some embodiments, after culturing in a static bioreactor or a non-static bioreactor, the liver organoids may be dissociated into single cells, and may be subsequently reconstituted and/or amplified via additional culturing steps in a static bioreactor or a non-static bioreactor, optionally a 3D rotating bioreactor. In some embodiments, the methods further comprise cryopreserving the liver organoids. In some embodiments, cryopreserving the liver organoid comprises slow freeze or vitrification cryopreservation, optionally wherein the liver organoid can be cryopreserved with chroman, emlicarbazen (emricasan), polyamines, and trans-ISRIB (CEPT).

In some embodiments, the metaforegut cells and/or metaforegut endoderm cells have been derived from pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells. In some embodiments, the metaforegut cells and/or the metaforegut endoderm cells have been derived from a subject, optionally a subject suffering from a liver-related disease or disorder. In some embodiments, the method may be used in a Good Manufacturing Practice (GMP) compliant process. Embodiments of the present disclosure also include posterior foregut cells and/or posterior foregut endoderm cells or liver organoids produced by any of the foregoing methods.

Additional embodiments of the present disclosure include in vitro compositions comprising pluripotent stem cells, definitive endoderm, foregut endoderm, ventral foregut endoderm, and/or downstream hepatocyte types, and at least one exogenous tissue culture surface, at least one exogenous TGF-b pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator.

In some embodiments, the composition comprises a metaforegut cell and/or a metaforegut endoderm cell, and the metaforegut cell and/or metaforegut endoderm cell is a dissociated metaforegut cell and/or metaforegut endoderm cell. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells can be greater than or equal to, exactly or about 1×10 ⁵, 2×10 ⁵, 3×10 ⁵, 4×10 ⁵, 5X 10 ⁵, 6X 10 ⁵, 7X 10 ⁵, 8X 10 ⁵, 9X 10 ⁵, Cell densities of 1 x 10 ⁶, 2 x 10 ⁶, 3 x 10 ⁶, 4 x 10 ⁶, or 5 x 10 ⁶ cells/cm ² of the surface area of the tissue culture surface, or any cell density in the range defined by any two of the foregoing cell densities. in some embodiments, the tissue culture surface may be coated with a basement membrane matrix or a component thereof. In some embodiments, the basement membrane matrix or component thereof does not comprise a non-human animal component that renders the basement membrane matrix or component thereof xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-swart (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel ^®、Cultrex^® or Geltrex ^®. In some embodiments, the basement membrane matrix or component thereof comprises human fibronectin, collagen IV, entactin, basement membrane glycans, fibrin, and/or hydrogels.

In some embodiments, at least a portion of the metaforegut cells and/or metaforegut endoderm cells can be spontaneously formed three-dimensional (3D) spheroids, optionally wherein the spheroids comprise a structure capable of having a single lumen, and/or wherein the mature liver organoid is free of hematopoietic tissue and acquired immune cells. In some embodiments, the TGF-b pathway inhibitor may be selected from the group consisting of A83-01, repSox, LY365947, and SB431542, optionally wherein the TGF-b pathway inhibitor comprises or is A83-01. In some embodiments, the TGF-b pathway inhibitor may have a concentration equal to or about 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the TGF-b pathway inhibitor has a concentration equal to or about 500 nM. In some embodiments, the FGF pathway activator can be selected from FGF1、FGF2、FGF3、FGF4、FGF4、FGF5、FGF6、FGF7、FGF8、FGF8、FGF9、FGF10、FGF11、FGF12、FGF13、FGF14、FGF15、FGF16、FGF17、FGF18、FGF19、FGF20、FGF21、FGF22 and FGF23, optionally wherein the FGF pathway activator comprises or is FGF2. In some embodiments, the FGF pathway activator can have a concentration equal to or about 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, or 10ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the FGF pathway activator has a concentration equal to or about 5 ng/mL. In some embodiments, the Wnt pathway activator may be selected from Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11、Wnt16、BML 284、IQ-1、WAY 262611、CHIR99021、CHIR 98014、AZD2858、BIO、AR-A014418、SB 216763、SB 415286、 aloxin, indirubin, altbolone, kenparone, lithium chloride, TDZD 8, and TWS119, optionally wherein the Wnt pathway activator comprises or is CHIR99021. In some embodiments, the Wnt pathway activator may have a concentration equal to or about 1 μΜ, 1.5 μΜ,2 μΜ, 2.5 μΜ,3 μΜ, 3.5 μΜ,4 μΜ, 4.5 μΜ,5 μΜ, 5.5 μΜ,6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ, or 8 μΜ, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the Wnt pathway activator has a concentration equal to or about 3 μΜ. In some embodiments, the VEGF pathway activator may be selected from VEGF or GS4012, optionally wherein the VEGF pathway activator comprises or is VEGF. In some embodiments, the VEGF pathway activator may have a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the VEGF pathway activator has a concentration equal to or about 10 ng/mL. In some embodiments, the composition further comprises exogenous EGF, or the composition does not comprise exogenous EGF. In some embodiments, EGF may have a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein EGF has a concentration equal to or about 20 ng/mL. In some embodiments, the composition further comprises exogenous ascorbic acid and/or transgene-produced ascorbic acid, or the composition does not comprise exogenous ascorbic acid and/or transgene-produced ascorbic acid. In some embodiments, the ascorbic acid may have a concentration equal to or about 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, or 100 μg/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ascorbic acid has a concentration equal to or about 50 μg/mL. In some embodiments, the composition may further comprise a ROCK inhibitor, or may be cultured in a medium that does not comprise a ROCK inhibitor, optionally wherein the ROCK inhibitor comprises or is Y-27632. In some embodiments, the ROCK inhibitor may have a concentration equal to or about 1 μΜ,2 μΜ,3 μΜ,4 μΜ,5 μΜ, 6 μΜ,7 μΜ,8 μΜ,9 μΜ,10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ,16 μΜ, 17 μΜ,18 μΜ, 19 μΜ, or 20 μΜ, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ROCK inhibitor has a concentration equal to or about 10 μΜ.

In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, ventral foregut endoderm and/or downstream liver cells can differentiate from stem cells. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, ventral foregut endoderm and/or downstream liver cells can differentiate from induced pluripotent stem cells. In some embodiments, the posterior foregut cells and/or posterior foregut endoderm cells, definitive endoderm, ventral foregut endoderm and/or downstream liver cells have been passaged less than 4 times. In some embodiments, the cells comprise or consist essentially of metaforegut cells and/or metaforegut endoderm cells. In some embodiments, the TGF-b pathway inhibitor may be A83-01, the FGF pathway activator may be FGF2, the Wnt pathway activator may be CHIR99021, the VEGF pathway activator may be VEGF, and the ROCK inhibitor may be Y-27632.

Additional embodiments of the present disclosure include liver organoids produced by any of the foregoing methods.

Additional embodiments of the present disclosure include in vitro compositions comprising a) metaintestinal cells and/or metaintestinal endoderm cells, liver organoids and/or mature liver organoids, and b) a culture medium, wherein the culture medium optionally comprises a hepatocyte culture medium and is optionally supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine and a corticosteroid, and wherein the composition optionally further comprises c) a retinoic acid pathway activator. In some embodiments, the cMET tyrosine kinase receptor agonist may be selected from Hepatocyte Growth Factor (HGF), PG-001, fugonidone, tetronium Lei Walai, recombinant InlB321 protein, and an agonistic c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokine may be selected from the group consisting of IL-6, oncostatin M (OSM), leukemia Inhibitory Factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokine (CLC). In some embodiments, the corticosteroid may be selected from dexamethasone, beclomethasone, betamethasone, flucortisone, halometasone, and mometasone. In some embodiments, the medium may be supplemented with HGF, OSM, and dexamethasone. In some embodiments, the medium may be supplemented with dexamethasone. In some embodiments, the retinoic acid pathway activator may be selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally wherein the retinoic acid pathway activator comprises or is retinoic acid. In some embodiments, the retinoic acid pathway activator may be at a concentration of 1.0µM、1.1µM、1.2µM、1.3µM、1.4µM、1.5µM、1.6µM、1.7µM、1.8µM、1.9µM、2.0µM、2.1µM、2.2µM、2.3µM、2.4µM、2.5µM、2.6µM、2.7µM、2.8µM、2.9µM or 3.0 μm, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the retinoic acid pathway activator has a concentration equal to or about 2.0 μm. In some embodiments, the HGF may have a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the HGF has a concentration equal to or about 10 ng/mL. In some embodiments, the OSM may have a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the OSM has a concentration equal to or about 20 ng/mL. In some embodiments, dexamethasone may have a concentration equal to or about 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, or 200nM, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein dexamethasone has a concentration equal to or about 100 nM. in some embodiments, the composition does not include exogenous EGF.

In some embodiments, the composition further comprises a low concentration of exogenous bilirubin, optionally wherein the low concentration of bilirubin is at or near human fetal physiological bilirubin concentration. In some embodiments, bilirubin may be, may be about, may be less than, or may be less than about ：0.1mg/L、0.2mg/L、0.3mg/L、0.4mg/L、0.5mg/L、0.6mg/L、0.7mg/L、0.8mg/L、0.9mg/L、1mg/L、1.25mg/L、1.5mg/L、1.75mg/L、2.0mg/L、2.25mg/L、2.5mg/L、2.75mg/L or 3.0mg/L, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 0.1mg/L to 3mg/L, 0.5mg/L to 2.0mg/L, 0.5mg/L to 1.5mg/L, 0.3mg/L to 2.5mg/L, or 0.5mg/L to 1.75 mg/L), or 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, or 1mg/L, or any range defined by any two of the foregoing concentrations (e.g., 0.1mg/L to 1mg/L, 0.1mg/L to 0.5mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L or 0.7 mg/L). In some embodiments, the composition comprises a mature liver organoid and the mature liver organoid exhibits a luminal protrusion resembling a biliary tract, and/or has a single lumen and generally spherically shaped structure, and/or wherein the mature liver organoid is free of hematopoietic tissue and acquired immune cells. In some embodiments, mature liver organoids may express reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to liver organoids not contacted with low doses of bilirubin. In some embodiments, mature liver organoids may express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to liver organoids not contacted with low doses of bilirubin. In some embodiments, the mature liver organoid can express CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof. In some embodiments, mature liver organoids may exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids not contacted with low doses of bilirubin.

Additional embodiments of the present disclosure include in vitro compositions comprising mature liver organoids wherein cells of the mature liver organoids are contacted with low doses of bilirubin, optionally wherein the low doses of bilirubin are provided exogenously, and the mature liver organoids exhibit luminal protrusions resembling bile canaliculi, and/or have a single lumen and generally spherically shaped structure, and/or wherein the mature liver organoids are free of hematopoietic tissues and acquired immune cells. In some embodiments, mature liver organoids may express reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to liver organoids in which cells are not contacted with low doses of bilirubin. In some embodiments, mature liver organoids may express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to liver organoids in which cells are not contacted with low doses of bilirubin. In some embodiments, the mature liver organoid can express CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof. In some embodiments, mature liver organoids may exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids in which cells are not contacted with low doses of bilirubin.

In some embodiments, the composition further comprises a hyperbilirubinemia liver organoid, wherein the hyperbilirubinemia liver organoid cells are contacted with a high concentration and/or a second concentration of bilirubin. In some embodiments, the high/second concentration of bilirubin is, is about, is greater than or is greater than about ：2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or is greater than 20mg/L, or is any concentration within a range defined by any two of the foregoing concentrations (e.g., 2mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L), or 4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or is any concentration within a range defined by any two of the foregoing concentrations (e.g., 4mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L). In some embodiments, hyperbilirubinemia liver organoids may express elevated levels of UGT1A1 or NRF2, or both, relative to liver organoids not treated with high/second concentrations of bilirubin. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells, liver organoids and/or mature liver organoids comprise a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA encoding a functional GULO protein or both, wherein the metaforegut cells and/or metaforegut endoderm cells, liver organoids and/or mature liver organoids are capable of synthesizing ascorbic acid. In some embodiments, the functional GULO protein is murine GULO (mGULO). In some embodiments, optionally using a tetracycline-inducible system, genes encoding functional GULO proteins may be conditionally expressed. In some embodiments, CRISPR can be used to engineer metaforegut cells and/or metaforegut endoderm cells, liver organoids, and/or mature liver organoids to include genes encoding functional GULO proteins. In some embodiments, the gene encoding the functional GULO protein or mRNA, or both, is introduced into the liver organoid by transfection. In some embodiments, a liver organoid and/or mature liver organoid comprising functional GULO protein may express increased levels of NRF2 relative to a liver organoid and/or mature liver organoid not comprising functional GULO protein. in some embodiments, a liver organoid and/or mature liver organoid comprising functional GULO protein may express reduced levels of IL1B, IL or TNFa or any combination thereof relative to a liver organoid and/or mature liver organoid not comprising functional GULO protein. In some embodiments, a liver organoid and/or mature liver organoid comprising functional GULO protein may exhibit reduced caspase-3 activity relative to a liver organoid and/or mature liver organoid not comprising functional GULO protein. In some embodiments, liver organoids and/or mature liver organoids comprising functional GULO protein may express increased levels of ALB relative to liver organoids and/or mature liver organoids not comprising functional GULO protein. in some embodiments, the liver organoids and/or mature liver organoids comprising functional GULO protein may resemble periportal liver tissue and express periportal liver markers. In some embodiments, the periportal liver marker may include FAH, ALB, PAH, CPS a1, HGD, or any combination thereof. In some embodiments, a liver organoid and/or mature liver organoid comprising a functional GULO protein may exhibit increased CYP3A4 and CYP1A2 activity relative to a liver organoid and/or mature liver organoid not comprising a functional GULO protein. In some embodiments, liver organoids and/or mature liver organoids comprising functional GULO protein may exhibit increased bilirubin conjugation activity relative to liver organoids and/or mature liver organoids not comprising functional GULO protein. In some embodiments, liver organoids and/or mature liver organoids comprising functional GULO protein may exhibit increased viability in culture relative to liver organoids and/or mature liver organoids not comprising functional GULO protein. In some embodiments, the liver organoid and/or mature liver organoid has been differentiated from a pluripotent stem cell comprising a functional GULO protein and/or a gene or mRNA encoding a functional GULO protein or both, whereby the pluripotent stem cell is capable of synthesizing ascorbic acid.

Additional embodiments of the present disclosure include methods of administering the foregoing liver organoids or compositions to a subject in need thereof, as well as methods for treating liver-related diseases or disorders in a subject in need thereof, comprising administering to the subject one or more of the foregoing liver organoids or compositions.

In some embodiments, the liver organoids have been produced by cells derived from a subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells. In some embodiments, administering comprises transplanting a liver organoid or composition into the subject. In some embodiments, the liver-related disease or disorder includes one or more types of liver dysfunction and/or failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic disease, autoimmune liver disease, wilson's disease, metabolic-related fatty liver disease, hyperammonemia, hyperbilirubinemia, crigler-Najjar syndrome, urea cycle disorder, walman disease, liver cancer, hepatoblastoma, metabolic dysfunction-related liver disease (MASLD), metALD, metabolic dysfunction-related steatohepatitis (MASH), drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic disease, liver cyst, acetaminophen acute liver injury, and/or alcohol-related liver disease. In some embodiments, liver dysfunction and/or failure includes hyperammonemia and/or hyperbilirubinemia, or metabolic disease includes non-alcoholic fatty liver disease (NAFLD), or non-alcoholic fatty liver disease (NAFLD) includes metabolic dysfunction-related steatohepatitis (MASH), or hepatitis includes hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, TT, and/or autoimmune hepatitis. In some embodiments, the subject may have a reduced serum bilirubin and/or ammonia levels, and/or increased serum protein after transplantation. In some embodiments, the subject may have improved symptoms of biliary stricture and/or liver regeneration after transplantation. In some embodiments, the subject may have increased survival after transplantation. In some embodiments, the liver organoid is implanted onto the liver of the subject. In some embodiments, the liver organoids have been treated with an Amino Acid (AA) supplement. In some embodiments, the liver organoids have been treated with an Amino Acid (AA) supplement for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more prior to transplantation. In some embodiments, the liver-related disease or disorder comprises acetaminophen acute liver injury. In some embodiments, the method may be used in a Good Manufacturing Practice (GMP) compliant process.

Additional embodiments of the present disclosure include methods for screening comprising contacting the foregoing liver organoids with a candidate compound or composition and assessing the effect of the candidate compound or composition on the liver organoids. In some embodiments, the liver organoid may be a model of a liver-related disease or disorder, and determining the effect of the candidate compound or composition on the liver organoid comprises assessing the effect of the candidate compound or composition on the liver-related disease or disorder. In some embodiments, the liver organoids have been produced by cells derived from a subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells. In some embodiments, the subject has a liver-related disease or disorder. In some embodiments, the method may be used in a Good Manufacturing Practice (GMP) compliant process.

Additional embodiments of the present disclosure include compositions comprising liquid components that supplement amino acids according to table 3. Additional embodiments include compositions comprising mixtures of growth factors according to the embodiments of table 1 or table 2.

Additional embodiments of the present disclosure include compositions comprising a liquid component of supplemental amino acids comprising, by volume, just or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of a non-essential amino acid solution (containing just or about 890mg/L alanine, 1320mg/L asparagine, 1330mg/L aspartic acid, 750mg/L glycine, 105mg/L serine, 1150mg/L proline, and 1470mg/L glutamic acid), just or about 4%, by volume, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% essential amino acid solution (containing exactly or about 6320mg/L arginine, 1200mg/L cysteine, 2100mg/L histidine, 2620mg/L isoleucine, 2620mg/L leucine, 3625mg/L lysine, 755mg/L methionine, 1650mg/L phenylalanine, 2380mg/L threonine, 510mg/L tryptophan, 1800mg/L tyrosine and 2340mg/L valine) and exactly or about 65% by volume, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% of hepatocyte medium (HCM) and is also supplemented with exactly or about 5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL、21mg/mL、22mg/mL、23mg/mL、24mg/mL、25mg/mL、26mg/mL、27mg/mL、28mg/mL、29mg/mL、30mg/mL、31mg/mL、32mg/mL、33mg/mL、34mg/mL or 35mg/mL glycine. In some embodiments, the composition comprises a liquid component of supplemental amino acids comprising a solution of just or about 14% of non-essential amino acids (containing just or about 890mg/L alanine, 1320mg/L asparagine, 1330mg/L aspartic acid, 750mg/L glycine, 105mg/L serine, 1150mg/L proline and 1470mg/L glutamic acid), a solution of just or about 6% of essential amino acids (containing just or about 6320mg/L arginine, 1200mg/L cysteine, 2100mg/L histidine, and, 2620mg/L isoleucine, 2620mg/L leucine, 3625mg/L lysine, 755mg/L methionine, 1650mg/L phenylalanine, 2380mg/L threonine, 510mg/L tryptophan, 1800mg/L tyrosine and 2340mg/L valine) and a hepatocyte medium (HCM) of exactly or about 80% by volume, and is also supplemented with exactly or about 20g/L glycine. In some embodiments, the pH is between about pH6 to 8, or between pH6.5 to 7.5, or is just or about pH 7.0. In some embodiments, the composition further comprises Hepatocyte Growth Factor (HGF), oncostatin M, dexamethasone, and/or ascorbic acid. In some embodiments, the composition further comprises liver lineage committed cells differentiated from definitive endoderm cells using retinoic acid. In some embodiments, the liver lineage committed cells are characterized as liver organoids.

In some embodiments, liver organoids may be characterized as secreting increased levels of albumin and urea relative to liver organoids included in HCM without amino acid supplements. In some embodiments, liver organoids may be characterized as expressing increased levels of liver maturation-related gene expression relative to liver organoids included in HCM without amino acid supplements. In some embodiments, a liver organoid may be characterized as expressing reduced levels of vimentin relative to a liver organoid included in an HCM without an amino acid supplement.

In some embodiments, the composition does not include a non-human animal component that renders the base film matrix or component thereof xenogeneic to humans. In some embodiments, the composition does not include murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, matrigel ^®、Cultrex^®, and/or Geltrex ^®.

Additional embodiments of the present disclosure include in vitro hyperbilirubinemia liver organoids comprising naturally occurring and/or engineered mutations in UDP glucuronyl transferase family 1 member A1 (UGT 1 A1). In some embodiments, the hyperbilirubinemia liver organoids are produced by contacting precursor cells, precursor liver organoids, and/or precursor mature liver organoids with exogenous bilirubin for at least two rounds. In some embodiments, the hyperbilirubinemia liver organoids are of clonal origin and/or derived from ipscs.

Additional embodiments of the present disclosure include cryopreserved compositions comprising liver organoids, chroman 1, emlicarbazin, polyamines, and trans-ISRIB (CEPT), and/or cryopreserved compositions comprising mature liver organoids, chroman, emlicarbazin, polyamines, and trans-ISRIB (CEPT), and/or cryopreserved compositions comprising hyperbilirubinemia liver organoids, chroman, emlicarbazin, polyamines, and trans-ISRIB (CEPT).

Additional embodiments of the present disclosure include kits comprising means for performing any of the foregoing methods, and/or kits comprising a composition or means for producing any of the foregoing compositions, and/or kits for producing any of the foregoing liver organoids. Additional embodiments of the present disclosure include the use of these methods, compositions or kits as medicaments, tools for the treatment and/or prevention of diseases, diagnostic tools and/or medical research.

Drawings

In addition to the features described herein, additional features and variations will become apparent from the following description of the drawings and exemplary embodiments. It should be understood that these drawings depict embodiments and are not intended to limit the scope.

Fig. 1A depicts an embodiment of a schematic diagram for cryopreserving liver organoids and optional subsequent steps.

Fig. 1B depicts an embodiment of fluorescence microscopy images of liver organoids that have been frozen and thawed (as compared to non-frozen organoids) using slow freezing and vitrification cryopreservation methods to observe the abundance of living cells (labeled with calcein AM) and dead cells (labeled with ethidium homodimer-1).

Fig. 1C depicts an embodiment of quantification of albumin secretion from a liver organoid that has been frozen and thawed using slow freeze and vitrification cryopreservation methods (as compared to a non-frozen organoid).

FIG. 2A depicts an embodiment of RT-qPCR quantification of ALB, CYP3A4, PCK1 and G6PC expression in liver organoids cultured with the addition of Amino Acid (AA) supplemented medium at different times of culture.

Figure 2B depicts an embodiment of quantification of albumin secretion and urea production in liver organoids cultured with AA supplementation medium added at different times of culture.

FIG. 2C depicts an embodiment of a schematic representation of activity, such as PCK1 activity, in liver organoids grown in the presence of AA supplemented media using a luciferase reporter method with living cells or cell lysates.

Fig. 2D depicts an embodiment of bright field microscopy images of organoids grown in AA supplemented media and standard hepatocyte media (HCM).

Figure 2E depicts an embodiment of quantification of PCK1 expression measured by luciferase reporter in liver organoids grown in AA supplemented medium compared to liver organoids grown in standard hepatocyte medium in live cell and cell lysate assays.

Fig. 3A depicts an embodiment of a schematic diagram of two-dimensional (2D) hepatocyte differentiation from pluripotent stem cells and cultures using AA supplemented media added at different times of culture.

Fig. 3B depicts an embodiment of a bright field image of a 2D hepatocyte culture grown with AA supplementation medium added at different times of the culture.

Fig. 3C depicts an embodiment of quantification of lactate production in 2D hepatocytes grown in AA supplemented media compared to those grown in standard hepatocyte media.

Figure 3D depicts an embodiment of quantification of albumin secretion in 2D hepatocytes grown in AA supplemented media added at different times of culture compared to those grown in standard hepatocyte media.

Fig. 3E depicts an embodiment of quantification of albumin secretion in 2D hepatocytes grown chronically in AA supplemented media, wherein the albumin secretion rate is normalized to the rate of hepatocytes grown in standard hepatocytes media.

FIG. 3F depicts an embodiment of RT-qPCR quantification of gene expression of ALB, E-cadherin, CYP3A4, G6PC, PKM and PCK1 in 2D hepatocytes grown in AA supplemented media added at different times of culture compared to those grown in standard hepatocytes media.

Fig. 3G depicts an embodiment of fluorescence and bright field microscopy images of 2D hepatocytes engineered to express mScarlet under PCK1 reporter factors, compared to those grown in standard hepatocyte media with or without insulin starvation, compared to those grown in AA supplemented media with or without insulin starvation.

Fig. 3H depicts an embodiment of fluorescence microscopy images to detect expression of EpCAM (epithelial cells), vimentin (mesenchymal cells) and DAPI or hnf4α (nuclei) in 2D hepatocytes grown in AA supplemented media compared to those grown in standard hepatocytes media.

Fig. 4A depicts an embodiment of a schematic of culturing a liver organoid without the use of Matrigel ^® or other basement membrane matrix with heterogeneous components.

Fig. 4B depicts an embodiment showing a bright field image of spontaneous 3D spheroids formed from a single layer of foregut cells, where the spheroids can be transferred to mature into liver organoids without the use of Matrigel ^® or other basement membrane matrix with heterogeneous composition.

Fig. 4C depicts an embodiment of bright field and fluorescence microscopy images showing that organoids grown in the absence of Matrigel ^® exhibit normal organoid morphology.

Fig. 4D depicts an embodiment of quantification of albumin secretion compared between organoids grown in the absence of Matrigel ^®, organoids grown in Matrigel ^® according to previous protocols, and 2D hepatocyte cultures.

FIG. 4E depicts an embodiment of RT-qPCR quantification of ALB, AFP, HNF. Alpha., RBP4 and AAT expression between organoids grown in the absence of Matrigel ^®, organoids grown in Matrigel ^® according to the previous protocol, and 2D hepatocyte cultures.

Fig. 5A depicts an embodiment of a schematic diagram for the passage and expansion of foregut cells for large-scale organoid production following differentiation of pluripotent stem cells.

Fig. 5B depicts an embodiment showing the growth of foregut cells (days 1-7) and spontaneous formation of spheroids when plated on laminin or Matrigel ^® -coated plates.

Fig. 5C depicts an embodiment of a bright field microscope image showing further growth of foregut cells (days 8-10) and complete formation of spheroids, where cells plated on laminin coated plates appear to cause more efficient spheroid formation than cells plated on Matrigel ^® coated plates.

Fig. 5D depicts an embodiment showing bright field microscopy images of growth of foregut cells (days 1-5) and spontaneous formation of spheroids when plated on laminin coated plates at different plating densities.

Fig. 5E depicts an embodiment showing bright field microscopy images of additional spheroids generated by additional culture when spheroids are collected from initial spontaneous formation of anterior intestinal cells.

Fig. 5F depicts an embodiment of bright field microscopy images and quantification of total potential cell numbers obtainable by multiple passages of initially differentiated foregut cells from pluripotent stem cells. Spheroids are formed during passage 1-3, rather than from passage 4 foregut cells.

FIG. 5G depicts an embodiment of RT-qPCR quantification of CDX2, FOXA2, AFP, VIM, SOX, HNF4α and ALB expression in foregut cells from passages 1-4.

Fig. 5H depicts an embodiment of a schematic representation of liver organogenesis starting from pluripotent stem cells and including passaging for large-scale foregut cells.

Fig. 5I depicts an embodiment of a schematic diagram starting from pluripotent stem cells and including liver organogenesis for large-scale foregut cell passages, and aggregating foregut cells using a device to improve uniformity of organoid size and shape.

Fig. 6A depicts an embodiment of a schematic of a 3D rotational culture of liver organoids grown in the absence of Matrigel ^®.

Fig. 6B depicts an embodiment showing a bright field image of liver organoids that can be grown using 3D rotational culture in the absence of Matrigel ^®.

FIG. 6C depicts an embodiment of RT-qPCR quantification of AFP, HNF4α, FOXA2, ALB, CDX2 and VIM in liver organoids grown in 3D rotation culture compared to generation 1 foregut cells.

Figure 7A depicts an embodiment of a schematic diagram for producing HLO and maturation with low doses of bilirubin.

Fig. 7B depicts an embodiment of a bright field image of HLO treated with low dose bilirubin (1 mg/L) compared to a control, and a lumen profile using ImageJ, wherein the arrows indicate a lumen protrusion similar to the bile canaliculi found in human liver.

FIG. 7C depicts an embodiment of a comparison of lumen size and roundness of control and 1mg/L bilirubin treated HLO.

FIG. 7D depicts an embodiment of RT-qPCR of control organoids and organoids treated with 1mg/L bilirubin and maturation marker genes (ALB, NANOG, SLC A2, HO-1, AFP and CDX 2) compared to human liver samples.

FIG. 7E depicts an embodiment of CYP3A4 and CYP1A2 activity assays in response to rifampicin (rifampicin) and omeprazole (omeprazole) in control and 1mg/L bilirubin treated HLO (RLU: relative light units; CTG: cellTiter-Glo assay).

FIGS. 7F-7H depict embodiments of immunofluorescence of mature liver enzymes and transporters in 1mg/L bilirubin treated liver organoids. Fig. 7F depicts the detection of CYP2E1 and MRP 3. Fig. 7G depicts the detection of CYP7A1 and MRP 1. Fig. 7H depicts detection of PROX1 and OATP 2.

Fig. 8A depicts an embodiment of a bright field image of ascorbic acid depleted HLO at day 15 compared to a control.

Fig. 8B depicts an embodiment of an exemplary workflow of generating mGULO iPSC.

FIG. 8C depicts an embodiment of a linear diagram of the synthetic mGULO-mCherry gene designed for expression of mGULO in a hiPSC under the doxycycline-activated TetOn system and a vector diagram of the pAAVS-Ndi-CRISPRi (Gen 1) plasmid for cloning the mGULO gene into a hiPSC.

Fig. 8D depicts an embodiment of a schematic diagram of the generation of HLO using ipscs modified to express TetOn mGULO.

Fig. 8E depicts an embodiment of bright field and fluorescent images of mCherry expression in doxycycline (Dox) treated mGULO HLO compared to control HLO.

Fig. 8F depicts an embodiment of bright field and fluorescent images of mCherry expression in mGULO HLO with or without Dox treatment at day 18.

FIG. 8G depicts an embodiment of ELISA for mGULO protein expression and cellular antioxidant concentration in mGULO HLO treated with Dox (10 ng/mL or 100 ng/mL) compared to control HLO.

Fig. 8H depicts an embodiment of RT-qPCR of inflammatory and detoxifying marker genes (NRF 2, 1L1B, IL, and tnfa) in ascorbic acid depleted Dox treated mGULO HLO compared to ascorbic acid depleted control or mGULO HLO.

Fig. 8I depicts an embodiment of a caspase 3 activity assay of Dox-treated mGULO HLO for ascorbic acid depletion compared to control or mGULO HLO for ascorbic acid depletion.

Fig. 8J depicts an embodiment of a heat map from RNA-seq showing that Dox treated mGULO HLO expresses a periportal marker compared to control HLO.

FIG. 8K depicts an embodiment of gene up-regulation by functional classification, showing that there is an excessive representation of periportal access in mGULO HLO of Dox treatment.

Fig. 8L depicts an embodiment of bright field images of mGULO and control HLOs with or without 1mg/L bilirubin treatment, and lumen contours using ImageJ, with arrows indicating lumen protrusions similar to the bile canaliculi found in human liver.

FIG. 8M depicts an embodiment of a comparison of lumen size and roundness of mGULO HLO or control HLO treated with Dox with or without 1mg/L bilirubin treatment.

FIG. 8N depicts an embodiment of quantification of albumin expression in a Dox treated mGULO HLO or control HLO with or without 1mg/L bilirubin treatment.

FIG. 8O depicts an embodiment of a bright field image of mGULO HLO treated with bilirubin and different concentrations of Dox (0 ng/mL, 10ng/mL, 100ng/mL, or 1000ng/mL Dox).

Figure 8P depicts an embodiment of the CYP3A4 and CYP1A2 activity assays in control or Dox treated mGULO HLO in response to rifampicin and omeprazole with 1mg/L bilirubin.

Figure 8Q depicts an embodiment of UnaG assay showing a loss of fluorescence, indicating bilirubin conjugation even in the presence of deep yellow bilirubin.

Fig. 8R depicts an embodiment of UnaG assay of mGULO organoids treated with Dox compared to a control.

Fig. 8S depicts an embodiment of quantification of the total percentage of active organoids and organoids carrying conjugated bilirubin in a Dox treated mGULO organoid compared to a control.

Fig. 9A depicts an embodiment of a schematic diagram for producing HLO and treatment with different concentrations of bilirubin.

FIG. 9B depicts an embodiment of bright field images after 1 day and 4 days of treatment of HLO with bilirubin (0 mg/L-10 mg/L).

FIG. 9C depicts an embodiment of RT-qPCR of UGT1A1 and NRF2 genes of organoids treated with different concentrations of bilirubin compared to untreated organoids.

Fig. 9D depicts an embodiment of an overview of a patient with Crigler-Najjar syndrome (CNS) from which CNS ipscs are produced. DNA sequencing of the patient revealed a nonsense mutation in UGT1A1 gene c.858c > a (p.cys280x).

Fig. 9E depicts an embodiment showing fluorescence images of CNS ipscs derived from patients with Crigler-Najjar syndrome expressing classical pluripotency markers Sox2 and Oct 4.

Fig. 9F depicts an embodiment showing that CNS ipscs can differentiate into bright field images of Definitive Endoderm (DE) and liver organoids (hLO) according to a standard protocol.

Fig. 9G depicts an embodiment showing fluorescence images of the liver organoid expression proliferation marker Ki67, liver specific marker AFP and epithelial marker ECAD produced by CNS ipscs.

Figure 9H depicts an embodiment of bright field images of CNS HLOs treated with bilirubin (10 mg/L) and controls (0 mg/L bilirubin) after 1 day and 4 days, showing that these HLOs suffer from bilirubin toxicity.

Fig. 9I depicts an embodiment of bright field images of CNS HLOs and CNS HLOs that have been transfected with UGT1A1mRNA 10 days after treatment with bilirubin (10 mg/L).

Fig. 9J depicts an embodiment of a bilirubin assay that measures unconjugated bilirubin (UCB) and Conjugated Bilirubin (CB) in the HLO of fig. 9I.

FIG. 9K depicts an embodiment of a bilirubin assay measuring unconjugated bilirubin (UCB) and Conjugated Bilirubin (CB) in mGULO HLO treated with 10mg/L bilirubin and Dox (0 ng/mL, 10ng/mL, 100ng/mL, or 1000 ng/mL).

FIG. 10A depicts an embodiment of bright field images of liver organoids treated with 10mg/L bilirubin and the glucocorticoid agonist hydrocortisone (HC; 1. Mu.M or 5. Mu.M) or dexamethasone (Dex; 1. Mu.M or 5. Mu.M).

Fig. 10B depicts an embodiment of a bilirubin assay that measures unconjugated bilirubin and conjugated bilirubin in the liver organoids of fig. 10A.

FIG. 10C depicts an embodiment of bright field images of liver organoids treated with 10mg/L bilirubin and the glucocorticoid antagonist ketoconazole (KCZ; 1. Mu.M or 5. Mu.M) or mifepristone (Mif; 1. Mu.M or 5. Mu.M).

Fig. 10D depicts an embodiment of a bilirubin assay that measures unconjugated bilirubin and conjugated bilirubin in the liver organoids of fig. 10C.

FIG. 10E depicts an embodiment of RT-qPCR of UGT1A1 and NRF2 genes for organoids treated with 10mg/L bilirubin as compared to organoids treated with 10mg/L bilirubin and hydrocortisone, dexamethasone, ketoconazole, or mifepristone.

FIG. 10F depicts an embodiment of a comparison of enrichment pathways obtained from RNA sequencing between organoids treated with 10mg/L bilirubin and 1. Mu.M mifepristone, as compared to a control, and a GSEA map comparing enriched ROS and xenobiotic metabolism.

FIG. 10G depicts an embodiment of a Venn diagram (VENN DIAGRAM) showing genes differentially expressed in ROS and xenobiotic metabolism.

FIG. 10H depicts embodiments of ChIP-PCR and ChIP-qPCR of organoids treated with 10mg/L bilirubin and 1. Mu.M mifepristone (Mife) or 1. Mu.M dexamethasone (Dex).

Fig. 11A depicts an embodiment of a workflow for positive displacement planting of HLOs in rodents.

Fig. 11B depicts an embodiment of an albumin ELISA in serum collected from a mGULO HLO or sham operated Gunn rat at various time points after transplantation.

Fig. 11C depicts an embodiment of a bilirubin assay for the Gunn rat of fig. 11B after implantation.

Fig. 11D depicts an embodiment of AST and ALT assays for the Gunn rats of fig. 11B after implantation.

FIG. 12A depicts an embodiment of an exemplary process of subjecting HLO induced by conventional methods to expansion culture in 3D bioreactor culture with or without Matrigel ^®.

Fig. 12B depicts an embodiment of HLO growth after 15 days of 3D bioreactor culture compared to conventional static culture.

Fig. 12C depicts an embodiment in which large HLOs were obtained in 3D bioreactor culture, even in groups where Matrigel ^® was not added.

Fig. 13 depicts an embodiment in which similar expression levels of E-cadherin, vimentin, and Prox1 were observed, indicating that HLOs obtained from 3D bioreactor cultures had comparable properties to those obtained from static cultures.

Fig. 14A depicts an embodiment in which HLOs induced by conventional methods are dissociated into single cells and passaged and reconstituted using a 3D bioreactor.

Fig. 14B depicts an embodiment of induction of uniform HLO via reconstitution of HLO in a 3D bioreactor after 6 days with and without Matrigel ^®.

Fig. 15A depicts an embodiment of a schematic of a model for rescue of acetaminophen acute liver injury by HLO transplantation.

Fig. 15B depicts an embodiment of an image of HLO for transplantation.

Fig. 15C depicts an embodiment of Kaplan-Meier survival curves for acute liver injury rescue by HLO transplantation.

Detailed Description

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like numerals generally identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

The following description of the various embodiments is merely exemplary and explanatory and is not to be construed as limiting or restricting in any way. Other embodiments, features, objects, and advantages of the present teachings will be apparent from the description and drawings, and from the claims.

The disclosure herein describes various embodiments in affirmative language. The present disclosure also includes embodiments in which subject matter, such as materials or materials, method steps and conditions, protocols or procedures, is excluded, in whole or in part.

It should be understood that any use of the subtitles herein is for organizational purposes and should not be construed as limiting the application of those subtitle features to the various embodiments herein. Each feature described herein is applicable to and useful in all of the various embodiments discussed herein, and all features described herein can be used in any desired combination, regardless of the particular example embodiments described herein. It should also be noted that the exemplary description of specific features is primarily intended to provide information and is not intended to limit the design, sub-features and functionality of the explicitly described features in any way.

SUMMARY

The development of methods for producing human liver organoids from pluripotent stem cells has enabled the study of liver function very similar to that of the native liver. In contrast to methods involving the combination of primary hepatocytes or differentiation from adult multipotent cells, differentiation of multipotent stem cells produces liver organoids with a rich variety of cell types, including hepatocytes, astrocytes, kupffer cells (Kupffer cells), and other epithelial and mesenchymal cell lineages that make up the normal liver.

However, since maintenance of pluripotent stem cells and formation of downstream intermediates are sensitive, previous culture methods typically involve the use of basement membrane matrices that mimic niches that promote cell proliferation. Common basement membrane matrices used in laboratory settings are those isolated from murine Engelbreth-Holm-switch (EHS) sarcoma cells. While these matrices provide great utility in cell culture, their presence prevents subsequent use in humans because they contain xenogenic animal components and may also potentially carry pathogens. Thus, there is a continuing process to develop cell culture methods that do not involve the use of heterogeneous basement membrane matrices.

Provided herein are methods for culturing organoids (such as liver organoids) without the use of Matrigel ^® or other heterogeneous basement membrane matrices. Other improvements for culturing liver organoids are also disclosed, such as methods of supplementing growth medium and expanding cells for larger scale manufacturing beyond laboratory culture limits.

In some embodiments, human Liver Organoids (HLOs), such as those produced according to the methods disclosed herein, are used to model liver-related diseases and disorders. For example, HLOs produced according to the methods disclosed herein may be used to model hyperbilirubinemia by treating them with different concentrations of bilirubin.

{ Update with any further independent claim embodiment once the claims are agreed/finalized }

The incidence of liver disease increases at a faster rate. Hyperbilirubinemia (NH) of newborns is a condition that worsens the health of newborns. It affects 60% of all newborns and causes 114,000 deaths worldwide each year. Currently, the only treatment for NH involves 12 hours of phototherapy or exchange of blood, but these can cause other complications. Thus, an effective and scalable model system for these liver diseases has now become a necessary condition for understanding the molecular mechanisms behind it and developing potential therapies.

There are currently two major model organisms used to model NH, gunn rats and UGT1A1 knockout mice. However, these models lack the key human proteins (OATP family) and epigenetic controls involved in bilirubin metabolism. Recent studies have revealed that many aspects of UGT1A1 regulation do not convert well between species. Genetic regulation of UGT1A1 gene as a rate-limiting enzyme in bilirubin metabolism is significantly different in humans. Gunn rats with UGT defects are quite difficult to maintain and require special regulation. Furthermore, UGT1 KO mouse models exhibit lethal hyperbilirubinemia and live for only a few weeks. Many treatments have been tested with these models, but most fail.

Breeding model organisms such as mice and rats requires months of work and planning and the chances of obtaining the desired genotype are relatively low. Furthermore, model organisms show a high degree of variation in response to biochemical disturbances over several generations. These rodents also risk losing the desired genotype when bred for long periods of time and also require complex training and procedures to model the disease and evaluate the efficacy of the treatment. Thus, there is an urgent need to develop more measurable models to understand the kinetics of bilirubin metabolism.

HLOs, such as those produced according to the methods disclosed herein, are easy to use and vary very little from lot to lot. A large number of HLOs can be produced within a few weeks. With these properties, several drugs were tested over a short time span to identify key pathways involved in bilirubin metabolism. Thus, liver organoids are useful models for studying diseases and conditions associated with dysfunctional bilirubin metabolism, such as jaundice, crigler-Najjar syndrome, gilbert's syndrome, dubin-Johnson syndrome, or Rotor syndrome.

These HLOs may be derived from induced pluripotent stem cells (ipscs) from a patient source, wherein the patient may be healthy or have a diseased condition and are identical in genetic content to the corresponding patient. They express most liver markers expressed during the prenatal stage of development. Furthermore, they are cloned and thus respond similarly to external stimuli and biochemical disturbances. These HLOs are highly scalable and easy to handle, allowing screening methods to test a large number of drugs and small molecules.

Although abnormal metabolism of bilirubin can lead to disease, bilirubin is an important metabolite during early fetal development and acts as a metabolic hormone in adults to play an antioxidant role. The HLO model prepared by previous methods that do not involve the use of bilirubin is similar to immature tissue and expresses fetal markers and intestinal markers. Thus, maturation of HLO is induced by using low doses of bilirubin (mimicking normal fetal physiological levels) during the culturing steps disclosed herein, as also disclosed herein. Modulation of the glucocorticoid receptor pathway in these HLOs, such as treatment with mifepristone and ketoconazole, improves conjugation and metabolism of bilirubin.

Vitamin C is also essential for normal fetal development and is involved in the formation of perihepatic periportal regions. L-gulonolactone oxidase (GULO) is a naturally occurring enzyme that synthesizes vitamin C, but this enzyme is nonfunctional in humans and some other animals such as guinea pigs, requiring exogenous vitamin C supplementation (usually by dietary supplementation). As shown in guinea pig animal models, vitamin C deficiency causes significant metabolic disorders.

Genetic modifications in iPSC cell lines are easier compared to model organisms, and they can be easily maintained for longer periods before differentiating into organoids. With this, iPSC-derived organoids (such as those produced according to the methods disclosed herein) expressing functional L-gulonolactone oxidase (GULO), such as murine GULO (mGULO), were produced. When ipscs and organoids are of human origin, expression of functional L-gulonolactones allows for ascorbic acid synthesis, which is generally inactive in humans. These mGULO organoids exhibit increased efficiency in conjugating bilirubin and exhibit improved viability when treated with bilirubin. The production of ascorbic acid in mGULO organoids reduces oxidative stress in organoids and drives expression of NRF2, a major regulator of the cellular detoxification pathway, thereby promoting expression of UGT1A1 that catalyzes bilirubin conjugation. These mGULO organoids are genetically identical to the patients from which they were derived and encompass aspects of human bilirubin metabolism. Thus, these organoids can be used as model systems for elucidating the mechanisms of liver diseases and disorders (such as NH) to develop and develop therapeutic treatments thereof.

Definition of terms

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like numerals generally identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs when read in light of this disclosure. For purposes of this disclosure, the following terms are explained as follows.

The disclosure herein describes various embodiments in affirmative language. The present disclosure also includes embodiments in which subject matter, such as materials or materials, method steps and conditions, protocols or procedures, is excluded, in whole or in part.

The articles "a" and "an" are used herein to refer to one or more than one (e.g., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

"About" means an amount, level, value, number, frequency, percentage, size, quantity, weight, or length that varies by up to 10% relative to a reference amount, level, value, number, frequency, percentage, size, quantity, weight, or length.

The term "or" is used in the claims to mean "and/or" unless explicitly indicated to mean only the alternatives or alternatives are mutually exclusive, but the disclosure supports the definition of only the alternatives and "and/or". For example, "x, y, and/or z" may refer to "x alone," y alone, "" z alone, "" x, y, and z, "" x and y, or z, "" x or (y and z) "or" x or y or z. It is specifically contemplated that x, y or z may be explicitly excluded from the embodiments. As used herein, "another" may mean at least a second or more.

The term "a" or "an" means more than one.

As used herein, the term "plurality" may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

As used herein, the term "set" means one or more. For example, a group of items includes one or more items.

As used herein, the phrase "at least one," when used with a list of items, means that different combinations of one or more of the listed items may be used, and that only one of the items in the list may be required. An item may be a particular object, thing, step, operation, procedure, or category. In other words, in the alternative, by "at least one of the items" is meant that any combination of items or numbers of items from a list may be used, but not all items in the list may be required. For example, but not limited to, "at least one of item A, item B, or item C" means item A, and item B, item A, item B, and item C, or item A and item C. In some cases, "at least one of item a, item B, or item C" means, but is not limited to, two items a, one item B, and ten items C, four items B, and seven items C, or some other suitable combination.

As used herein, "substantially" means sufficient for the intended purpose. Thus, the term "substantially" allows for minor, insignificant changes to absolute or perfect conditions, dimensions, measurements, results, etc., such as would be expected by one of ordinary skill in the art, without significantly affecting the overall performance. When used with respect to a numerical value or parameter or characteristic that may be expressed as a numerical value, substantially means within ten percent.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. "consisting of" is meant to include, but not be limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of" means that the listed elements are essential or necessary, and that no other elements can be present. "consisting essentially of" is meant to encompass any element listed after the phrase and is limited to other elements that do not interfere with or facilitate the activities or actions specified for the listed elements in this disclosure. Thus, the phrase "consisting essentially of" means that the listed elements are necessary or mandatory, but other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the listed elements.

Reference throughout this specification to "one embodiment," "an embodiment," "one particular embodiment," "a related embodiment," "an embodiment," "one additional embodiment," or "another embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in various embodiments.

As used herein, the terms "individual," "subject," or "patient" have their ordinary and customary meaning as understood in the present specification, and refer to a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or a bird, such as a chicken, as well as any other vertebrate or invertebrate. The term "mammal" is used in its usual biological sense. Thus, mammals include, in particular, but are not limited to, primates, including apes (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, and the like.

As used herein, the term "treatment" or the like with respect to a disease or condition may refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or side effects attributable to the disease. For example, treatment may include an execution regimen, which may include administering one or more drugs to a patient in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include reducing the rate of disease progression, improving or moderating the disease state, and alleviating or improving prognosis. Alleviation may occur before signs or symptoms of a disease or condition and after their appearance. Thus, "treating" may include "preventing" a disease or an adverse condition. Furthermore, "treatment" does not require complete relief from signs or symptoms, does not require cure, and specifically includes regimens that have only marginal effects on the patient.

As used herein, "treatment" and "treatment" thus covers any treatment of a disease in a subject, particularly a human, and includes (a) preventing the occurrence of the disease in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., arresting its development, and (c) alleviating the disease, i.e., causing regression of the disease and/or alleviating one or more symptoms of the disease. "treating" may also encompass delivering an agent or administering therapy so as to provide a pharmacological effect even in the absence of a disease or condition.

The term "therapeutically effective" or "therapeutically effective amount" as used throughout the present application may refer to any amount effective to achieve a desired and/or beneficial effect, and/or to promote or enhance the health of a subject in terms of the medical treatment of a condition. This includes, but is not limited to, a decrease in the frequency or severity of one or more signs or symptoms of the disease. The effective amount may be administered in one or more administrations. In these methods, a therapeutically effective amount is an amount suitable for the therapeutic indication. Therapeutic indication means achieving any desired effect, such as one or more of moderating, improving, stabilizing, reversing, slowing or delaying disease progression, improving quality of life, or extending life. Such achievements may be measured by any suitable method, such as measuring tumor size or blood cell number, or any other suitable measurement.

As used herein, the term "effective amount" or "effective dose" has its ordinary and customary meaning as understood in accordance with the present specification and refers to the amount of the composition or compound that results in an observable effect. The actual dosage level of the active ingredient in the active compositions of the presently disclosed subject matter may be varied in order to administer an amount of the active composition or compound effective to achieve the desired response for a particular subject and/or application. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the composition, the formulation, the route of administration, the combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimum dose is administered and the dose is escalated to a minimum effective amount in the absence of dose limiting toxicity. Determination and adjustment of effective dosages, as well as evaluation of when and how such adjustments are made, are contemplated herein.

The term "disease state" as used herein may generally refer to a condition that affects the structure or function of an organism. The disease state may include, for example, a stage of disease progression.

As used herein, the term "assessing" may include any form of measuring and includes determining whether an element is present. The terms "determine," "measure," "evaluate," and "determine" are used interchangeably and may include quantitative determination and/or qualitative determination.

As used herein, unless otherwise indicated or clear from the context of a particular use, the terms "modulated" or "regulated" and "differentially regulated" may refer to up-regulation (i.e., activation or stimulation, e.g., by agonism or enhancement) and down-regulation (i.e., inhibition or repression, e.g., by antagonism, reduction or repression).

As used herein, the terms "function" and "functional" have their ordinary and customary meanings as understood in accordance with the present specification, and refer to biological, enzymatic or therapeutic functions.

As used herein, the term "marker" or "biomarker" may refer to any measurable substance taken as a sample from a subject, the presence of which indicates a certain phenomenon. Non-limiting examples of such phenomena may include disease states, conditions, or exposure to compounds or environmental conditions. In various embodiments described herein, the biomarkers can be used for diagnostic purposes (e.g., diagnosing a disease state, a health state, an asymptomatic state, a symptomatic state, etc.). The term "biomarker" may be used interchangeably with the term "marker". The term "marker" or "biomarker" may include biomolecules, e.g., nucleic acids, peptides, proteins, hormones, etc., whose presence or concentration may be detected and correlated with a known condition (such as a disease state). It may also be used to refer to a differentially expressed gene, the expression pattern of which may be used as part of a predictive, prognostic or diagnostic process in a health condition or disease state, or the differentially expressed gene may alternatively be used in a method of identifying a useful therapeutic or prophylactic therapy.

As used herein, the term "cell phenotype" may refer to any determinable, observable, and/or measurable characteristic associated with a population of cells.

As used herein, a "model" may include one or more in vitro or in vivo disease models, and the model may also include algorithms, one or more mathematical techniques, one or more machine learning algorithms, or a combination thereof. According to various embodiments as disclosed herein, the model may be used in the process and/or applied to the assay.

As used herein, a "process" may include one or more steps involving one or more features of one or more models as disclosed herein.

As used herein, the terms "function" and "functional" have their ordinary and customary meanings as understood from the specification, and may refer to biological functions, enzymatic functions, or therapeutic functions.

As used herein, the term "inhibit" has its ordinary and customary meaning as understood in accordance with the present specification, and may refer to a reduction or prevention of biological activity. The reduction may be a percentage of, about, at least about, no more than, or no more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount within a range defined by any two of the foregoing values. As used herein, the term "delay" has its ordinary and customary meaning as understood in accordance with this specification, and refers to slowing, delaying or deferring a biological event to a later time than would otherwise be expected. The delay may be a percentage delay of, about, at least about, no more than, or no more than about 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the foregoing values. The terms inhibit and delay do not necessarily denote 100% inhibition or delay. Partial suppression or delay may be achieved.

As used herein, the term "isolated" has its ordinary and customary meaning as understood in the present specification and refers to a substance and/or entity that has been (1) separated from at least some of its components with which it was associated when originally produced (whether in nature and/or in an experimental environment), and/or (2) produced, prepared, and/or manufactured by man. The isolated substance and/or entity may be separated (or comprise and/or span a range of values) from equal to, about, at least about, no more than, or no more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which it is originally associated. In some embodiments, the isolated agent is, is about, is at least about, does not exceed, or does not exceed about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100% or 100% pure (or comprises and/or spans a range of the foregoing values). As used herein, an "isolated" substance may be "pure" (e.g., substantially free of other components). As used herein, the term "isolated cell" may refer to a cell that is not comprised in a multicellular organism or tissue.

As used herein, "in vivo" is given its ordinary and customary meaning as understood in the present specification and refers to performing the method inside a living organism (typically animals, mammals, including humans and plants) rather than a tissue extract or dead organism.

As used herein, "ex vivo" is given its ordinary and customary meaning as understood in accordance with the present specification, and refers to a method performed outside a living organism with little change in natural conditions.

As used herein, "in vitro" is given its ordinary and customary meaning as understood in the present specification and refers to performing the method outside biological conditions, for example in a culture dish or tube.

As used herein, the term "nucleic acid" or "nucleic acid molecule" has its ordinary and customary meaning as understood in the present specification and refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments that occur naturally in cells, fragments produced by the Polymerase Chain Reaction (PCR), and fragments produced by any ligation, cleavage, endonuclease action, and exonuclease action. The nucleic acid molecule can be composed of monomers that are naturally occurring nucleotides (e.g., DNA and RNA) or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides) or a combination of both. Modified nucleotides may have alterations in the sugar moiety and/or in the pyrimidine or purine base moiety. Sugar modifications comprise, for example, substitution of one or more hydroxyl groups with halogen, alkyl, amine and azide groups, or the sugar may be functionalized as an ether or ester. In addition, the entire sugar moiety may be replaced by sterically and electronically similar structures (e.g., azasugar and carbocyclic sugar analogs). Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well known heterocyclic substituents. Nucleic acid monomers may be linked by phosphodiester linkages or analogues of such linkages. Analogs of phosphodiester linkages include phosphorothioates, phosphorodithioates, phosphoroselenates, phosphorodiselenates, phosphoroaniliothioates (phosphoanilothioates), phosphoroanilides (phosphoanilothioates) or phosphoramidates (phosphoramidate). The term "nucleic acid molecule" also encompasses so-called "peptide nucleic acids", which include naturally occurring or modified nucleobases attached to a polyamide backbone. The nucleic acid may be single-stranded or double-stranded. "oligonucleotide" may be used interchangeably with nucleic acid and may refer to double-stranded or single-stranded DNA or RNA. The one or more nucleic acids may be included in a nucleic acid vector or nucleic acid construct (e.g., plasmid, virus, retrovirus, lentivirus, phage, cosmid (fosmid), phagemid, bacterial Artificial Chromosome (BAC), yeast Artificial Chromosome (YAC), or Human Artificial Chromosome (HAC)) that may be used to amplify and/or express the one or more nucleic acids in various biological systems. Typically, the vector or construct will also contain elements including, but not limited to, a promoter, enhancer, terminator, inducer, ribosome binding site, translational start site, start codon, stop codon, polyadenylation signal, origin of replication, cloning site, multiple cloning site, restriction enzyme site, epitope, reporter gene, selectable marker, antibiotic selectable marker, targeting sequence, peptide purification tag or accessory gene, or any combination thereof.

A nucleic acid or nucleic acid molecule may include one or more sequences encoding different peptides, polypeptides, or proteins. The one or more sequences may be joined adjacently in the same nucleic acid or nucleic acid molecule, or have additional nucleic acids therebetween, such as a linker, repeat sequence, or restriction enzyme site, or any other sequence of length, about, at least about, no more than, or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or any length within a range defined by any two of the foregoing lengths. As used herein, the term "downstream" on a nucleic acid has its ordinary and customary meaning as understood in the present specification, and refers to a sequence that follows the 3' end of the preceding sequence on the strand comprising the coding sequence (sense strand) when the nucleic acid is double stranded. As used herein, the term "upstream" on a nucleic acid has its ordinary and customary meaning as understood in the present specification and refers to a sequence preceding the 5' end of the subsequent sequence on the strand comprising the coding sequence (sense strand) when the nucleic acid is double stranded. As used herein, the term "grouping" has its ordinary and customary meaning on nucleic acids as understood in the specification and refers to a sequence of two or more additional nucleic acids (e.g., a linker, repeat sequence, or restriction enzyme site) occurring directly nearby or in between, or any other sequence of length, about, at least about, no more than or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length within a range defined by any two of the foregoing lengths, but generally without having a functional or catalytic polypeptide or protein domain encoding sequence therebetween.

Nucleic acids described herein include nucleobases. The main, canonical, natural or unmodified bases are adenine, cytosine, guanine, thymine and uracil. Other nucleobases include, but are not limited to, purine, pyrimidine, modified nucleobases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5, 6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

As used herein, the terms "peptide," "polypeptide," and "protein" have their ordinary and customary meanings as understood in the specification, and refer to macromolecules that include amino acids linked by peptide bonds. Many functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymes, structures, transport, defense, hormones, or signaling. Peptides, polypeptides and proteins are typically (but not always) produced biologically from ribosomal complexes through the use of nucleic acid templates, although chemical synthesis is also useful. By using nucleic acid templates, peptide, polypeptide and protein mutations, such as substitutions, deletions, truncations, additions, replications or fusions of more than one peptide, polypeptide or protein, may be made. These fusions of more than one peptide, polypeptide or protein may be adjacent to each other in the same molecule, or have additional amino acids (e.g., linker, repeat sequence, epitope or tag) therebetween, or any other sequence that is, is at least about, is not more than, or is not more than about 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、150、200 or 300 bases long, or any length within a range defined by any two of the foregoing lengths. As used herein, the term "downstream" has its ordinary and customary meaning on polypeptides as understood in accordance with the specification and refers to sequences following the C-terminus of the preceding sequence. As used herein, the term "upstream" has its ordinary and customary meaning on polypeptides as understood in accordance with the specification, and refers to a sequence preceding the N-terminus of the subsequent sequence.

As used herein, the term "purity" of any given substance, compound or material has its ordinary and customary meaning as understood in accordance with this specification and refers to the actual abundance of the substance, compound or material relative to the expected abundance. For example, a substance, compound, or material may have a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, including all decimal amounts therebetween. Purity may be affected by unwanted impurities including, but not limited to, nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, vehicles, or contaminants, or any combination thereof. In some embodiments, the substance, compound or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process-related components, mycoplasma, pyrogens, bacterial endotoxins, and foreign materials. Purity may be measured using techniques including, but not limited to, electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetric or titration, or any combination thereof.

As used herein, the term "yield" of any given substance, compound or material has its ordinary and customary meaning as understood from the specification, and refers to the actual total amount of the substance, compound or material relative to the intended total amount. For example, the yield of a substance, compound, or material is, at least about, no more than, or no more than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total amount expected, including all decimal amounts therebetween. In any production step, the yield may be affected by the efficiency of the reaction or process, unwanted side reactions, degradation, the quality of the input substance, compound or material or the loss of the desired substance, compound or material.

As used herein, "pharmaceutically acceptable" has its ordinary and customary meaning as understood in the specification and refers to carriers, excipients and/or stabilizers that are non-toxic or have an acceptable level of toxicity to the cells or mammals to which they are exposed at the dosages and concentrations employed. As used herein, "pharmaceutically acceptable" diluents "," excipients "and/or" carriers "have their ordinary and customary meaning as understood in the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, the pharmaceutically acceptable diluents, excipients and/or carriers are approved by a regulatory agency of the federal, a state government or other regulatory agency, or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans, and non-human mammals, such as cats and dogs. The term diluent, excipient, and/or "carrier" may refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluents, excipients and/or carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions, and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and/or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. Physiologically acceptable carriers may also include one or more of antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt forming counterions such as sodium, and nonionic surfactants such as TWEEN ^®, polyethylene glycol (PEG) and PLURONICS ^®. The composition may also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, slow release formulations, and the like. The formulation should be suitable for the mode of administration.

Cryoprotectants are cell composition additives that improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryoprotectants include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methylformamide, dimethylformamide, glycerol 3-phosphate, proline, sorbitol, diethylene glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. Cryoprotectants may be used as part of a cryopreservation medium that contains other components, such as nutrients (e.g., albumin, serum, bovine serum, fetal bovine serum [ FCS ]), to enhance survivability of cells after thawing. In these cryopreservation media, the at least one cryoprotectant may be found at a concentration of, about, at least about, no more than or no more than about 0.01%、0.05%、0.1%、0.5%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、30%、40%、50%、60%、70%、80% or 90%, or any percentage within a range defined by any two of the foregoing numbers.

Other excipients having the desired properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediamine tetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate, sugars, glucose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be residual amounts or contaminants in the manufacturing process including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, beta-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof. The amount of excipient may be present in the composition in a percentage that is, is about, is at least about, does not exceed or does not exceed about 0%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、100% w/w or any weight percent within the range defined by any two of the numbers described above.

The term "pharmaceutically acceptable salt" has its ordinary and customary meaning as understood in the specification and includes relatively non-toxic inorganic and organic acid or base addition salts of compositions or excipients, including but not limited to analgesics, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from inorganic acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Examples of suitable inorganic bases for salt formation include hydroxides, carbonates and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, such organic bases of this class may include, but are not limited to, mono-, di-and tri-alkylamines, including methylamine, dimethylamine and triethylamine, mono-, di-or tri-hydroxyalkylamines, including monoethanolamine, diethanolamine and triethanolamine, amino acids, including glycine, arginine and lysine, guanidine, N-methylglucamine, L-glutamine, N-methylpiperazine, morpholine, ethylenediamine, N-benzylphenethylamine, trimethylolethane.

The appropriate formulation depends on the route of administration selected. Techniques for formulating and administering the compounds described herein are known to those of skill in the art. There are a variety of techniques in the art for administering compounds including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intra-aural, epidural, extradermal, aerosol, parenteral delivery, including intramuscular, subcutaneous, intra-arterial, intravenous, portal intravenous, intra-articular, intradermal, intraperitoneal, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injection. The pharmaceutical composition will generally be adapted to the particular intended route of administration.

As used herein, a "carrier" has its ordinary and customary meaning as understood in the specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and/or incorporation of the compound into cells, tissues, and/or body organs.

As used herein, a "diluent" has its ordinary and customary meaning as understood in the specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, diluents may be used to increase the volume of a powerful drug that is too small in mass to be manufactured and/or administered. The diluent may also be a liquid for dissolving the drug for administration by injection, ingestion or inhalation. A common form of diluent in the art is an aqueous buffer solution such as, but not limited to, phosphate buffered saline that mimics the composition of human blood.

As used herein, the term "basement membrane matrix" or "extracellular matrix" has its ordinary and customary meaning as understood in the specification and refers to any biological or synthetic compound, substance or composition that enhances cell attachment and/or growth. Any extracellular matrix known in the art, as well as any mimetic or derivative thereof, may be used in the methods disclosed herein. Some examples of extracellular matrices or mimics or derivatives thereof include, but are not limited to, cell-based feeder layers, polymers, proteins, polypeptides, nucleic acids, carbohydrates, lipids, polylysines, polyornithines, collagens IV, gelatins, fibronectin, vitronectin, laminin-511, elastin, tenascin, heparan sulfate, integrins, entactin, osteopontin, basement membrane glycans, fibrin, basement membrane, matrigel ^®, hydrogels, PEI, WGA, or hyaluronic acid, or any combination thereof. Common basement membrane matrices used in the laboratory are those isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. However, these basement membrane matrices are derived from non-human animals and thus contain heterologous components that prevent their use in humans. They are also undefined, which may lead to variability in manufacturing, as well as potentially carrying pathogens. Thus, in some embodiments, methods for culturing cells may involve the use of synthetic and/or defined alternatives to these heterogeneous substrate membrane matrices. The use of a non-heterogeneous substrate membrane matrix or a mimetic or derivative thereof enables the manufacture of a biological product that is more suitable for human use.

The term "passaging" as used herein has its ordinary and customary meaning as understood from the specification and refers to a conventional process performed in a biological cell culture process to maintain an active cell population for a long period of time. Since cells are typically proliferative in cell culture, they undergo multiple mitotic cycles until they occupy the available space, which is typically the surface of a cell culture vessel (e.g., plate, dish, or flask) submerged under culture medium. For example, cells may be grown as a monolayer on the surface of a cell culture vessel. If the growing cells occupy the entire available space on the surface, they cannot proliferate further and may exhibit aging behavior. To continue cell growth (which may be done to maintain the viability and proliferative properties of the cells and/or to expand the number of cells for downstream purposes), the cells may be passaged by taking a portion of the cells and inoculating this portion onto a fresh surface in the culture medium (e.g., the fresh surface of a cell culture vessel). This fraction of cells will continue to proliferate and multiply until they occupy the available space on the new surface, where such passaging can be repeated continuously.

The microstructure of the liver consists of a polygonal structure called "liver lobules". Classically, these leaflets present a hexagonal structure, but other geometries are observed depending on the tissue specification. Each leaflet unit contains a sheet or layer of hepatocytes enclosed by a bundle of blood vessels surrounding an internal central vein and referred to as the hepatic trisomy, which consists of the portal vein, hepatic artery, and bile duct. Liver activity occurs when blood flows from the peripheral hepatic portal trisomy through hepatocytes and into the central vein to return to the circulatory system. Due to the asymmetric organization of these leaflets, the hepatocyte layer is divided into three regions. The cells in the "periportal region" (region 1) are closest to the hepatic trisomy and receive the most oxygenated blood, the pericentral region (region 3) is closest to the central vein and thus receives the least amount of oxygenated blood, and the transition region (region 2) is between regions 1 and 3. Due to this separation, each region of hepatocytes exhibits different activities. For example, the hepatocytes of region 1 are involved in oxidative liver functions such as gluconeogenesis and oxidative metabolism of fatty acids, while the hepatocytes of region 3 are involved in glycolysis, adipogenesis and cytochrome P450-mediated detoxification. In some embodiments, the liver organoids disclosed herein exhibit portal vein Zhou Yang characteristics similar to tissue found in the periportal region of the hepatic lobule, including the functionality and cellular marker characteristics of the periportal region.

The term "bilirubin" as used herein has its ordinary and customary meaning as understood in the specification and refers to naturally occurring metabolites produced by normal catabolic degradation of heme. Bilirubin is produced by the enzyme biliverdin reductase catalyzing biliverdin. In the liver, bilirubin is conjugated to glucuronic acid by a family of enzymes known as UDT-glucuronyltransferase (UGT). This conjugation makes bilirubin water-soluble, enabling it to be carried in bile to the small intestine and colon, whereby it is further metabolized into waste products. Dysfunctional bilirubin metabolism, particularly due to abnormal UGT function that prevents bilirubin binding, results in the accumulation of bilirubin and is associated with various diseases characterized by hyperbilirubinemia. However, it is worth noting that although excess bilirubin is detrimental, bilirubin also has antioxidant capacity and thus may have a beneficial effect in reducing oxidative damage in cells.

The term "hyperbilirubinemia" as used herein has its ordinary and customary meaning as understood from the specification, and refers to a condition in which bilirubin levels are elevated, bilirubin being a natural product of heme catabolism. Bilirubin is filtered from the blood by the liver and converted to a water-soluble intermediate, which is then released in the bile to the intestine, metabolized by the microbiota, and excreted as waste. In newborns, bilirubin levels initially cleared by the mother through the placenta may not be adequately cleared by the immature liver. Excessive levels of bilirubin can potentially cause severe neurological damage (nuclear jaundice). Hyperbilirubinemia may also be caused by diseases affecting the liver, such as hepatitis and cirrhosis, in adults. Neonatal hyperbilirubinemia is treated by phototherapy, or in extreme cases by blood transfusion, while adult treatment is aimed at the root cause.

As used herein, the terms "L-gulonolactone oxidase" and "GULO" have their ordinary and customary meanings as understood in the specification, and refer to enzymes that catalyze the production of L-wood-hex-3-gulonolactone and hydrogen peroxide. L-wood-hexa-3-gulonolactone is then spontaneously converted to ascorbic acid (vitamin C). Thus, this enzyme is involved in the biosynthesis of vitamin C, an essential nutrient involved in many biological functions, such as the use as cofactor for several important enzymes and as an antioxidant. Notably, humans and other subnasal primates, certain species of bats and guinea pigs have evolved to carry non-functional GULO genes. Thus, these organisms are unable to synthesize ascorbic acid and require the intake of vitamin C from the diet or supplement, where a deficiency of vitamin C can lead to scurvy. As used in the disclosure herein, a "functional GULO protein" is a GULO protein that has catalytic activity of L-gulonolactone to cause ascorbic acid production. In contrast, an "inactive" GULO protein or "nonfunctional" GULO protein is a protein that does not have catalytic activity to produce ascorbic acid. Human and human-derived cells contain nonfunctional GULO proteins and do not have the ability to synthesize ascorbic acid. However, as disclosed herein, human cells may be engineered to express functional GULO proteins to achieve ascorbic acid synthesis capability. These functional GULO proteins can be expressed in human cells (or other cells that are incapable of normally synthesizing ascorbic acid) by conventional cloning methods, such as genetically engineering cells to have genetic sequences encoding the functional GULO proteins.

As used herein, the term "% w/w" or "% wt/wt" has its ordinary and customary meaning as understood in the specification and refers to the ratio of the weight of an ingredient or agent to the total weight of the composition multiplied by 100. As used herein, the term "% v/v" or "% vol/vol" has its ordinary and customary meaning as understood in the specification and refers to the ratio of the volume of a liquid of a compound, substance, ingredient or medicament to the total volume of the liquid of the composition multiplied by 100.

As used herein, the term "exogenous" has its ordinary and customary meaning as understood in the specification and refers to external factors originating outside of a biological sample (e.g., cell population, organoid, etc.), rather than naturally occurring and/or produced by the biological sample itself. As used herein, exogenous components, agents, and/or conditions are components, agents, and/or conditions added to the compositions described herein, but this does not necessarily exclude the possibility that the same components, agents, and/or conditions also exist through functions endogenous to the biological sample.

The terms "liver organoid" and "hepatocyte organoid" are used interchangeably herein and refer to a population of cells that differentiate in vitro to form self-organizing structures, typically three-dimensional (3D), and include one or more functional cell types. Liver organoids differ from naturally occurring liver tissue in many ways. For example, in contrast to naturally occurring liver tissue, liver organoids may have a structure with a single lumen and generally spherical shape, and may include a non-natural basement membrane. The individual lumens of the liver organoids contain 3D tissue, but typically do not produce any hepatic lobular or chordal structures, as does naturally occurring liver tissue. Liver organoids are also generally free of hematopoietic tissues and acquired immune cell subsets, such as T cell lineages. Furthermore, liver organoids may have different outflow mechanisms compared to naturally occurring liver tissue, as liver organoids may have a three-dimensional structure with a luminal structure but no drainage mechanism. Furthermore, liver organoids are often unable to accept dietary input because they lack a tube and connected vascular channels.

The liver organoids may be derived from Pluripotent Stem Cells (PSC), including at least Embryonic Stem Cells (ESC) or Induced Pluripotent Stem Cells (iPSC). Liver organoids may also be formed from liver-derived stem cells. Typically, liver organoids may self-organize in a manner similar to what occurs in vivo by cell sorting and spatially restricted lineage typing, but are optionally targeted in vitro, e.g., by careful introduction of exogenous and/or endogenous differentiation factors and/or conditions as described herein, optionally through one or more targeting steps, optionally involving the introduction of one or more components.

As used herein, the term "mature liver organoid" refers to a liver organoid that continues to develop from the liver organoid to include, in various embodiments, a luminal protrusion resembling a bile duct and/or a structure having a single lumen and generally having a spherical shape. Mature liver organoids can exhibit lumens with smaller dimensions and reduced roundness compared to the lumens of liver organoids. In some embodiments, the mature liver organoids can be produced by adding exogenous bilirubin and/or amino acid supplements as described herein. In some embodiments, mature liver organoids may be characterized by reduced levels of AFP, CDX2, and/or NANOG relative to liver organoid expression, and/or by increased levels of ALB, SLC4A2, and/or HO-1 relative to liver organoid expression. In some embodiments, the mature liver organoids can be characterized as expressing CYP2E1, CYP7A1, PROXI, MRP3, and/or OATP2. In some embodiments, the mature liver organoids may exhibit increased levels of CYP3A4 and/or CYP1A2 protein and/or enzymatic activity relative to the liver organoids.

As used herein, the term "hyperbilirubinemia liver organoids" refers to liver organoids that have been exposed to high concentrations of bilirubin (typically provided exogenously through one or more administrations) to mimic the hyperbilirubinemia state. In some embodiments, the hyperbilirubinemia liver organoids comprise genetic abnormalities that alter bilirubin metabolism, such as resulting in increased levels of bilirubin anabolism and/or decreased levels of bilirubin catabolism. Hyperbilirubinemia liver organoids may be characterized as expressing elevated levels of UGT1A1 and/or NRF2 relative to liver organoids not exposed to high concentrations of bilirubin.

As used herein, the term "tissue culture surface" has its ordinary and customary meaning as understood in the specification and refers to a surface of a substrate on which cells can aggregate and/or adhere to promote cell growth, differentiation, and/or function.

As used herein, the term "engineered" refers to entities that are artificially produced, including cells, nucleic acids, polypeptides, vectors, and the like. In at least some instances, the engineered entity is synthetic and contains elements that are not naturally occurring or constructed in the manner in which it is used in the present disclosure. In certain embodiments, the construct and/or vector is engineered by recombinant nucleic acid technology, and the cell is engineered by transfection or transduction of the engineered vector. Cells may be engineered to express heterologous proteins that the cell does not naturally express, either because the heterologous proteins are recombinant or synthetic, or because the cell does not naturally express these proteins.

Stem cells

As used herein, the term "totipotent stem cell" (also referred to as a universal stem cell) has its ordinary and customary meaning as understood from the specification, and is a stem cell that can differentiate into embryonic and extra-embryonic cell types. Such cells can construct a complete, viable organism. These cells are fused from ova and sperm cells. Cells resulting from the first few divisions of fertilized eggs are also totipotent.

As used herein, the term "Embryonic Stem Cells (ESCs)", also commonly abbreviated as ES cells, has its ordinary and customary meaning as understood in the specification, and refers to cells that are pluripotent and derived from the inner cell mass of a blastocyst (i.e., early embryo).

As used herein, the term "Pluripotent Stem Cell (PSC)" has its ordinary and customary meaning as understood in the specification and encompasses any cell that can differentiate into almost all cell types of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (inner gastric wall, gastrointestinal tract, lung), mesoderm (muscle, bone, blood, genitourinary) and ectoderm (epidermal tissue and nervous system). PSC can be a progeny of inner cell mass cells of a preimplantation blastocyst, or obtained by inducing non-pluripotent cells, such as adult cells, by forcing expression of certain genes. The pluripotent stem cells may be derived from any suitable source. Examples of multipotent stem cell sources include mammalian sources, including human, rodent, pig and bovine.

As used herein, the term "induced pluripotent stem cells (ipscs)", also commonly abbreviated as iPS cells, has its ordinary and customary meaning as understood from the specification, and refers to pluripotent stem cell types artificially derived from normally non-pluripotent cells such as adult cells by inducing "forced" expression of certain genes. hiPSC refers to a human iPSC. In some methods known in the art, ipscs are obtained by transfecting certain stem cell-related genes into non-pluripotent cells such as adult fibroblasts. Transfection may be accomplished by viral transduction using a virus (e.g., retrovirus or lentivirus). The transfected genes may contain the main transcriptional regulatory factors Oct-3/4 (POU 5F 1) and Sox2, but other genes may also enhance induction efficiency. After 3 to 4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically, and are typically isolated by morphological selection, doubling time, or by reporter and antibiotic selection. As used herein, ipscs include first generation ipscs, second generation ipscs in mice, and human induced pluripotent stem cells. In some methods, a retrovirus system is used to convert human fibroblasts into pluripotent stem cells using four key genes (Oct 3/4, sox2, klf4, and c-Myc). In other methods, lentiviral systems are used to transform somatic cells with OCT4, SOX2, NANOG, and LIN 28. Genes induced in iPSC expression include, but are not limited to, oct-3/4 (POU 5 FI), certain members of the Sox gene family (e.g., sox1, sox2, sox3, and Sox 15), certain members of the Klf family (e.g., klf1L, klf2, klf4, and Klf 5), certain members of the Myc family (e.g., C-Myc, L-Myc, and N-Myc), nanog, L1N28, tert, fbx15, eras, ECAT15-1, ECAT15-2, tel1, β -catenin, ECAT1, esg1, dnmt3L, ECAT, gdf3, fthl17, sal14, rex1, UTF1, stella, stat3, grb2, prdm, nr5a1, nr5a2, or E-cadherin, or any combination thereof.

As used herein, the term "precursor cell" has its ordinary and customary meaning as understood in the specification and encompasses any cell from which one or more precursor cells acquire the ability to self-renew or differentiate into one or more specialized cell types that can be used in the methods described herein. In some embodiments, the precursor cells are pluripotent or have the ability to become pluripotent. In some embodiments, the precursor cells are treated with an external factor (e.g., a growth factor) to obtain pluripotency. In some embodiments, the precursor cells may be totipotent (or totipotent) stem cells, pluripotent stem cells (induced or non-induced), multipotent stem cells, oligopotent stem cells, and monopotent stem cells. In some embodiments, the precursor cells may be from an embryo, infant, child, or adult. In some embodiments, the precursor cells may be somatic cells that are subjected to a treatment such that pluripotency is conferred by genetic manipulation or protein/peptide treatment. Precursor cells include embryonic stem cells (embryonic stem cell, ESCs), embryonic cancer cells (embryonic carcinoma cell, EC), and ectodermal stem cells (epiblast stem cell, epiSC).

In some embodiments, one step is to obtain pluripotent stem cells or to obtain stem cells that can be induced to be pluripotent. In some embodiments, the pluripotent stem cells are derived from embryonic stem cells, which in turn are derived from totipotent cells of early mammalian embryos, and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are multipotent stem cells derived from the inner cell mass of the blastocyst of an early embryo. Methods for deriving embryonic stem cells from embryonic cells are well known in the art. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described in the present application, but those skilled in the art will appreciate that the methods and systems described herein are applicable to any stem cell.

Additional stem cells that may be used in accordance with embodiments of the present disclosure include, but are not limited to, databases hosted by the national stem cell library (NSCB) of the human embryo stem cell research center at the University of California (UCSF) in san francisco, the WISC cell library at the institute of Wi, the university of wisconsin stem cell and regenerative medicine center (UW-SCRMC), novocell, inc (san diego, california), CELLARTIS AB (goldburg, sweden), ES Cell International Pte Ltd (singapore), the university of israel Technion (israel sea), and those stem cells provided or described by the stem cell databases hosted by the university of prinston and pennsylvania. Exemplary embryonic stem cells that can be used in embodiments according to the present disclosure include, but are not limited to SA01（SA001）;SA02（SA002）;ES01（HES-1）;ES02（HES-2）;ES03（HES-3）;ES04（HES-4）;ES05（HES-5）;ES06（HES-6）;BG01（BGN-01）;BG02（BGN-02）;BG03（BGN-03）;TE03（13）;TE04（14）;TE06（16）;UCOI（HSF1）;UC06（HSF6）;WA01（HI）;WA07（H7）;WA09（H9）;WA13（H13）;WA14（H14）. exemplary human pluripotent cell lines including, but not limited to TkDA3-4、1231A3、317-D6、317-A4、CDH1、5-T-3、3-34-1、NAFLD27、NAFLD77、NAFLD150、WD90、WD91、WD92、L20012、C213、1383D6、FF or 317-12 cells.

In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term "directed differentiation" describes a process by which less specialized cells become a specific specialized target cell type. Specificity of a specific target cell type can be determined by any suitable method that can be used to define or alter the initial cell fate. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

In some embodiments, adenoviruses may be used to transport the four genes necessary to produce ipscs that are substantially identical to embryonic stem cells. Since adenovirus does not combine any of its own genes with the targeted host, the risk of tumor production is eliminated. In some embodiments, ipscs are generated using non-viral based techniques. In some embodiments, reprogramming may be accomplished by a plasmid without any viral transfection system at all, albeit at a very low efficiency. In other embodiments, direct delivery of the protein is used to generate ipscs, thus eliminating the need for viruses or genetic modifications. In some embodiments, it is possible to generate mouse iPSCs using similar methods by repeatedly treating cells with certain proteins introduced into the cells by polyarginine anchors sufficient to induce pluripotency. In some embodiments, expression of the pluripotency inducing gene may also be increased by treating the somatic cells with FGF2 under hypoxic conditions.

As used herein, the term "feeder cell" has its ordinary and customary meaning as understood in the specification and refers to a cell that supports the growth of pluripotent stem cells, such as by secretion of growth factors into a culture medium or display on the surface of the cell. Feeder cells are typically adherent cells and may have growth arrest. For example, feeder cells may be growth arrested by irradiation (e.g., gamma rays), mitomycin-C treatment, electrical pulsing, or mild chemical fixation (e.g., with formaldehyde or glutaraldehyde). However, feeder cells do not necessarily have to be growth arrested. Feeder cells can be used for purposes such as secretion of growth factors, display of growth factors on the cell surface, detoxification of culture media, or synthesis of extracellular matrix proteins. In some embodiments, the feeder cells are allogeneic or xenogeneic with the supported target stem cells, which may have an impact on downstream applications. In some embodiments, the feeder cells are mouse cells. In some embodiments, the feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76/7 cells, human fibroblasts, human foreskin fibroblasts, human dermis fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human oviduct epithelial cells. In some embodiments, conditioned medium prepared from feeder cells is used in place of or in combination with feeder cell co-cultures. In some embodiments, feeder cells are not used during proliferation of the target stem cells.

Differentiation of PSC

Known methods for preparing downstream cell types (such as definitive endoderm, foregut endoderm, hindgut endoderm, and/or liver lineages) from pluripotent cells (e.g., ipscs or ESCs) are suitable for use in some embodiments of the methods described herein. In some embodiments, the pluripotent cells are derived from morula. In some embodiments, the pluripotent stem cells are stem cells. The stem cells used in these methods may include, but are not limited to, embryonic stem cells or induced pluripotent stem cells. The embryonic stem cells may be derived from an intra-embryonic cell mass or from embryonic gonadal ridges. Embryonic stem cells may originate from a variety of animal species including, but not limited to, various mammalian species including humans. In some embodiments, human embryonic stem cells are used to produce definitive endoderm or other downstream cell types, such as the hindforegut, hindforegut endoderm, and/or liver lineages. In some embodiments, ipscs are used to generate definitive endoderm or other downstream cell types, such as the hindforegut, hindforegut endoderm, and/or liver lineages. In some embodiments, human ipscs (hipscs) are used to generate definitive endoderm or other downstream cell types, such as the posterior foregut, posterior foregut endoderm, and/or liver lineages.

In some embodiments, PSCs, such as ESCs and ipscs, undergo committed differentiation into embryonic germ layer cells, organ tissue progenitor cells, and then differentiate into tissue such as liver tissue or any other biological tissue. In some embodiments, the directed differentiation is performed in a stepwise manner to obtain each of the differentiated cell types, wherein molecules (e.g., growth factors, ligands, agonists, antagonists) are added sequentially as differentiation proceeds. In some embodiments, the directed differentiation is performed in a non-stepwise manner, wherein molecules (e.g., growth factors, ligands, agonists, antagonists) are added simultaneously. In some embodiments, directed differentiation is achieved by selectively activating certain signaling pathways in PSCs or any downstream cells.

In some embodiments, the embryonic stem cells or ipscs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a period of time that is, is about, is at least about, is not more than, or is not more than about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours, or any time within a range defined by any two of the foregoing times (e.g., 6 hours to 300 hours, 24 hours to 120 hours, 48 hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours). In some embodiments, more than one small molecule compound, activator, inhibitor, or growth factor is added. In these cases, more than one small molecule compound, activator, inhibitor, or growth factor may be added simultaneously or separately.

In some embodiments, the embryonic stem cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors at a concentration of, at least about, no more than, or no more than about 10ng/mL、20ng/mL、50ng/rnL、75ng/mL、100ng/mL、120ng/mL、150ng/mL、200ng/mL、500ng/mL、1000ng/mL、1200ng/mL、1500ng/mL、2000ng/mL、5000ng/mL、7000ng/mL、10000ng/mL or 15000ng/mL, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 10ng/mL to 15000ng/mL, 100ng/mL to 5000ng/mL, 500ng/mL to 2000ng/mL, 10ng/mL to 2000ng/mL, or 1000ng/mL to 15000 ng/mL). In some embodiments, the concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, the concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors varies during the course of the treatment. In some embodiments, more than one small molecule compound, activator, inhibitor, or growth factor is added. In these cases, the concentration of more than one small molecule compound, activator, inhibitor, or growth factor may be different.

In some embodiments, the ESC or iPSC is cultured in a growth medium that supports stem cell growth. In some embodiments, the ESC or iPSC is cultured in a stem cell growth medium. In some embodiments, the stem cell growth medium is RPMI 1640, DMEM/F12 or advanced DMEM/F12. In some embodiments, the stem cell growth medium comprises Fetal Bovine Serum (FBS). In some embodiments, the stem cell growth medium comprises FBS at a concentration of, about, at least about, no more than, or no more than about 0%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19% or 20%, or any percentage within a range (e.g., 0% to 20%, 0.2% to 10%, 2% to 5%, 0% to 5%, or 2% to 20%) defined by any two of the foregoing concentrations. In some embodiments, the stem cell growth medium does not contain a heterologous component. In some embodiments, the growth medium includes one or more small molecule compounds, activators, inhibitors, or growth factors.

In some embodiments, the pluripotent stem cells are prepared from somatic cells. In some embodiments, the pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, the pluripotent stem cells are cryopreserved. In some embodiments, the somatic cells are cryopreserved. In some embodiments, the pluripotent stem cells are prepared from PBMCs. In some embodiments, the human PSC is prepared from human PBMCs. In some embodiments, the pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, the PBMCs are grown on feeder cell substrates. In some embodiments, the PBMCs are grown on Mouse Embryonic Fibroblast (MEF) feeder cell substrates. In some embodiments, the PBMCs are grown on irradiated MEF feeder cell substrates.

In some embodiments, the stem cells are treated with one or more growth factors to differentiate into definitive endoderm cells. Such growth factors may include growth factors from the TGF-beta superfamily. In some embodiments, the one or more growth factors include the Nodal/activin and/or BMP subgroup of the TGF- β growth factor superfamily. In some embodiments, the one or more growth factors are selected from the group consisting of Nodal, activin A, activin B, BMP4, wnt3a, or a combination of any of these growth factors. In some embodiments, the stem cells are contacted with activin a. In some embodiments, the stem cells are contacted with activin a and BMP 4.

In some embodiments, definitive Endoderm (DE) can further undergo foreendoderm patterning, foregut specialization, and morphogenesis, depending on FGF, wnt, BMP or retinoic acid, or any combination thereof. In some embodiments, the human PSC is effectively directed to differentiate into liver epithelium and mesenchymal tissue in vitro. It should be appreciated that an agent such as a growth factor may be added to any stage of development to promote the formation of a particular type of liver tissue. In some embodiments, sirnas and/or shrnas targeting cellular components associated with FGF, wnt, BMP or retinoic acid signaling pathways are used to inhibit or activate these pathways.

Culture and expansion of definitive endoderm, foregut and downstream cell types

Methods of preparing liver organoids have been previously explored in, for example, ouchi et al "Modeling Steatohepatitis in Humans with Pluripotent Stem Cell-Derived Organoids" Cell Metabolism (2019) 30(2):374-384;Shinozawa et al "High-Fidelity Drug-Induced Fiver Injury Screen Using Human Pluripotent Stem Cell-Derived Organoids" Gastroenterology (2021) 160(3):831-846;PCT, disclose WO 2018/085615, WO 2018/191673, WO 2018/226267, WO 2019/126626, WO 2020/023245, WO 2020/069285 and WO 2021/262667, each of which is hereby expressly incorporated by reference in its entirety. The disclosure of liver organoid compositions and methods of making the same applies to Human Liver Organoids (HLOs) described herein.

In some embodiments, pluripotent stem cells, definitive endoderm, hindforegut endoderm, foregut endoderm, and/or downstream hepatocyte types are cultured and expanded as described herein. In some embodiments, pluripotent stem cells, definitive endoderm, hindforegut endoderm and/or foregut endoderm are cultured and expanded as described herein. In some embodiments, pluripotent stem cells, definitive endoderm, hind foregut and/or hind foregut endoderm cells are cultured and expanded as described herein. In some embodiments, foregut endoderm cells are cultured and expanded as described herein.

In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm, foregut endoderm and/or downstream hepatocyte type are contacted with a TGF-b pathway inhibitor. In some embodiments, the TGF-b pathway inhibitor comprises one or more of A83-01, repSox, LY365947, and SB 431542. In some embodiments, the cells are not treated with a TGF-b pathway inhibitor. The TGF-b pathway inhibitors provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm, and/or downstream hepatocyte type are contacted with an FGF pathway activator. In some embodiments, the FGF pathway activator comprises an FGF protein. In some embodiments, the FGF protein comprises a recombinant FGF protein. In some embodiments, the FGF pathway activator comprises one or more of ：FGF1、FGF2、FGF3、FGF4、FGF4、FGF5、FGF6、FGF7、FGF8、FGF8、FGF9、FGF10、FGF11、FGF12、FGF13、FGF14、FGF15（FGF19、FGF15/FGF19）、FGF16、FGF17、FGF18、FGF20、FGF21、FGF22 or FGF23 of the following. In some embodiments, the cells are not treated with an FGF pathway activator. The FGF pathway activators provided herein can be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

In some embodiments, the pluripotent stem cell, definitive endoderm, hind foregut endoderm, and/or downstream hepatocyte type is contacted with a Wnt pathway activator. In some embodiments, the Wnt pathway activator comprises a Wnt protein. In some embodiments, the Wnt protein comprises a recombinant Wnt protein. In some embodiments, the Wnt pathway activator comprises Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11、Wnt16、BML 284、IQ-1、WAY 262611 or any combination thereof. In some embodiments, the Wnt pathway activator comprises a GSK3 signaling pathway inhibitor. In some embodiments, the Wnt pathway activator comprises CHIR99021, CHIR 98014, AZD2858, BIO, AR-a014418, SB 216763, SB 415286, aloxin, indirubin, altbolone, kenparone, lithium chloride, TDZD, or TWS119, or any combination thereof. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the cells are not treated with a Wnt pathway activator. The Wnt pathway activators provided herein may be used in combination with any one of the other growth factors, pathway activators, or pathway inhibitors provided herein.

In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm, and/or downstream hepatocyte type are contacted with a VEGF pathway activator. In some embodiments, the VEGF pathway activator comprises one or more of VEGF or GS 4012. In some embodiments, the cells are not treated with a VEGF pathway activator. The VEGF pathway activators provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm, foregut endoderm and/or downstream hepatocyte type are contacted with an EGF pathway activator. In some embodiments, the EGF pathway activator comprises EGF. In some embodiments, the cells are not treated with an EGF pathway activator. The EGF pathway activators provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm, and/or downstream hepatocyte type are contacted with ascorbic acid. In some embodiments, the cells are not treated with ascorbic acid. The ascorbic acid provided herein may be used in combination with any of the other growth factors, pathway activators or pathway inhibitors provided herein.

In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm, foregut endoderm and/or downstream hepatocyte type are contacted with a BMP pathway activator or BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises BMP protein. In some embodiments, the BMP protein is a recombinant BMP protein. In some embodiments, the BMP pathway activator comprises BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, or IDE2, or any combination thereof. In some embodiments, the BMP pathway inhibitor comprises Noggin, repSox, LY364947,364947, LDN-193189, SB431542, or any combination thereof. In some embodiments, the cells are not treated with BMP pathway activators or BMP pathway inhibitors. The BMP pathway activators or BMP pathway inhibitors provided herein can be used in combination with any of the other growth factors, pathway activators or pathway inhibitors provided herein.

In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm, and/or downstream hepatocyte type are contacted with a retinoic acid pathway activator. In some embodiments, the retinoic acid pathway activator comprises retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, or AM580, or any combination thereof. In some embodiments, the cells are not treated with a retinoic acid pathway activator. The retinoic acid pathway activators provided herein may be used in combination with any of the other growth factors, pathway activators or pathway inhibitors provided herein.

In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm, foregut endoderm and/or downstream hepatocyte types are converted to hepatocyte types via a "one-step process". In some embodiments, the pluripotent stem cells are transformed into a hepatocyte type via a "one-step" method. For example, one or more molecules that can differentiate pluripotent stem cells into DE cultures (e.g., activin a) are combined with additional molecules that can promote directed differentiation of DE cultures (e.g., FGF4, CHIR99021, RA) to directly treat pluripotent stem cells.

In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm, foregut endoderm and/or downstream hepatocyte types are expanded in the cell culture. In some embodiments, pluripotent stem cells (e.g., ESC and/or iPSC), definitive endoderm, hindforegut endoderm, and/or downstream hepatocyte types are expanded in cell culture. In some embodiments, the pluripotent stem cells, definitive endoderm, hindforegut endoderm and/or downstream hepatocyte types are expanded in the basement membrane matrix. In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm and/or downstream hepatocyte types are expanded in Matrigel ^®. In some embodiments, the pluripotent stem cells, definitive endoderm, hindforegut endoderm and/or downstream hepatocyte types are expanded in a basement membrane matrix that does not comprise non-human animal components. In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm and/or downstream hepatocyte types are expanded in a non-xenogenic basement membrane matrix. In some embodiments, the pluripotent stem cells, definitive endoderm, hind foregut endoderm and/or downstream hepatocyte types are not expanded in Matrigel ^®. In some embodiments, the pluripotent stem cells, definitive endoderm, hindforegut endoderm and/or downstream hepatocyte types are expanded in laminin, collagen IV, entactin, basement membrane glycans, fibrin and/or hydrogels. In some embodiments, pluripotent stem cells, definitive endoderm, hind foregut endoderm and/or downstream hepatocyte types are expanded in a cell culture comprising a ROCK inhibitor (e.g., Y-27632).

In some embodiments, pluripotent stem cells (e.g., ESC and/or iPSC) are differentiated into definitive endoderm cells. In some embodiments, the pluripotent stem cells are differentiated into definitive endoderm cells by contacting the pluripotent stem cells with activin A, BMP, or both. In some embodiments, the pluripotent stem cells are contacted with activin a at a concentration that is, is about, is at least about, does not exceed or does not exceed about 10ng/mL、20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL、100ng/mL、110ng/mL、120ng/mL、130ng/mL、140ng/mL、150ng/mL、160ng/mL、170ng/mL、180ng/mL、190ng/mL or 200ng/mL, or is any concentration within a range defined by any two of the foregoing concentrations (e.g., 10ng/mL to 200ng/mL, 10ng/mL to 100ng/mL, 100ng/mL to 200ng/mL, or 50ng/mL to 150 ng/mL). In some embodiments, the pluripotent stem cells are contacted with activin A at a concentration of 100ng/mL or about 100 ng/mL. In some embodiments, the pluripotent stem cells are contacted with BMP4 at a concentration that is, is about, is at least about, is not more than or is not more than about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL、100ng/mL、110ng/mL、120ng/mL、130ng/mL、140ng/mL、150ng/mL、160ng/mL、170ng/mL、180ng/mL、190ng/mL or 200ng/mL, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 1ng/mL to 200ng/mL, 1ng/mL to 100ng/mL, 25ng/mL to 200ng/mL, 1ng/mL to 80ng/mL, or 25ng/mL to 100 ng/mL). In some embodiments, the pluripotent stem cells are contacted with BMP4 at a concentration of 50ng/mL or about 50 ng/mL.

Provided herein are methods for expanding metaintestinal cells. An exemplary method is shown in fig. 5H without limitation to specific characteristics, such as time and growth conditions illustrated in the figures. In some embodiments, the methods comprise a) dissociating a monolayer of metaforegut cells into metaforegut cells and/or metaforegut endoderm cells, b) seeding the metaforegut cells and/or metaforegut endoderm cells onto a tissue culture surface, and c) culturing the metaforegut cells and/or metaforegut endoderm cells with a TGF-b pathway inhibitor, a FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are further cultured with ascorbic acid. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are not cultured with ascorbic acid. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are further cultured with EGF. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are not cultured with EGF. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are further cultured with a ROCK inhibitor. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are not cultured with a ROCK inhibitor. In some embodiments, enzymatic and/or mechanical dissociation is used to dissociate the metaforegut cell monolayer into metaforegut cells and/or metaforegut endoderm cells. In some embodiments, enzymatic dissociation may involve the use of any conventional enzymatic dissociation solution commonly known in the art, such as Accutase, accumax, trypsin/EDTA, collagenase, dispase, trypEE Express, or TRYPLE SELECT. In some embodiments, mechanical dissociation may involve disrupting the cells using a pipette, microchannel, or other device with a well of appropriate size to mechanically shear the cell group without disrupting individual cells. In some embodiments, the posterior foregut cells and/or the posterior foregut endoderm cells are seeded onto the surface of the tissue container at a cell density of, about, at least about, no more than, or no more than about 1×10 ⁵, 2×10 ⁵, 3×10 ⁵, a cell density of, 4X 10 ⁵, 5X 10 ⁵, 6X 10 ⁵, 7X 10 ⁵, 8X 10 ⁵, 9×10 ⁵ 1X 10 ⁶ 2X 10 ⁶, 3X 10 ⁶, The surface area of the tissue culture surface of 4X 10 ⁶ or 5X 10 ⁶ cells/cm ², or a range defined by any two of the foregoing cell densities (e.g., 1X 10 ⁵ -5X 10 ⁶, 1X 10 ⁵ -5X 10 ⁵, 5X 10 ⁵ to 5X 10 ⁶ or 3X 10 ⁵ to 7X 10 ⁵ cells/cm ² surface area of tissue culture surface). In some embodiments, the metaintestinal cells and/or metaforeintestinal endoderm cells are seeded onto the tissue container surface at a cell density equal to or about 5 x 10 ⁵ cells/cm ² of surface area of the tissue culture surface. In some embodiments, the tissue culture surface is coated with a basement membrane matrix or a component thereof. In some embodiments, the base film matrix or component thereof does not comprise a non-human animal component, such that the base film matrix or component thereof is non-xenogeneic with humans. In some embodiments, the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the base film matrix or component thereof is not Matrigel ^®、Cultrex^® or Geltrex ^®. In some embodiments, the basement membrane matrix or component thereof comprises human fibronectin, collagen IV, entactin, basement membrane glycans, fibrin, and/or hydrogels or other substances that are not xenogeneic to humans. In some embodiments, the basement membrane matrix or component thereof is or comprises laminin. In some embodiments, the basement membrane matrix or component thereof is or comprises laminin-511. In some embodiments, the metaintestinal cells and/or metaforeintestinal endoderm cells are cultured until three-dimensional (3D) spheroids are spontaneously formed. in some embodiments, the metaforegut cells and/or metaforegut endoderm cells are cultured for a number of days, about, at least about, no more than, or no more than about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days, or a range defined by any two of the preceding days (e.g., 4 days-6 days, 2-35 days, 2-15 days, 20-35 days, or 10-20 days). in some embodiments, the metaintestinal cells and/or metaforeintestinal endoderm cells are cultured for at least 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days, or for at least any number of days within a range defined by any two of the preceding days (e.g., 4 days-6 days, 4 days-35 days, 4 days-15 days, 20 days-35 days, or 10 days-20 days).

In some embodiments of the methods provided herein, the TGF-b pathway inhibitor is selected from the group consisting of A83-01, repSox, LY365947, and SB431542. In some embodiments, the TGF-b pathway inhibitor is A83-01. In some embodiments, the TGF-b pathway inhibitor is provided at a concentration that is, is about, is at least about, is no more than, or is no more than about 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 500ng/mL, 600ng/mL, 700ng/mL, 800ng/mL, 900ng/mL, or 1000ng/mL, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 100nM-1000nM, 100nM-500nM, 500nM-1000nM, or 300nM-700 nM). In some embodiments, the TGF-b pathway inhibitor is provided at a concentration equal to or about 500 nM.

In some embodiments of the methods provided herein, the FGF pathway activator is selected from the group consisting of ：FGF1、FGF2、FGF3、FGF4、FGF4、FGF5、FGF6、FGF7、FGF8、FGF8、FGF9、FGF10、FGF11、FGF12、FGF13、FGF14、FGF15、FGF16、FGF17、FGF18、FGF19、FGF20、FGF21、FGF22 and FGF23. In some embodiments, the FGF pathway activator is FGF2. In some embodiments, the FGF pathway activator is provided at a concentration that is, is about, is at least about, does not exceed or does not exceed about 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, or 10ng/mL, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 1ng/mL-10ng/mL, 1ng/mL-5ng/mL, 5ng/mL-10ng/mL, or 3ng/mL-7 ng/mL). In some embodiments, the FGF pathway activator is provided at a concentration equal to or about 5 ng/mL.

In some embodiments of the methods provided herein, the Wnt pathway activator is selected from the group consisting of ：Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11、Wnt16、BML 284、IQ-1、WAY 262611、CHIR99021、CHIR 98014、AZD2858、BIO、AR-A014418、SB 216763、SB 415286、 aloxin, indirubin, altbolone, kenarone, lithium chloride, TDZD, and TWS119. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the Wnt pathway activator is provided at a concentration of, at about, at least about, no more than, or no more than about 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, or 8 μm, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 1 μm-8 μm, 1 μm-3 μm, 3 μm-8 μm, or 2 μm-4 μm). In some embodiments, the Wnt pathway activator is provided at a concentration equal to or about 3 μm.

In some embodiments of the methods provided herein, the VEGF pathway activator is selected from the group consisting of VEGF or GS4012. In some embodiments, the VEGF pathway activator is VEGF. In some embodiments, the VEGF pathway activator is provided at a concentration that is, is about, is at least about, does not exceed or does not exceed about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 1ng/mL-20ng/mL, 1ng/mL-10ng/mL, 10ng/mL-20ng/mL, or 5ng/mL-15 ng/mL). In some embodiments, the VEGF pathway activator is provided at a concentration equal to or about 10 ng/mL.

In some embodiments of the methods provided herein, the metaforegut cells and/or the metaforegut endoderm cells of step c) are cultured in a medium further comprising EGF. In some embodiments, EGF is provided at a concentration that is, is about, is at least about, does not exceed or does not exceed about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 10ng/mL-30ng/mL, 10ng/mL-20ng/mL, 20ng/mL-30ng/mL, or 15ng/mL-25 ng/mL). In some embodiments, EGF is provided at a concentration equal to or about 20 ng/mL. In some embodiments, the metaintestinal cells and/or metaintestinal endoderm cells of step c) are cultured in a medium that does not comprise EGF.

In some embodiments of the methods provided herein, the metaforegut cells and/or the metaforegut endoderm cells of step c) are cultured in a medium further comprising ascorbic acid. In some embodiments, the ascorbic acid is provided at a concentration that is, is at least about, does not exceed or does not exceed about 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, or 100 μg/mL, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 10 μg/mL-100 μg/mL, 10 μg/mL-50 μg/mL, 50 μg/mL-100 μg/mL, or 30 μg/mL-70 μg/mL). In some embodiments, the ascorbic acid is provided at a concentration equal to or about 50 μg/mL. In some embodiments, the metaintestinal cells and/or metaintestinal endoderm cells of step c) are cultured in a medium that does not comprise ascorbic acid.

In some embodiments of the methods provided herein, the metaforegut cells and/or the metaforegut endoderm cells of step c) are cultured with an inhibitor in a medium further comprising a ROCK inhibitor. In some embodiments, the ROCK inhibitor is provided at a concentration of, at about, at least about, no more than, or no more than about 1 μΜ,2 μΜ,3 μΜ,4 μΜ, 5 μΜ,6 μΜ,7 μΜ, 8 μΜ, 9 μΜ,10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, or 20 μΜ, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 1 μΜ -20 μΜ,1 μΜ -10 μΜ,10 μΜ -20 μΜ, or 5 μΜ -15 μΜ). In some embodiments, the ROCK inhibitor is provided at a concentration equal to or about 10 μg/mL. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the metaintestinal cells and/or metaintestinal endoderm cells of step c) are cultured in a medium that does not comprise a ROCK inhibitor.

In some embodiments, the metaforegut cells and/or metaforegut endoderm cells of the methods provided herein can be cultured for multiple generations. In some embodiments, steps a) -c) of the methods provided herein are repeated with the cells of step c). In some embodiments, the methods further comprise passaging the cells of step c) one or more times. In some embodiments, passage of the cells of step c) to the metaforegut cells and/or metaforegut endoderm cells does not spontaneously form spheroids. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are passaged and cultured for a number of days, about, at least about, no more than, or no more than about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or any number of days within a range defined by any two of the preceding days (e.g., 2 days-10 days, 2 days-4 days, 2 days-6 days, 4 days-10 days, 6 days-10 days, 4 days-6 days, or 3 days-7 days), and then the metaforegut cells and/or metaforegut endoderm cells are passaged again. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are passaged and cultured for at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or any number of days within a range defined by any two of the preceding days (e.g., 2 days-10 days, 2 days-4 days, 2 days-6 days, 4 days-10 days, 6 days-10 days, 4 days-6 days, or 3 days-7 days), and then the metaforegut cells and/or metaforegut endoderm cells are passaged again. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are passaged and cultured for 4 days, 5 days, or 6 days, and then the metaforegut cells and/or metaforegut endoderm cells are passaged again. In some embodiments, no more than 1, 2, or 3 cell passages are performed. In some embodiments, the total yield of metaforegut cells and/or metaforegut endoderm cells (which may be in spheroid form) according to the methods provided herein involving multiple passages is 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, or 1000-fold, or any fold within a range defined by any two of the foregoing (e.g., 50-1000-fold, 50-200-fold, 1000-fold, or 100-fold 300-fold) obtained in cell culture.

In some embodiments, the methods further comprise collecting the metaforegut cells and/or the metaforegut endoderm cells and differentiating the metaforegut cells and/or the metaforegut endoderm cells into a liver organoid. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are cultured until spontaneously forming a three-dimensional (3D) spheroid, optionally wherein the spheroid comprises a structure having a single lumen, and the metaforegut cells and/or metaforegut endoderm cells are collected from the spheroid. In some embodiments, the methods further comprise dissociating the spheroids into individual pellets of metaforegut cells and/or metaforegut endoderm cells and/or metaforegut endoderm cells prior to the differentiating step. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are collected from the metaforegut cell monolayer prior to the differentiating step by dissociating the metaforegut cell monolayer into individual metaforegut cells and/or metaforegut endoderm cells and/or a mass of metaforegut cells and/or metaforegut endoderm cells.

Provided herein in some embodiments are methods for expanding metaforegut cells and/or metaforegut endoderm and/or foregut endoderm cells. In some embodiments, the methods comprise a) dissociating a monolayer of metaforegut cells into metaforegut cells and/or metaforegut endoderm cells, b) seeding the metaforegut cells and/or metaforegut endoderm cells onto a tissue culture surface, and c) culturing the metaforegut cells and/or metaforegut endoderm cells with a TGF-b pathway inhibitor, a FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator. In some embodiments, enzymatic and/or mechanical dissociation is used to dissociate the metaforegut cell monolayer into metaforegut cells and/or metaforegut endoderm cells. In some embodiments, enzymatic dissociation may involve the use of any conventional enzymatic dissociation solution commonly known in the art, such as Accutase, accumax, trypsin/EDTA, collagenase, dispase, trypLE Express, or TRYPLE SELECT. In some embodiments, mechanical dissociation may involve disrupting the cells using a pipette, microchannel, or other device with a well of appropriate size to mechanically shear the cell group without disrupting individual cells. In some embodiments, the posterior foregut cells and/or the posterior foregut endoderm cells are seeded onto the surface of the tissue container at a cell density of, about, at least about, no more than, or no more than about 1×10 ⁵, 2×10 ⁵, 3×10 ⁵, a cell density of, 4X 10 ⁵, 5X 10 ⁵, 6X 10 ⁵, 7X 10 ⁵, 8X 10 ⁵, 9×10 ⁵ 1X 10 ⁶ 2X 10 ⁶, 3X 10 ⁶, The surface area of the tissue culture surface of 4X 10 ⁶ or 5X 10 ⁶ cells/cm ², or a range defined by any two of the foregoing cell densities (e.g., 1X 10 ⁵ -5X 10 ⁶, 1X 10 ⁵ -5X 10 ⁵, 5X 10 ⁵ to 5X 10 ⁶ or 3X 10 ⁵ to 7X 10 ⁵ cells/cm ² surface area of tissue culture surface). In some embodiments, the metaintestinal cells and/or metaforeintestinal endoderm cells are seeded onto the tissue container surface at a cell density equal to or about 5 x 10 ⁵ cells/cm ² of surface area of the tissue culture surface. In some embodiments, the tissue culture surface is coated with a basement membrane matrix or a component thereof. In some embodiments, the base film matrix or component thereof does not comprise a non-human animal component, such that the base film matrix or component thereof is non-xenogeneic with humans. In some embodiments, the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the base film matrix or component thereof is not Matrigel ^®、Cultrex^® or Geltrex ^®. In some embodiments, the basement membrane matrix or component thereof comprises human fibronectin, collagen IV, entactin, basement membrane glycans, fibrin, and/or hydrogels or other substances that are not xenogeneic to humans. In some embodiments, the basement membrane matrix or component thereof is or comprises laminin. In some embodiments, the basement membrane matrix or component thereof is or comprises laminin-511. In some embodiments, the metaintestinal cells and/or metaforeintestinal endoderm cells are cultured until three-dimensional (3D) spheroids are spontaneously formed. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells are cultured for a number of days, about, at least about, no more than, or no more than about 2, 3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, or a range defined by any two of the preceding days (e.g., 2-35 days, 2-15 days, 20-35 days, or 10-20 days). In some embodiments, the metaintestinal cells and/or metaforeintestinal endoderm cells are cultured for at least 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days, or at least any number of days within a range defined by any two of the preceding days (e.g., 4 days-35 days, 4 days-15 days, 20 days-35 days, or 10 days-20 days). In some embodiments, steps a) -c) of the methods provided herein are repeated with the cells of step c). In some embodiments, the methods further comprise passaging the cells of step c) one or more times. In some embodiments, passage of the cells of step c) to the metaforegut cells and/or metaforegut endoderm cells does not spontaneously form spheroids. In some embodiments, no more than 1,2, or 3 cell passages are performed. in some embodiments, the total yield of metaforegut cells and/or metaforegut endoderm cells (which may be in spheroid form) according to the methods provided herein involving multiple passages is 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 220-fold, 240-fold, 250-fold, 260-fold, and/or the like of the total yield of metaforegut cells and/or metaforegut endoderm cells obtained in cell culture without passages 270-fold, 280-fold, 290-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, or 1000-fold, or any fold within a range defined by any two of the foregoing (e.g., 50-fold to 1000-fold, 50-fold to 200-fold, 200-fold to 1000-fold, or 100-fold to 300-fold). In some embodiments, table 1 provides exemplary concentration ranges for each growth factor used in the methods of the invention. In embodiments of the methods, any concentration of a growth factor or range thereof under a certain "sub-embodiment" may be used in combination with the concentration of other growth factors or ranges thereof under the same or different "sub-embodiments". Thus, the combination is not limited to combinations under the same "sub-embodiment". For example, the presence of each of a TGF-b pathway inhibitor, FGF pathway activator, wnt pathway activator, and VEGF pathway activator, as defined under any "sub-embodiment" may be combined with the presence of EGF (any one of EGF "sub-embodiments" 1-3) or the absence of EGF ("sub-embodiment" 4). The same applies to any combination of the listed growth factors, whether they are present or absent in the methods practiced herein. Exemplary, non-limiting formulations are listed in table 2. For each of the ranges provided in tables 1 and 2, this should be construed to also include any concentration within the defined range. For example, a 100nM-1000nM TGF-b pathway inhibitor should be interpreted to include a TGF-b pathway inhibitor of 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM, or any concentration within a range defined by any two of the foregoing concentrations. exemplary concentrations within the limited range are provided throughout the present disclosure. In some embodiments, the TGF-b pathway inhibitor is A83-01. In some embodiments, the FGF pathway activator is FGF2. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the VEGF pathway activator is VEGF. In some embodiments, the ROCK inhibitor is Y-27632.

TABLE 1 exemplary combinations of growth factors for expanded metaintestinal cells

TABLE 2 exemplary combinations

Also disclosed herein are metaforegut cells and/or metaforegut endoderm cells produced by the methods provided herein.

Gene editing

In some embodiments, the iPSC, definitive endoderm cells, posterior foregut spheroids, or organoids are genetically modified or edited according to methods known in the art. Gene editing using CRISPR nucleases such as Cas9 is explored, for example, in PCT publication WO 2013/176772、WO 2014/093595、WO 2014/0193622、WO 14/093655、WO 2014/993712、WO 2014-093661、WO 2014/204728、WO 2014/2004729、WO 2015/071474、WO 2016/115326、WO 2016/141224、WO 2017/023803 and WO 2017/070633, each of which is expressly incorporated herein by reference in its entirety.

Methods and compositions for preparing liver organoids

Provided herein are methods and compositions for differentiating metaforegut cells and/or metaforegut endoderm into liver organoids. In some embodiments, the methods comprise i) contacting the posterior foregut cells and/or the posterior foregut endoderm cells and/or the foregut endoderm cells with a retinoic acid pathway activator, optionally in the form of spheroids, optionally in the form of individual cells or clusters of cells dissociated from the spheroids, and/or optionally aggregated cells in a microwell or other device described herein, optionally wherein the spheroids comprise a structure having a single lumen, and ii) contacting the cells of step i) with a medium comprising Hepatocyte Growth Factor (HGF), oncostatin M (OSM), and dexamethasone (e.g., hepatocyte medium (HCM)) for a period of time, thereby differentiating the posterior foregut cells and/or the posterior foregut endoderm cells and/or the foregut endoderm cells into liver organoids. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells and/or foregut endoderm cells can be any of the metaforegut cells and/or metaforegut endoderm cells and/or foregut endoderm cells disclosed herein. In some embodiments of the present invention, in some embodiments, the metaforegut cells and/or metaforegut endoderm cells are in the form of spheroids or individual metaforegut cells and/or metaforegut endoderm cells and/or a pellet of metaforegut cells and/or metaforegut endoderm cells derived from dissociated spheroids. In some embodiments, the metaforegut cells and/or metaforegut endoderm cells and/or foregut endoderm cells can be produced by a method that does not involve the use of a heterogeneous basement membrane matrix. In some embodiments, the posterior foregut cells and/or the posterior foregut endoderm cells and/or the foregut endoderm cells may be those generally known in the art.

In some embodiments of the methods provided herein, the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580. In some embodiments, the retinoic acid pathway activator is retinoic acid. In some embodiments, the retinoic acid pathway activator is provided at a concentration that is, is about, is at least about, does not exceed or does not exceed about 1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM or 3.0 μm, or is any concentration within a range defined by any two of the foregoing concentrations (e.g., 1.0 μm-3.0 μm, 1.0 μm-2.0 μm, 2.0 μm-3.0 μm, or 1.5 μm-2.5 μm). In some embodiments, the retinoic acid pathway activator is provided at a concentration equal to or about 2.0 μm.

In some embodiments, the culture medium (e.g., hepatocyte culture medium) is supplemented with cMET tyrosine kinase receptor agonists, interleukin 6 (IL-6) family cytokines, and/or corticosteroids. In some embodiments, the cMET tyrosine kinase receptor agonist is selected from the group consisting of HGF, PG-001, fugonidone, te Lei Walai phenanthrene, recombinant InlB protein, and an agonistic c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokine is selected from the group consisting of IL-6, OSM, leukemia Inhibitory Factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokine (CLC). In some embodiments, the corticosteroid is selected from the group consisting of dexamethasone, beclomethasone, betamethasone, fluke, halometasone, and mometasone. In some embodiments, the medium (e.g., hepatocyte medium) is supplemented with HGF, OSM, and dexamethasone. In some embodiments, the medium (e.g., hepatocyte medium) is supplemented with dexamethasone.

In some embodiments of the methods provided herein, the HGF is provided at a concentration that is, is about, is at least about, does not exceed or does not exceed about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 1ng/mL to 20ng/mL, 1ng/mL to 10ng/mL, 10ng/mL to 20ng/mL, or 5ng/mL to 15 ng/mL). In some embodiments, HGF is provided at a concentration equal to or about 10 ng/mL.

In some embodiments of the methods provided herein, the OSM is provided at a concentration that is, is about, is at least about, does not exceed or does not exceed about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or is any concentration within a range defined by any two of the foregoing concentrations (e.g., 10ng/mL to 30ng/mL, 10ng/mL to 20ng/mL, 20ng/mL to 30ng/mL, or 15ng/mL to 25 ng/mL). In some embodiments, OSM is provided at a concentration equal to or about 20 ng/mL.

In some embodiments of the methods provided herein, dexamethasone is provided at a concentration that is, is about, is at least about, is no more than, or is no more than about 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, or 200nM, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 50nM-200nM, 50nM-100nM, 100nM-200nM, or 50nM-150 nM). In some embodiments, dexamethasone is provided at a concentration equal to or about 100 nM.

In some embodiments of the methods provided herein, the cells of step i) and/or step ii) are contacted in a medium further comprising EGF. In some embodiments, EGF is provided at a concentration that is, is about, is at least about, does not exceed or does not exceed about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 10ng/mL-30ng/mL, 10ng/mL-20ng/mL, 20ng/mL-30ng/mL, or 15ng/mL-25 ng/mL). In some embodiments, EGF is provided at a concentration equal to or about 20 ng/mL. In some embodiments, the cells of step i) and/or step ii) are contacted in a medium that does not comprise EGF.

In some embodiments of the methods provided herein, the cells of step ii) are cultured in growth medium supplemented with non-essential amino acids, and glycine. In some embodiments, the post-supplementation growth medium comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of non-essential amino acids by total volume, or within a range defined by any two of the foregoing values, e.g., 7% -9%, 6% -10%, 5% -12%, 8% -14%, 10% -15%, 4% -15%, 15% -17%, 13% -19%, 12% -24%, or 10% -25%, etc. in some embodiments, the growth medium after supplementation is about 6% -10%, 8% -14%, 10% -15%, 4% -15%, 15% -17%, 13% -19%, 12% -24% or 10% -25%, or about 4%, 6%, 8%, 10%, 12%, 14% or 16% of non-essential amino acids by total volume. In some embodiments, the post-supplementation growth medium comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% essential amino acids by total volume, or essential amino acids within a range defined by any two of the foregoing values, e.g., 7% -9%, 6% -10%, 5% -12%, 8% -14%, 10% -15%, 4% -15%, 15% -17%, 13% -19%, 12% -24%, or 10% -25%. In some embodiments, the growth medium after supplementation is about 6% -10%, 8% -14%, 10% -15%, 4% -15%, 15% -17%, 13% -19%, 4%, 6%, 8%, 10%, 12%, 14% or 16% essential amino acids by total volume. In some embodiments, the supplemental glycine is provided at a concentration that is, is about, is at least about, does not exceed or does not exceed about 5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL、21mg/mL、22mg/mL、23mg/mL、24mg/mL、25mg/mL、26mg/mL、27mg/mL、28mg/mL、29mg/mL、30mg/mL、31mg/mL、32mg/mL、33mg/mL、34mg/mL or 35mg/mL, or is any concentration within a range defined by any two of the foregoing concentrations (e.g., 19mg/mL-21mg/mL, 18mg/mL-22mg/mL, 16mg/mL-24mg/mL, 10mg/mL-30mg/mL, or 5mg/mL-34 mg/mL). In some embodiments, the supplemental glycine is provided at a concentration equal to or about 18mg/mL to 22mg/mL or 20 mg/mL. In some embodiments, the growth medium is any standard growth medium commonly used for cell culture and combinations thereof. For example, the growth medium is eagle Minimum Essential Medium (MEM), eagle minimum essential medium with alpha modification (a-MEM), eagle Basal Medium (BME), dulbeck Modified Eagle Medium (DMEM), or hepatocyte medium (HCM). For exemplary purposes only, table 3 depicts standard nonessential amino acid and essential amino acid concentrations for MEM, and in some embodiments provided herein, exemplary concentrations of these amino acids after supplementation. In some embodiments, the supplemented media concentration is ± 10%, ± 5% or ± 1% of the values listed in table 3.

TABLE 3 exemplary amounts of amino acid supplements

In some embodiments of the methods provided herein, the cells of step ii) are further contacted with a low/first concentration of bilirubin, wherein the liver organoid formed is a mature liver organoid. In some embodiments, the low/first concentration of bilirubin is human fetal physiological concentration of bilirubin. In some embodiments, the low/first concentration of bilirubin is, is about, is less than, or is less than about 0.1mg/L to 1mg/L, 0.5mg/L to 1mg/L, or 1mg/L. In some embodiments, the low concentration/first concentration of bilirubin is, is about, is less than, or is less than about 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, or 1mg/L, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 0.1mg/L to 1mg/L, 0.1mg/L to 0.5mg/L, 0.5mg/L to 1mg/L, 0.3mg/L to 0.7mg/L, or 0.4mg/L to 0.6 mg/L). In some embodiments, the low/first concentration of bilirubin is, is about, is less than, or is less than about 0.1mg/L to 3mg/L, 0.5mg/L to 3mg/L, or 3mg/L. In some embodiments, the low concentration/first concentration of bilirubin is, is about, is less than, or is less than about 0.1mg/L、0.2mg/L、0.3mg/L、0.4mg/L、0.5mg/L、0.6mg/L、0.7mg/L、0.8mg/L、0.9mg/L、1mg/L、1.25mg/L、1.5mg/L、1.75mg/L、2.0mg/L、2.25mg/L、2.5mg/L、2.75mg/L or 3.0mg/L, or is any concentration within a range defined by any two of the foregoing concentrations (e.g., 0.1mg/L to 3mg/L, 0.5mg/L to 2.0mg/L, 0.5mg/L to 1.5mg/L, 0.3mg/L to 2.5mg/L, or 0.5mg/L to 1.75 mg/L). In some embodiments, the mature liver organoids exhibit luminal protrusions resembling bile canaliculi, and/or have a single lumen and generally spherical-shaped structures. In some embodiments, the mature liver organoid expresses reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to a liver organoid not contacted with the low/first dose of bilirubin. In some embodiments, the mature liver organoid expresses increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to a liver organoid not contacted with the low/first dose of bilirubin. In some embodiments, the mature liver organoid expresses CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof. In some embodiments, the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids not contacted with low/first doses of bilirubin.

In some embodiments of the methods provided herein, the cells of step ii) are further contacted with a high/second concentration of bilirubin, wherein the liver organoid formed is a hyperbilirubinemia liver organoid. In some embodiments, when used in the methods disclosed herein, the liver organoid is, is about, is at least, or is at least about 18 days old, 19 days old, 20 days old, 21 days old, 22 days old, 23 days old, 24 days old, 25 days old, 26 days old, 27 days old, 28 days old, 29 days old, 30 days old, 31 days old, 32 days old, 33 days old, 34 days old, or 35 days old, or a range defined by any two of the foregoing values, e.g., 18 days-35 days, 18 days-30 days, 20 days-25 days, or 18 days-25 days. In some embodiments, the high/second concentration of bilirubin is, is about, is greater than, or is greater than about 2mg/L to 10mg/L, 5mg/L to 10mg/L, or 20mg/L. In some embodiments, the high/second concentration of bilirubin is, is about, greater than, or greater than about 2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 2mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L). In some embodiments, the liver organoid is contacted with high/second concentration bilirubin for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, or a range defined by any two of the foregoing values, e.g., 1 day-10 days, 1 day-5 days, 3 days-8 days, 5 days-10 days, or 7 days-10 days, to form a hyperbilirubinemia liver organoid. In some embodiments, hyperbilirubinemia liver organoids express elevated levels of UGT1A1 or NRF2, or both, relative to liver organoids not treated with high/second concentrations of bilirubin.

In some embodiments of the methods provided herein, the liver organoid comprises a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA encoding a functional GULO protein or both, wherein the liver organoid is capable of synthesizing ascorbic acid. In some embodiments, the functional GULO protein is mGULO. However, the functionality GULO may alternatively be derived from any other animal species comprising a functional GULO protein. In some embodiments, the gene encoding the functional GULO protein is conditionally expressed. In some embodiments, the gene is conditionally expressed using a tetracycline-inducible system or any other system commonly known in the art for conditional expression. In some embodiments, the liver organoids are engineered with genes encoding functional GULO proteins using CRISPR or any other genetic engineering method generally known in the art. In some embodiments, the gene encoding the functional GULO protein or mRNA, or both, is introduced into the mature liver organoid by transfection. In some embodiments, a liver organoid comprising functional GULO protein expresses increased levels of NRF2 relative to a liver organoid that does not comprise functional GULO protein. In some embodiments, the liver organoid comprising functional GULO protein expresses reduced levels of IL1B, IL6 or TNFa or any combination thereof, relative to a liver organoid not comprising functional GULO protein, optionally when cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid. In some embodiments, the liver organoids comprising functional GULO protein exhibit reduced caspase-3 activity relative to liver organoids not comprising functional GULO protein, optionally when cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid. In some embodiments, the liver organoid comprising functional GULO protein expresses increased levels of ALB relative to a liver organoid that does not comprise functional GULO protein. In some embodiments, the liver organoid comprising the functional GULO protein is similar to and expresses a periportal liver marker. In some embodiments, the periportal marker comprises FAH, ALB, PAH, CPS a 1, HGD, or any combination thereof. In some embodiments, a liver organoid comprising functional GULO protein exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid that does not comprise functional GULO protein. In some embodiments, a liver organoid comprising a functional GULO protein exhibits increased bilirubin conjugation activity relative to a liver organoid that does not comprise a functional GULO protein. In some embodiments, a liver organoid comprising functional GULO protein exhibits increased viability in culture relative to a liver organoid that does not comprise functional GULO protein. In some embodiments, the liver organoid has been differentiated from a pluripotent stem cell comprising a functional GULO protein and/or a gene or mRNA encoding a functional GULO protein or both, whereby the pluripotent stem cell is capable of synthesizing ascorbic acid.

In some embodiments of the methods provided herein, the liver organoid comprises an inactive UGT1A1 gene, wherein the liver organoid is a model of Crigler-Najjar syndrome.

In some embodiments of the methods provided herein, the methods further comprise aggregating the posterior foregut cells and/or the posterior foregut endoderm cells and/or the foregut endoderm cells in a microwell or other device (e.g., aggresell) prior to step i). An exemplary schematic for culturing liver organoids from, for example, posterior foregut cells and/or posterior foregut endoderm cells aggregated in microwells is shown in fig. 5I. In some embodiments, each aggregate of metaintestinal cells and/or metaintestinal endoderm cells comprises about 250, about 500, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 7500, about 8000, about 8500, about 9000, about 9500, or about 10000 metaintestinal cells and/or metaintestinal endoderm cells, or any number of metaintestinal cells and/or metaintestinal endoderm cells within a range defined by any two of the foregoing cell numbers. In some embodiments, the aggregated foregut cells and/or foregut endoderm cells produce liver organoids of more uniform size. Additional information regarding methods of aggregating metaintestinal cells and/or metaintestinal endoderm cells to produce liver organoids of more uniform size, such as with microwells or other devices (such as aggresell), can be found in PCT publication WO 2021/030373, which is hereby expressly incorporated by reference in its entirety.

In some embodiments of the methods provided herein, the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or a component thereof. In some embodiments, the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof that is heterologous to the human. In some embodiments, the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the cells of step i) and/or step ii) are not contacted with Matrigel ^®、Cultrex^® or Geltrex ^®.

In some embodiments of the methods provided herein, the cells of step i) and/or step ii) are cultured in a non-static bioreactor. In some embodiments, the cells of step i) and/or step ii) are cultured in a rotating bioreactor.

In some embodiments of the methods provided herein, the methods further comprise cryopreserving the liver organoids. In some embodiments, cryopreserving the liver organoid comprises slow freeze or vitrification cryopreservation. In some embodiments, liver organoids are cryopreserved with chroman a, emlicarbazepine, polyamines, and trans-ISRIB (CEPT). In some embodiments chroman 1 is provided at a concentration equal to or about 50 nM. In some embodiments, the emlicarbazepine is provided at a concentration equal to or about 5 μm. In some embodiments, the polyamine is provided at a concentration equal to or about 1:1000. In some embodiments, trans-ISRIB is provided at a concentration equal to or about 7 μm.

Disclosed herein are liver organoids produced by the methods disclosed herein.

When applied to any of the cells disclosed herein, such as pluripotent stem cells, definitive endoderm, hindforegut endoderm, and/or liver organoids, the cells may be derived from a patient. In some embodiments, the patient has liver disease. In some embodiments, the definitive endoderm, the hind foregut endoderm, and/or the liver organoids can be derived from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells.

Application method

Also disclosed are methods comprising administering any of the liver organoids disclosed herein to a subject in need thereof. Also disclosed herein are methods for treating a liver-related disease or disorder in a subject in need thereof. In some embodiments, the methods comprise administering to the subject any liver organoids disclosed herein. In some embodiments, the liver organoids have been produced by cells derived from the subject. In some embodiments, the cells derived from the subject are induced pluripotent stem cells.

Also disclosed herein are methods for screening. In some embodiments, the methods comprise contacting any liver organoid disclosed herein with a candidate compound or composition, and assessing the effect of the candidate compound or composition on the liver organoid. In some embodiments, the liver organoid is a model of a liver-related disease or disorder, and assessing the effect of the candidate compound or composition on the liver organoid comprises assessing the effect of the candidate compound or composition on the liver-related disease or disorder. In some embodiments, the liver organoids have been produced by cells derived from the subject. In some embodiments, the cells derived from the subject are induced pluripotent stem cells. In some embodiments, the subject has a liver-related disease or disorder.

Liver-related diseases and disorders

Liver organoids of the present disclosure may be used to treat and/or study or model liver-related diseases and disorders. In some embodiments, the methods comprise administering any liver organoids or liver cells disclosed herein. Also disclosed herein are liver organoids or liver cells as disclosed herein for use in the manufacture of a medicament for treating a liver-related disease or disorder. Also disclosed herein is a liver organoid or hepatocyte disclosed herein for use in treating a liver-related disease or disorder in a subject in need thereof.

Liver-related diseases and conditions related to the present disclosure may include conditions such as liver dysfunction and/or failure (e.g., hyperammonemia and/or hyperbilirubinemia, etc.), hepatitis (e.g., hepatitis a, hepatitis b, hepatitis c, hepatitis b, hepatitis e, hepatitis G, TT and/or autoimmune hepatitis, etc.), viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic diseases (e.g., metabolic dysfunction-related liver disease (MASLD), metALD, non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-related fatty hepatitis (MASH), etc.), autoimmune liver disease, wilson's disease, metabolic-related fatty liver disease, hyperammonemia, hyperbilirubinemia, crigler-Najjar syndrome, urea circulatory disorder, walman disease, liver cancer, hepatoblastoma, drug-induced liver injury (DILI), glycogen storage disease, alcoholic disease, liver disease, and/or storage-related cyst. Those of skill in the art will appreciate other liver-related diseases and conditions that the liver organoids disclosed herein may have an associated relationship.

For example, a liver organoid may be transplanted into a subject having liver dysfunction and/or failure, wherein the transplanted liver organoid is implanted onto the liver of the subject. Following transplantation, the subject may have reduced serum bilirubin and/or ammonia levels, and/or increased serum protein proteins, and/or improved bile duct stenosis and/or liver regeneration symptoms, and may also have increased survival rates.

For example, these liver organoids may be used in an in vitro human model system for studying hepatocyte function and developmental divergence, studying liver related diseases, identifying and/or screening therapeutic targets, and/or identifying therapeutic compounds and/or compositions effective in treating liver related diseases or disorders. Thus, the liver organoids of the present disclosure may allow for new developments in liver disease treatment and research.

Composition and method for producing the same

In some embodiments, provided herein are also compositions for performing any of the methods disclosed herein. In some embodiments, provided herein are also compositions produced according to the processes provided in any of the methods disclosed herein. It is expressly contemplated that in certain embodiments, any of the methods or compositions described herein may be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

In some embodiments, provided herein are compositions comprising metaforegut cells and/or metaforegut endoderm cells and/or liver organoids produced using any one or more of the methods provided herein. In some embodiments, provided herein are compositions comprising at least a portion of metaforegut cells and/or metaforegut endoderm cells that have spontaneously formed three-dimensional (3D) spheroids, optionally wherein the spheroids comprise a structure having a single lumen.

In some embodiments, the compositions provided herein are in vitro compositions produced outside of a multicellular living organism. In some embodiments, the compositions provided herein can be introduced into a multicellular living organism. In some embodiments, the compositions provided herein comprise exogenously provided components, agents, and/or conditions. In some embodiments, the compositions provided herein comprise exogenously provided components, agents, and/or conditions that mimic the in vivo properties required to induce specific cellular differentiation and/or organoid tissue.

In some embodiments, provided herein are in vitro compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, at least one exogenous tissue culture surface, at least one exogenous TGF-b pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator. In some embodiments, the composition may further comprise an endogenous TGF-b pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and/or a VEGF pathway activator. In some embodiments, the composition comprises metaforegut cells and/or metaforegut endoderm cells that have been dissociated from monolayers and/or spheroids. In some embodiments, the composition comprises metaforegut cells and/or metaforegut endoderm cells having a cell density greater than or equal to, just or about 1×10 ⁵, 2×10 ⁵, 3×10 ⁵, 4×10 ⁵, a cell density of, 5X 10 ⁵, 6X 10 ⁵, 7X 10 ⁵, 8X 10 ⁵, 9X 10 ⁵, 1X 10 ⁶, 2X 10 ⁶, 3X 10 ⁶, 4X 10 ⁶, or 5X 10 ⁶ cells/cm ² of surface area of tissue culture surface, or any cell density in the range defined by any two of the foregoing cell densities.

In some embodiments, provided herein are compositions comprising a tissue culture surface coated with a basement membrane matrix or a component thereof. In some embodiments, the base film matrix or component thereof does not comprise a non-human animal component. In some embodiments, the base film matrix or component thereof does not comprise a non-human animal component that makes the base film matrix or component thereof xenogeneic to humans. In some embodiments, the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-switch (EHS) sarcoma cells, is not Matrigel ^®, is not Cultrex ^®, and/or is not Geltrex ^®. In some embodiments, the basement membrane matrix or component thereof comprises human fibronectin, collagen IV, entactin, basement membrane glycans, fibrin, and/or hydrogels.

In some embodiments, provided herein are compositions comprising exogenous TGF-b pathway inhibitors. In some embodiments, the exogenous TGF-b pathway inhibitor comprises, consists essentially of, or consists of a83-01, repox, LY365947, and/or SB 431542. In some embodiments, the exogenous TGF-b pathway inhibitor comprises, consists essentially of, or consists of TGF-b pathway inhibitor a 83-01. In some embodiments, the composition comprises a TGF-b pathway inhibitor at a concentration equal to or about 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM, or at any concentration within a range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises a TGF-b pathway inhibitor at a concentration equal to or about 500 nM.

In some embodiments, provided herein are compositions comprising exogenous FGF pathway activators. In some embodiments, the exogenous FGF pathway activator comprising, consisting essentially of, or consisting of ：FGF1、FGF2、FGF3、FGF4、FGF4、FGF5、FGF6、FGF7、FGF8、FGF8、FGF9、FGF10、FGF11、FGF12、FGF13、FGF14、FGF15、FGF16、FGF17、FGF18、FGF19、FGF20、FGF21、FGF22 and/or FGF23 is included in the composition. In some embodiments, the exogenous FGF pathway activator comprises, consists essentially of, or consists of FGF 2. In some embodiments, the composition comprises FGF pathway activator at a concentration equal to or about 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, or 10ng/mL, or any concentration within a range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises FGF pathway activator at a concentration equal to or about 5 ng/mL.

In some embodiments, provided herein are compositions comprising exogenous Wnt pathway activators. In some embodiments, the exogenous Wnt pathway activator comprising, consisting essentially of, or consisting of ：Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11、Wnt16、BML 284、IQ-1、WAY 262611、CHIR99021、CHIR 98014、AZD2858、BIO、AR-A014418、SB 216763、SB 415286、 aloxin, indirubin, altbolone, kenarone, lithium chloride, TDZD, and/or TWS119. In some embodiments, the exogenous Wnt pathway activator comprising, consisting essentially of, or consisting of CHIR 99021. In some embodiments, the composition comprises a Wnt pathway activator at a concentration equal to or about 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, or 8 μm, or at any concentration within a range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises Wnt pathway activator at a concentration equal to or about 3 μm.

In some embodiments, provided herein are compositions comprising exogenous VEGF pathway activators. In some embodiments, the composition comprises, consists essentially of, or consists of an exogenous VEGF pathway activator comprising VEGF and/or GS4012. In some embodiments, the exogenous VEGF pathway activator comprising, consisting essentially of, or consisting of the composition comprises VEGF. In some embodiments, the composition comprises a VEGF pathway activator at a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL or any concentration within a range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises a concentration of VEGF pathway activator at or about 10 ng/mL.

In some embodiments, provided herein are compositions comprising exogenous EGF. In some embodiments, provided herein are compositions that do not include exogenous EGF. In some embodiments, provided herein are compositions comprising EGF at a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or any concentration within a range defined by any two of the foregoing concentrations. In some embodiments, provided herein are compositions comprising EGF at a concentration of equal to or about 20 ng/mL.

In some embodiments, provided herein are compositions comprising exogenous ascorbic acid and/or transgenically produced ascorbic acid. In some embodiments, provided herein are compositions that do not include exogenous ascorbic acid and/or transgenically produced ascorbic acid. In some embodiments, provided herein are compositions comprising ascorbic acid at a concentration equal to or about 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, or 100 μg/mL, or any concentration within a range defined by any two of the foregoing concentrations. In some embodiments, provided herein are compositions comprising ascorbic acid at a concentration equal to or about 50 μg/mL.

In some embodiments, provided herein are compositions comprising ROCK inhibitors. In some embodiments, provided herein are compositions that do not include a ROCK inhibitor. In some embodiments, the ROCK inhibitor comprises, consists essentially of, or consists of Y-27632. In some embodiments, provided herein are compositions comprising a ROCK inhibitor at a concentration equal to or about 1 μΜ,2 μΜ,3 μΜ,4 μΜ,5 μΜ,6 μΜ, 7 μΜ,8 μΜ, 9 μΜ, 10 μΜ, 11 μΜ, 12 μΜ,13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ or 20 μΜ or any in the range defined by any two of the foregoing concentrations. In some embodiments, provided herein are compositions comprising a ROCK inhibitor at a concentration equal to or about 10 μm.

In some embodiments, provided herein are compositions comprising metaforegut cells and/or metaforegut endoderm cells that have been differentiated from stem cells and/or are differentiating from stem cells. In some embodiments, provided herein are compositions comprising metaforegut cells and/or metaforegut endoderm cells that have been differentiated from and/or are differentiating from induced pluripotent stem cells. In some embodiments, provided herein are compositions comprising metaforegut cells and/or metaforegut endoderm cells that have been passaged 1,2, or 3 times. In some embodiments, provided herein are compositions comprising metaforegut cells and/or metaforegut endoderm cells that have been passaged less than 4 times.

In some embodiments, provided herein are compositions comprising A83-01, FGF2, CHIR99021, VEGF, and/or Y-27632, optionally further comprising iPSC, PSC, and/or metaforegut cells and/or metaforegut endoderm cells.

In some embodiments, provided herein are compositions comprising a) a metaintestinal cell and/or a metaintestinal endoderm cell, a liver organoid and/or a mature liver organoid, and b) a culture medium, wherein the culture medium optionally comprises a hepatocyte culture medium and is optionally supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine and a corticosteroid, and wherein the composition optionally further comprises c) a retinoic acid pathway activator. In some embodiments, the compositions provided herein comprise a cMET tyrosine kinase receptor agonist. In some embodiments, the compositions provided herein comprise, consist essentially of, or consist of a Hepatocyte Growth Factor (HGF), PG-001, fugonidone, tetanus Lei Walai phenanthrene, recombinant InlB protein, and/or an agonistic c-Met antibody (e.g., LMH 85).

In some embodiments, provided herein are compositions comprising an IL-6 family cytokine. In some embodiments, the IL-6 family cytokine comprises, consists essentially of, or consists of IL-6, oncostatin M (OSM), leukemia Inhibitory Factor (LIF), cardiac neurotrophin-1, ciliary neurotrophic factor (CTNF), and/or cardiac dystrophin-like cytokine (CLC).

In some embodiments, provided herein are compositions comprising a corticosteroid. In some embodiments, the corticosteroid comprises, consists essentially of, or consists of dexamethasone, beclomethasone, betamethasone, flucortisone, halometasone, and/or mometasone.

In some embodiments, provided herein are compositions comprising hepatocyte media supplemented with HGF, OSM, and/or dexamethasone. In some embodiments, provided herein are compositions comprising a hepatocyte medium supplemented with dexamethasone. In some embodiments, provided herein are compositions comprising hepatocyte media supplemented with HGF. In some embodiments, provided herein are compositions comprising an OSM-supplemented hepatocyte medium.

In some embodiments, provided herein are compositions comprising retinoic acid pathway activators. In some embodiments, the retinoic acid pathway activator comprises, consists essentially of, or consists of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and/or AM580. In some embodiments, the retinoic acid pathway activator comprises, consists essentially of, or consists of retinoic acid. In some embodiments, the composition comprises retinoic acid pathway activator at a concentration equal to or about 1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM or 3.0 μm or at any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises retinoic acid pathway activator at a concentration equal to or about 2.0 μm.

In some embodiments, the composition comprises HGF. In some embodiments, the composition comprises HGF at a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL or any concentration within a range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises HGF at a concentration equal to or about 10 ng/mL.

In some embodiments, the composition comprises OSM. In some embodiments, the composition comprises OSM at a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or any concentration within a range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises OSM at a concentration equal to or about 20 ng/mL.

In some embodiments, the composition comprises dexamethasone. In some embodiments, the composition comprises dexamethasone at a concentration equal to or about 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, or 200nM, or at any concentration within a range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises dexamethasone at a concentration equal to or about 100 nM.

In some embodiments, the composition comprises exogenous bilirubin. In some embodiments, the composition comprises both exogenous bilirubin and endogenous bilirubin. In some embodiments, the composition comprises a low concentration of exogenous bilirubin. In some embodiments, the low concentration of exogenous bilirubin is at or near human fetal physiological bilirubin concentration. Human fetal bilirubin levels are considered to be generally about 1mg/L (0.1 mg/dL), which rises rapidly to 3mg/L-10mg/L (0.3 mg/dL-1.0 mg/dL) 24 hours after birth. In some embodiments, the composition comprises exogenous and/or endogenous bilirubin at, or below about ：0.1mg/L、0.2mg/L、0.3mg/L、0.4mg/L、0.5mg/L、0.6mg/L、0.7mg/L、0.8mg/L、0.9mg/L、1mg/L、1.25mg/L、1.5mg/L、1.75mg/L、2.0mg/L、2.25mg/L、2.5mg/L、2.75mg/L or 3.0mg/L, or at a range defined by any two of the foregoing concentrations (e.g., 0.1mg/L to 3mg/L, 0.5mg/L to 2.0mg/L, 0.5mg/L to 1.5mg/L, 0.3mg/L to 2.5mg/L, or 0.5mg/L to 1.75 mg/L), or at least any concentration within 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, or 1mg/L, or at a range defined by any two of the foregoing concentrations (e.g., 0.1mg/L to 1.5mg/L, 0.3mg/L, 0.5mg/L to 1.75 mg/L), or at least any two of the foregoing concentrations. In some embodiments, the composition comprises exogenous bilirubin at a concentration of, at, or below about ：0.1mg/L、0.2mg/L、0.3mg/L、0.4mg/L、0.5mg/L、0.6mg/L、0.7mg/L、0.8mg/L、0.9mg/L、1mg/L、1.25mg/L、1.5mg/L、1.75mg/L、2.0mg/L、2.25mg/L、2.5mg/L、2.75mg/L or 3.0mg/L, or in a range defined by any two of the foregoing concentrations (e.g., 0.1mg/L to 3mg/L, 0.5mg/L to 2.0mg/L, 0.5mg/L to 1.5mg/L, 0.3mg/L to 2.5mg/L, or 0.5mg/L to 1.75 mg/L), or in a range of 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, or 1mg/L, or in a range defined by any two of the foregoing concentrations (e.g., 0.1mg/L to 1mg/L, 0.1mg/L to 2.5mg/L, or 0.5mg/L to 1.75 mg/L), or in a range of 0.1mg/L, 0.5mg/L, 0.4mg/L, 0.6mg/L, 0.7mg/L, or 0.5 mg/L.

In some embodiments, provided herein are compositions comprising mature liver organoids. In some embodiments, provided herein are compositions comprising mature liver organoids that exhibit luminal protrusions resembling bile canaliculi, and/or structures having a single lumen and generally having a spherical shape. In some embodiments, provided herein are compositions comprising mature liver organoids produced by contact with low doses of exogenous bilirubin. In some embodiments, provided herein are compositions comprising mature liver organoids that express reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to liver organoids that have not been contacted with low doses of bilirubin. In some embodiments, compositions comprising mature liver organoids are provided that express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to liver organoids not contacted with low doses of bilirubin. In some embodiments, compositions are provided that comprise mature liver organoids that express CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof. In some embodiments, compositions are provided that comprise mature liver organoids that exhibit increased CYP3A4 and/or CYP1A2 activity relative to liver organoids that have not been contacted with low doses of bilirubin. In some embodiments, provided herein are compositions comprising mature liver organoids wherein cells of the mature liver organoids are contacted with low doses of exogenous bilirubin and the mature liver organoids exhibit luminal protrusions resembling bile canaliculi, and/or have a single lumen and generally spherically shaped structures. In some embodiments, provided herein are compositions comprising mature liver organoids that express reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to liver organoids in which cells are not contacted with low doses of bilirubin. In some embodiments, provided herein are compositions comprising mature liver organoids that express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to liver organoids in which cells are not contacted with low doses of bilirubin. In some embodiments, provided herein are compositions comprising mature liver organoids that express CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof. In some embodiments, provided herein are compositions comprising mature liver organoids that exhibit increased CYP3A4 and/or CYP1A2 protein levels and/or enzymatic activity relative to liver organoids in which cells are not contacted with low doses of bilirubin.

In some embodiments, provided herein are compositions comprising hyperbilirubinemia liver organoids, wherein hyperbilirubinemia liver organoid cells are contacted with a high concentration and/or a second concentration of bilirubin. In some embodiments, provided herein are compositions comprising hyperbilirubinemia liver organoids, wherein the high and/or second concentration of bilirubin is, is about, greater than or greater than about ：2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 2mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L), or 4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 4mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L). In some embodiments, provided herein are compositions comprising hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoids express elevated levels of UGT1A1 or NRF2, or both, relative to liver organoids not treated with high/second concentrations of bilirubin.

In some embodiments, provided herein are also compositions comprising a metaforegut cell and/or a metaforegut endoderm cell, a liver organoid and/or a mature liver organoid engineered to comprise a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA encoding a functional GULO protein or both, wherein the metaforegut cell and/or the metaforegut endoderm cell, the liver organoid and/or the mature liver organoid are capable of synthesizing ascorbic acid. In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids, and/or mature liver organoids engineered to express a functional GULO protein, wherein the functional GULO protein is murine GULO (mGULO). In some embodiments, the gene encoding the functional GULO protein is conditionally expressed. In some embodiments, the gene encoding the functional GULO protein is constitutively expressed. In some embodiments, the gene encoding the functional GULO protein is conditionally expressed using a tetracycline-inducible system.

In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids engineered to comprise a gene encoding a functional GULO protein using CRISPR-mediated knock-in. In some embodiments, provided herein are compositions comprising a metaforegut cell and/or a metaforegut endoderm cell, a liver organoid, and/or a mature liver organoid comprising a functional GULO encoding a functional GULO protein or mRNA or both, wherein the functional gene is introduced into the metaforegut cell and/or the metaforegut endoderm cell, the liver organoid, the mature liver organoid, and/or the precursor cell by transfection. In some embodiments, provided herein are compositions comprising a posterior foregut cell and/or a posterior foregut endoderm cell, a liver organoid, and/or a mature liver organoid engineered to comprise a gene encoding a functional GULO protein using an adenovirus-mediated gene transfection procedure. In some embodiments, provided herein are compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids engineered to comprise a gene encoding a functional GULO protein using an adeno-associated virus-mediated gene transfection procedure.

In some embodiments, the compositions provided herein comprise a liver organoid and/or mature liver organoid comprising a functional GULO protein, wherein the liver organoid and/or mature liver organoid expresses increased levels of NRF2 relative to a liver organoid and/or mature liver organoid that does not comprise a functional GULO protein. In some embodiments, the compositions provided herein comprise a liver organoid and/or mature liver organoid comprising a functional GULO protein, wherein the liver organoid and/or mature liver organoid expresses reduced levels of IL1B, IL6 or TNFa or any combination thereof relative to a liver organoid and/or mature liver organoid that does not comprise a functional GULO protein. In some embodiments, the liver organoid and/or mature liver organoid comprising functional GULO protein exhibits reduced caspase-3 activity relative to a liver organoid and/or mature liver organoid not comprising functional GULO protein. In some embodiments, liver organoids and/or mature liver organoids comprising functional GULO protein express increased levels of ALB relative to liver organoids and/or mature liver organoids not comprising functional GULO protein. In some embodiments, the liver organoid and/or mature liver organoid comprising the functional GULO protein is similar to periportal liver tissue and/or expresses periportal liver markers. In some embodiments, the periportal liver marker comprises or consists of FAH, ALB, PAH, CPS1, HGD, or any combination thereof, FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, a liver organoid and/or mature liver organoid comprising a functional GULO protein exhibits increased CYP3A4 and/or CYP1A2 protein levels and/or enzymatic activity relative to a liver organoid and/or mature liver organoid not comprising a functional GULO protein. In some embodiments, a liver organoid and/or mature liver organoid comprising a functional GULO protein exhibits increased bilirubin conjugation activity relative to a liver organoid and/or mature liver organoid that does not comprise a functional GULO protein. In some embodiments, the liver organoid and/or mature liver organoid comprising functional GULO protein exhibits increased viability in culture relative to a liver organoid and/or mature liver organoid that does not comprise functional GULO protein. In some embodiments, the liver organoid and/or mature liver organoid has been differentiated from a pluripotent stem cell comprising a functional GULO protein and/or a gene or mRNA encoding a functional GULO protein or both, whereby the pluripotent stem cell is capable of synthesizing ascorbic acid.

In some embodiments, provided herein are also compositions according to table 1, table 2, or table 3.

In some embodiments, provided herein are compositions comprising a liquid component of supplemental amino acids comprising, by volume, a solution of just or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of non-essential amino acids (containing just or about 890mg/L alanine, 1320mg/L asparagine, 1330mg/L aspartic acid, 750mg/L glycine, 105mg/L serine, 1150mg/L proline, and 1470mg/L glutamic acid), a solution of just or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of essential amino acids (containing just or about 6320mg/L arginine, 1200mg/L cysteine, 2100mg/L histidine, 2620mg/L isoleucine, 2620mg/L leucine, 3625mg/L lysine, 755mg/L methionine, 1650mg/L phenylalanine, 2380mg/L threonine, 510mg/L tryptophan, 1800mg/L serine, and 1470mg/L glutamic acid), and just or about 80%, by volume, and also comprising, by volume, just or about 67%, 58%, 80%, 86%, 75%, 80%, 75%, and also 75%, 80%, 75%, 80%, or 75%, 80% of the medium.

In some embodiments, provided herein are compositions comprising a liquid component of supplemental amino acids comprising a solution of non-essential amino acids (containing exactly or about 890mg/L alanine, 1320mg/L asparagine, 1330mg/L aspartic acid, 750mg/L glycine, 105mg/L serine, 1150mg/L proline, and 1470mg/L glutamic acid), a solution of essential amino acids (containing exactly or about 6320mg/L arginine, 1200mg/L cysteine, 2100mg/L histidine, 2620mg/L isoleucine, 2620mg/L leucine, 3625mg/L lysine, 755mg/L methionine, 1650mg/L phenylalanine, 2380mg/L threonine, 510mg/L tryptophan, 1800mg/L tyrosine, and 2340mg/L valine) at exactly or about 6% by volume, and also supplemented with exactly or about 20g/L glycine at the liver cell culture medium (m). In some embodiments, the compositions provided herein can have a pH of between about pH 6 to 8, or between pH 6.5 to 7.5, or just or about pH 7.0. In some embodiments, the compositions provided herein comprise Hepatocyte Growth Factor (HGF), oncostatin M, dexamethasone, and/or ascorbic acid.

In some embodiments, the compositions provided herein comprise liver lineage committed cells differentiated from definitive endoderm cells using retinoic acid. In some embodiments, the compositions provided herein comprise liver lineage committed cells characterized as liver organoids. In some embodiments, the compositions provided herein comprise a liver organoid characterized by secretion of increased levels of albumin and/or urea relative to a liver organoid comprised in HCM without an amino acid supplement. In some embodiments, the compositions provided herein comprise a liver organoid characterized by an increased level of expression of a liver maturation-related gene relative to a liver organoid comprised in HCM without an amino acid supplement. In some embodiments, the compositions provided herein comprise a liver organoid characterized as expressing reduced levels of vimentin relative to a liver organoid comprised in an HCM without an amino acid supplement. In some embodiments, the compositions provided herein specifically do not comprise a non-human animal basement membrane matrix or components thereof. In some embodiments, the compositions provided herein specifically do not comprise murine Engelbreth-Holm-switch (EHS) sarcoma cells, matrigel ^®、Cultrex^®, and/or Geltrex ^®.

Also provided herein, in some embodiments, are liver organoids comprising mutations naturally occurring and/or engineered in UDP glucuronyltransferase family 1 member A1 (UGT 1 A1). In some embodiments, provided herein are hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoids are produced by contacting precursor cells, precursor liver organoids, and/or precursor mature liver organoids with exogenous bilirubin for at least two rounds. In some embodiments, provided herein are hyperbilirubinemia liver organoids produced by clonally derived cells and/or ipscs.

In some embodiments, provided herein are cryopreserved compositions comprising liver organoids, chroman 1, emlicarbazepine, polyamines, and trans-ISRIB (CEPT). In some embodiments, provided herein are cryopreserved compositions comprising mature liver organoids, chroman a1, emlicarbazepine, polyamines, and trans-ISRIB (CEPT). In some embodiments, provided herein are cryopreserved compositions comprising hyperbilirubinemia liver organoids, chroman, emlicarbazin, polyamines, and trans-ISRIB (CEPT).

Pharmaceutical compositions according to various embodiments of the present disclosure may include one or more additional pharmaceutically acceptable components, which may include carriers, excipients, and/or stabilizers that are non-toxic or have an acceptable level of toxicity to the cells or mammals to which they are exposed at the dosages and concentrations employed. As used herein, "pharmaceutically acceptable" diluents "," excipients "and/or" carriers "have their ordinary and customary meaning as understood in the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, the pharmaceutically acceptable diluents, excipients and/or carriers are approved by a regulatory agency of the federal, a state government or other regulatory agency, or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans, and non-human mammals, such as cats and dogs. The term diluent, excipient, and/or "carrier" may refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluents, excipients and/or carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions, and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and/or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. Physiologically acceptable carriers may also include one or more of antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt forming counterions such as sodium, and nonionic surfactants such as TWEEN ^®, polyethylene glycol (PEG) and PLURONICS ^®. The composition may also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, slow release formulations, and the like. The formulation should be suitable for the mode of administration.

Other excipients having the desired properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediamine tetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate, sugars, glucose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be residual amounts or contaminants in the manufacturing process including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, b-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof. The amount of excipient may be present in the composition in a percentage that is, is about, is at least about, does not exceed or does not exceed about 0%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、100% w/w or any weight percent within the range defined by any two of the numbers described above.

The pharmaceutical compositions may include one or more "pharmaceutically acceptable salts" which may include relatively non-toxic inorganic and organic acid or base addition salts of the composition or excipient, including but not limited to analgesics, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from inorganic acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Examples of suitable inorganic bases for salt formation include hydroxides, carbonates and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, such organic bases of this class may include, but are not limited to, mono-, di-and tri-alkylamines, including methylamine, dimethylamine and triethylamine, mono-, di-or tri-hydroxyalkylamines, including monoethanolamine, diethanolamine and triethanolamine, amino acids, including glycine, arginine and lysine, guanidine, N-methylglucamine, L-glutamine, N-methylpiperazine, morpholine, ethylenediamine, N-benzylphenethylamine, trimethylolethane.

The appropriate formulation depends on the route of administration selected. Techniques for formulating and administering the compounds described herein are known to those of skill in the art. There are a variety of techniques in the art for administering compounds including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intra-aural, epidural, extradermal, aerosol, parenteral delivery, including intramuscular, subcutaneous, intra-arterial, intravenous, portal intravenous, intra-articular, intradermal, intraperitoneal, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injection. The pharmaceutical composition will generally be adapted to the particular intended route of administration.

As used herein, a "carrier" has its ordinary and customary meaning as understood in the specification and may refer to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and/or incorporation of the compound into a cell, tissue, and/or body organ.

As used herein, a "diluent" has its ordinary and customary meaning as understood in the specification and may refer to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, diluents may be used to increase the volume of a powerful drug that is too small in mass to be manufactured and/or administered. The diluent may also be a liquid for dissolving the drug for administration by injection, ingestion or inhalation. A common form of diluent in the art is an aqueous buffer solution such as, but not limited to, phosphate buffered saline that mimics the composition of human blood.

Dosage and route of administration

Embodiments of the present disclosure may include methods of administering or treating animals, which may involve administering an amount of at least one treatment effective to treat a disease, condition, or disorder that an organism has or is suspected of having or being susceptible to, or producing a desired physiological effect. In some embodiments, the disease, condition, or disorder may be a liver-related disease or disorder.

In some embodiments, the at least one treatment may include a composition or pharmaceutical composition that may be administered to an animal (e.g., a mammal, a primate, a monkey, or a human) in an amount of about 0.005mg/kg body weight to about 50mg/kg body weight, about 0.01mg/kg body weight to about 15mg/kg body weight, about 0.1mg/kg body weight to about 10mg/kg body weight, about 0.5mg/kg body weight to about 7mg/kg body weight, about 0.005mg/kg, about 0.01mg/kg, about 0.05mg/kg, about 0.1mg/kg, about 0.5mg/kg, about 1mg/kg, about 3mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 7mg/kg, about 7.5mg/kg, about 8mg/kg, about 10mg/kg, about 12mg/kg, or about 15 mg/kg. In some conditions, the dose may be about 0.5mg/kg of human body weight or about 6.5mg/kg of human body weight. In some cases, some subjects (e.g., mammals, mice, rabbits, cats, pigs, or dogs) may be administered at a dose of about 0.005mg/kg body weight to about 50mg/kg body weight, about 0.01mg/kg body weight to about 15mg/kg body weight, about 0.1mg/kg body weight to about 10mg/kg body weight, about 0.5mg/kg body weight to about 7mg/kg body weight, about 0.005mg/kg, about 0.01mg/kg, about 0.05mg/kg, about 0.1mg/kg, about 1mg/kg, about 5mg/kg, about 10mg/kg, about 20mg/kg, about 30mg/kg, about 40mg/kg, about 50mg/kg, about 80mg/kg, about 100mg/kg, or about 150 mg/kg. Of course, those skilled in the art will appreciate that many concentrations can be employed in the methods of the present disclosure, and that any number of concentrations will be able to be adjusted and tested, in part, using the guidance provided herein, in order to find a concentration that achieves the desired result in a given situation. In some embodiments, the dose or therapeutically effective dose of a compound disclosed herein will be a dose sufficient to achieve a plasma concentration of the compound or active metabolite thereof within the ranges set forth herein, e.g., about 1nM to 10nM, 10nM to 100nM, 0.1 μΜ to 1 μΜ,1 μΜ to 10 μΜ,10 μΜ to 100 μΜ, 100 μΜ to 200 μΜ, 200 μΜ to 500 μΜ or even 500 μΜ to 1000 μΜ, preferably about 1nM to 10nM, 10nM to 100nM or 0.1 μΜ to 1 μΜ.

In other embodiments, the treatment may be administered in combination with one or more other therapeutic agents for a given disease, condition, or disorder.

The compounds and pharmaceutical compositions are preferably prepared and administered in dosage units. Solid dosage units are tablets, capsules and suppositories. For treatment of a subject, different daily doses may be used depending on the activity of the compound, the mode of administration, the nature and severity of the disease or condition, the age and weight of the subject.

However, in some cases, higher or lower daily doses may be appropriate. Daily dosage administration may be carried out by single administration in the form of individual dosage units or several smaller dosage units as well as by multiple administration of sub-divided doses at specific intervals.

The treatment may be administered locally or systemically in a therapeutically effective dose. Of course, the amount effective for such use will depend on the severity of the disease or condition as well as the weight and overall state of the subject. In general, the dosage used in vitro may provide useful guidance on the amount of pharmaceutical composition that may be used for in situ administration, and animal models may be used to determine an effective dosage for treating a particular disorder.

Various considerations are described, for example, in Langer, 1990, science, 249: 1527;Goodman and Gilman's (eds.), 1990, id. Each of which is incorporated herein by reference for all purposes. The parenteral dosage of the active agent may be converted to a corresponding oral dosage by multiplying the parenteral dosage by an appropriate conversion factor. For general use, the parenteral dose in mg/mL is multiplied by 1.8 = the corresponding oral dose in milligrams ("mg"). For oncology applications, the parenteral dose in mg/mL is multiplied by 1.6 = the corresponding oral dose in mg. The average adult weight was about 70kg. See, e.g., miller-Keane, 1992, encyclopedia & Dictionary of Medicine, nursing & ALLIED HEALTH, 5 th edition, (w.b. samaders co.), pages 1708 and 1651.

However, it will be appreciated that the specific dosage level for any particular patient will depend on a variety of factors including the activity of the particular compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

In some embodiments, administration may include a combination of one or more therapeutic unit doses with a pharmaceutically acceptable carrier, and may additionally include other pharmaceutically acceptable agents, medicaments, carriers, adjuvants, diluents, and excipients. In certain embodiments, the carrier, vehicle, or excipient may facilitate administration, delivery, and/or improve preservation of the composition. In other embodiments, the one or more carriers include, but are not limited to, saline solutions, such as physiological saline, ringer's solution, PBS (phosphate buffered saline), and generally mixtures of various salts, including potassium salts and phosphate salts, with or without sugar additives such as glucose. The carrier may include aqueous and nonaqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the body fluid of the intended recipient, and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. In other embodiments, the one or more excipients may include, but are not limited to, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Nontoxic auxiliary substances such as wetting agents, buffers or emulsifying agents may also be added to the compositions. Oral formulations may include commonly employed excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. [00275] The amount of active ingredient in a unit dosage formulation may vary or be adjusted from 0.1mg to 10000mg, more typically from 1.0mg to 1000mg, and most typically from 10mg to 500mg, depending on the particular application and potency of the active ingredient. The composition may also contain other compatible therapeutic agents, if desired.

The treatment may be administered to the subject by any number of suitable routes of administration or formulations. Treatments, such as immunotherapy, may also be used to treat various diseases in a subject. Subjects include, but are not limited to, mammals, primates, monkeys (e.g., macaque, rhesus monkey, or cynomolgus monkey), humans, dogs, cats, cows, pigs, birds (e.g., chickens), mice, rabbits, and rats. In certain embodiments described herein, the subject is a human.

The route of administration of the therapeutic compounds described herein may be any suitable route. The route of administration may be, but is not limited to, oral, parenteral, dermal, nasal, rectal, vaginal and ocular. In other embodiments, the route of administration may be parenteral, mucosal, intravenous, subcutaneous, topical, intradermal, oral, sublingual, intranasal, or intramuscular. The choice of route of administration may depend on the nature of the compound (e.g., the physical and chemical nature of the compound) as well as the age and weight of the animal, the particular disease (e.g., cancer type) and the severity of the disease (e.g., stage or severity of cancer). Of course, combinations of routes of administration may be administered as desired.

Some embodiments of the present disclosure include methods for providing a treatment to a subject comprising one or more administrations of one or more compositions, which may be the same or different if there is more than one administration.

Toxicity of

The ratio between the toxicity and therapeutic effect of a particular treatment is its therapeutic index and can be expressed as the ratio between the LD50 (the amount of compound lethal in 50% of the population) and the ED50 (the amount of compound effective in 50% of the population). Compounds exhibiting high therapeutic indices are preferred. Therapeutic index data obtained from in vitro assays, cell culture assays, and/or animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds is preferably within a range of plasma concentrations including the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. See, e.g., fingl et al, in THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, chapter 1, page 1, 1975. The exact formulation, route of administration, and dosage may be selected by the individual practitioner depending on the condition of the patient and the particular method of using the compound. For in vitro formulations, the exact formulation and dosage may be selected by the individual practitioner depending on the condition of the patient and the particular method of using the compound.

Kit for detecting a substance in a sample

In some embodiments, also disclosed herein are kits that provide means for performing any of the methods described herein. In some embodiments, also disclosed herein are kits comprising any of the compositions or means for producing a composition described herein.

In some embodiments, the kit may be prepared from readily available components and reagents. For example, such kits may comprise any one or more of enzymes, reaction tubes, buffers, detergents, primers, probes, antibodies, cell culture media, differentiation inducing agents, amino acid mixtures/supplements, engineered constructs and/or polynucleotides, transcription inducers, bilirubin, ascorbic acid, retinoic acid pathway activators, corticosteroids, cMET tyrosine kinase receptor agonists, IL-6 family cytokines, TGF-b pathway inhibitors, FGF pathway activators, wnt pathway activators, VEGF pathway activators, ROCK inhibitors, and/or cells. In some embodiments, the components and reagents may be packaged together in any combination, and/or may be packaged separately. In some embodiments, the kit may include components and reagents concentrated to a higher working concentration than disclosed herein or provided herein. In some embodiments, the individual components may also be provided in the kit in concentrated amounts, and in some aspects, the components are provided separately in the same concentration as they are in solutions with other components. In some embodiments, the concentration of the component may be provided at 1x, 2x, 5x, 10x, or 20x or more. In some embodiments, the kit may comprise components that may be packaged individually or placed in containers such as tubes, bottles, vials, syringes, or other suitable container means.

In some embodiments, the kit is contained in a container. The kit may further comprise instructions for using the kit to assess expression and/or differentiation of the cells. The reagents in the kit for measuring expression and/or determining differentiation may comprise a plurality of PCR probes and/or primers for qRT-PCR and/or a plurality of antibodies or fragments thereof for assessing expression of biomarkers suitable for classifying the cell status.

In some embodiments, the kit is produced using Good Manufacturing Practice (GMP) and is GMP-compliant.

Having described the embodiments in detail, it will be apparent that modifications, variations and equivalent embodiments are possible without departing from the scope of the embodiments defined in the appended claims. Further, it should be understood that all examples in this disclosure are provided as non-limiting examples.

Examples

The following non-limiting examples are provided to further illustrate the embodiments disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent examples of modes for carrying out the invention that have been found to function well in the practice of the embodiments and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the embodiments disclosed herein.

Example 1 protocol for freezing liver organoids

Cryopreservation protocol using CELLBANKER ^® a 1

CELLBANKER ^® 1 (AMSBIO) containing 50nmchroman 1, 5 μM of emlicarbazepine, 1:1000 polyamine and 7 μM of trans-ISRIB (CEPT) was kept on ice or at 4 ℃.

Day 20-24 Human Liver Organoids (HLOs) were collected from 6-well plates and filtered through 150 μm screens into 50mL conical tubes (total volume should not exceed about 25 mL). Approximately 20mL of cold PBS was added to achieve a final volume of approximately 45 mL. The suspension was pipetted up and down five times to wash the organoids. The tube was then centrifuged at 300×g for 5 minutes at 4 ℃ to pellet the HLO. The supernatant was aspirated and washed again with 10-20 mL fresh PBS, centrifuged and aspiration repeated. Washed HLO was resuspended in CELLBANKER ^® 1+cept freezing medium, 2 freezing vials per well of 6-well plate, each freezing vial having 0.75mL of freezing medium suspension. The frozen vials were then transferred to-80 ℃ freezer containers for 24 hours and then transferred to liquid nitrogen for storage for longer periods.

Cryopreservation protocol using Cell Reservoir One Vitrify

Cell Reservoir One Vitrify (Nacalai) containing CEPT was kept on ice or at 4 ℃.

The same HLO collection and washing steps as described above were performed. Washed HLO was resuspended in Cell Reservoir One Vitrify +cept freezing medium and rapidly transferred to cryopreservation tubes. The tube was immersed for 10 seconds at 2/3 height in liquid nitrogen using forceps, and then the tube was completely immersed. This step should be performed in 60 seconds or more. The tube was then transferred to a liquid nitrogen storage tank for long term storage.

CELLBANKER ^® 1 thawing protocol 1

Standard thawing procedures may be performed. Briefly, the frozen vials were immersed in 37 ℃ water for up to 2 minutes until a small amount of solid medium remained in the vials. The preheated medium was added to the vial and the contents transferred to a conical tube with fresh medium. The suspension was pipetted to wash the thawed HLO and then centrifuged at 300×g for 5 minutes at room temperature. The medium was aspirated and the HLO was resuspended in fresh medium containing CEPT (and optionally Matrige ^® a 1).

Cell Reservoir One Vitrify thawing protocol

10ML of cell culture medium was pre-heated in a centrifuge tube at 37 ℃. The sterile plates were also preheated in a 37 ℃ incubator. The cryopreservation tube containing the frozen cells was removed from the liquid nitrogen reservoir, the cap was removed, and all liquid nitrogen in the tube was discarded. HLO was rapidly thawed by adding more than 800 μl of pre-warmed cell culture medium to the tube and pipetting several times. The larger the volume used, the faster the sample thaws. An appropriate volume of medium should be added depending on the size of the tube. The thawed cell suspension is then transferred to a sterile centrifuge tube. The cryopreservation tube was washed with fresh cell culture medium and the thawed cell suspension was added. The HLO was centrifuged at 300 Xg for 5 minutes at room temperature. The medium was aspirated and the HLO was resuspended in fresh medium containing CEPT (and optionally Matrigel ^®).

EXAMPLE 2 freezing and thawing of liver organoids cultures

Human liver organoids (day 22 of culture) were frozen in CELLBANKER ^® a or Vitrify (24 hours at-80 ℃ C., then transferred to liquid nitrogen storage). An exemplary scheme is provided in example 1.

For testing, organoids were thawed and immediately placed into hepatocyte medium. Live/dead staining was performed on the same day as thawing. In addition, the culture medium on the day of thawing and after 48 hours was collected to examine albumin secretion. A schematic diagram of this situation is provided in fig. 1A.

Fig. 1B shows live/dead staining results of thawed liver organoids. Organoids were stained with calcein AM (which marks living cells), ethidium homodimer-1 (which marks dead cells) and NucBlue (which marks nuclei). Human liver organoids show less damage after short-term freezing, indicating that freezing is a viable method of transporting organoids. The viability of the organoids subjected to vitrification (e.g., using Cell Reservoir One Vitrify) was slightly higher than that of organoids cryopreserved by slow freezing (e.g., using CELLBANKER ^® 1).

Fig. 1C shows the results of measuring Albumin (ALB) secretion of a thawed liver organoid according to the protocol provided in example 1, as compared to an unfrozen liver organoid control. After slow freezing or vitrification cryopreservation, liver organoids do not exhibit albumin secretion immediately after thawing. However, liver organoids preserved by vitrification cryopreservation recovered ALB secretion function by up to 70% compared to unfrozen controls after 3 days of recovery culture.

Example 3.2D and 3D liver organoids are enhanced in maturation

Methods for promoting maturation of liver organoids in culture have been explored. Previous liver organoid growth methods used hepatocyte media and tested for supplementation of this media with additional amino acids. Amino Acid (AA) supplemented media was prepared as a mixture of a 14% non-essential amino acid solution (100×, containing alanine, asparagine, aspartic acid, glycine, serine, proline, and glutamic acid; thermoFisher, grand Island, NY), a 6% essential amino acid solution (50×, containing arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine; thermoFisher, grand Island, NY), and 80% hepatocyte media (HCM) (Hepatocyte Culture Medium BulletKit; lonza, walkersville, MD), which was neutralized to pH 7.0 using NaOH, and further supplemented with 20g/L glycine. Optionally, the hepatocyte medium may be supplemented with hepatocyte growth factor, including but not limited to Hepatocyte Growth Factor (HGF), oncostatin M, and/or dexamethasone.

Liver organoids were prepared from definitive endoderm differentiation and induction of liver lineage commitment using retinoic acid according to conventional methods, but were cultured in AA supplemented media for 8, 10 or 12 days after retinoic acid induction.

Expression of liver-specific genes Albumin (ALB), cytochrome P450 family 3 subfamily a member 4 (CYP 3 A4), phosphoenolpyruvate carboxylase 1 (PCK 1) and glucose-6-phosphate catalytic subunit (G6 PC) was quantified by RT-qPCR using liver organoids cultured in HCM without AA supplement as controls (fig. 2A). Liver organoids cultured in AA supplemented media for 8 days, 10 days, and 12 days exhibited enhanced expression of all these genes relative to control liver organoids.

ALB secretion and urea production in liver organoids cultured in AA supplemented medium for 8 days, 10 days and 12 days (and 14 days for urea quantification) following retinoic acid induction were also quantified compared to liver organoids cultured in HCM without AA supplement as a control (fig. 2B). Prolonged exposure to AA supplementation medium (12 days or 14 days) resulted in enhanced albumin secretion and urea production compared to control liver organoids.

Pluripotent stem cells are engineered with a luciferase reporter factor driven by the PCK1 promoter to enable measurement of gluconeogenesis and differentiation into liver organoids. Exemplary PCK1 luciferase reporter factors may be found in PCT publication WO 2021/2626676, which is hereby expressly incorporated by reference in its entirety. PCK1 expression in these liver organoids with this construct can be quantified by luciferase activity, either with living cells or after lysis. A schematic of this process is provided in fig. 2C.

Liver organoids were grown in AA supplemented medium or in HCM without AA supplement as controls. Bright field images of these organoids are shown in fig. 2D. Liver organoids grown in AA supplemented media exhibited increased PCK1 expression compared to controls as measured by living cells and luciferase activity after lysis (fig. 2E).

The use of AA supplementation media can also be extended from 3D organoids to two-dimensional (2D) hepatocyte cultures from multipotent stem cell differentiation. To test the effect of the medium on 2D hepatocyte cultures, AA supplemented medium was used to replace HCM without AA supplement at different times of culture (counting from initial pluripotent stem cell differentiation, day 12, 14, 16, 18, 21 or 34 of culture) after liver induction. A schematic representation of this process is shown in fig. 3A, where a is HCM alone, B is hcm+aa starting from day 12, and C-G replaces HCM with hcm+aa on days 14, 16, 18, 21 and 34, respectively.

Bright field images of 2D hepatocyte cultures grown at different time points supplemented with AA supplementation medium are shown in fig. 3B. Cells under all conditions showed normal hepatocyte morphology.

Lactic acid production was compared for 2D hepatocyte cultures grown in AA supplemented medium or HCM without AA supplement (fig. 3C). For each time point, fresh medium was changed the day before and sampled 24 hours later for testing. Hepatocytes grown in AA supplemented media exhibited reduced lactate, indicating a shift in metabolism from glucose-dependent metabolism to pyruvate-dependent metabolism relative to cells grown in conventional hepatocyte growth media.

Albumin secretion was quantified in 2D hepatocyte cultures after replacement of HCM without AA supplement with AA supplementation medium at various time points (fig. 3D). Hepatocytes supplemented with AA supplemented media showed maximum albumin secretion after 12 days of culture. Increased albumin secretion was observed over even longer culture durations relative to control hepatocytes (fig. 3E).

Gene expression of ALB, E-cadherin (E-cad), CYP3A4, G6PC, pyruvate kinase M1/2 (PKM) and PCK1 was quantified in day 58 2D hepatocytes grown in HCM without AA supplement or in AA supplemented media added at different times of culture (fig. 3F). Overall, AA supplements enhance liver maturation and gene expression. Interestingly, PCK1 and G6PC expression (repressed by insulin normally added in the medium) decreased with increasing duration of AA supplementation, suggesting that AA addition normalizes liver organoid metabolism.

The pluripotent stem cells were engineered to express mScarlet under the PCK1 promoter that served as a reporter of gluconeogenesis, and differentiated into hepatocytes in 2D culture. Hepatocytes were grown in HCM or AA supplemented media without AA supplements. Cells were also optionally incubated for 24 hours without insulin (in DMEM/F12 supplemented with 0.2% BSA) prior to fluorescent quantification. FIG. 3G shows mScarlet fluorescence images of these hepatocytes. Cells grown in AA supplemented media showed an increase in mScarlet expression driven by the PCK1 promoter 24 hours after insulin starvation, indicating that these cells showed increased metabolism compared to the control.

EpCAM (epithelial marker), vimentin (mesenchymal marker) and DAPI or hepatocyte nuclear factor 4α (hnf4α) (nuclei) of day 57 2D hepatocyte cultures grown in HCM without AA supplement or with AA supplement medium replaced on day 12 were probed (fig. 3H). Although hepatocytes cultured in HCM without AA supplement differentiated from pluripotent stem cells exhibited vimentin-positive mesenchymal cells, the absence of such vimentin signals in cells cultured with AA supplemented medium suggests that culturing cells in AA supplemented medium potentially reduced mesenchymal cells and enriched hepatocytes.

Example 4 production of human organs without Matrigel ^® entrapment

The cultivation of 3D human liver organoids without Matrigel ^® containing heterogeneous components is briefly explored in PCT publication WO 2018/085615, which is hereby expressly incorporated by reference in its entirety. A schematic of such a scheme is depicted in fig. 4A. The metaforegut cells and/or metaforegut endoderm cells differentiated from pluripotent stem cells spontaneously form three-dimensional structures upon retinoic acid induction and subsequent liver culture conditions (e.g., contact with hepatocyte growth factor, oncostatin M, and dexamethasone). These three-dimensional structures can be pipetted from the original cell layer and further cultured to grow into liver organoids (fig. 4B). These liver organoids grown in the absence of Matrigel ^® exhibited normal organoid morphology (fig. 4C) and expected liver function, such as albumin secretion (fig. 4D) and expression of ALB, alpha Fetoprotein (AFP), hnf4α, retinol binding protein 4 (RBP 4) and α1 antitrypsin (AAT) (fig. 4E).

Organoids grown without Matrigel ^® are advantageous because they do not grow with xenogeneic animal components and thus can be used for human applications. Furthermore, they can grow to relatively large sizes that are easier to handle. However, these organoids may have larger dimensional changes than those grown in Matrigel ^®, may exhibit abnormal morphology (because Matrigel ^® helps maintain the 3D form), and may be less suitable for cryopreservation (which may reduce the scale-up for manufacturing purposes). In addition, conventional protocols involve the use of Matrigel ^® during culture steps other than organoid maturation, such as pluripotent stem cell culture.

To overcome one or more of these potential drawbacks, alternative methods for culturing organoids without Matrigel ^® or other basal membrane matrices with heterogeneous components have been explored.

Methods of culturing foregut cells after initial induction using a combination of FGF2, EGF, VEGF, CHIR99201,99201 and a83-01 are briefly explored in PCT publication WO 2018/191673. Modified versions of this combination with or without EGF may be used to expand foregut cells that result from differentiation of definitive endoderm cells in the presence of an FGF pathway activator (e.g., FGF 4) and a Wnt pathway activator (e.g., CHIR 99021).

A schematic of the expansion of foregut cells prior to subsequent differentiation into the hepatic lineage is depicted in fig. 5A. After induction of definitive endoderm into the foregut, cells can be dissociated into single cells using standard enzymatic dissociation solutions (such as Accutase) and then passaged in EP medium (advanced DMEM/F12, B27/N2/HEPES/Glutamax, 5ng/mL FGF2, 10ng/mL VEGF, 3 μm CHIR99021, 500nm a83-01 and 50 μg/mL ascorbic acid) optionally supplemented with 10 μ M Y-27632 (ROCK inhibitor).

The ability of these dissociated foregut cells to grow on a substrate other than Matrigel ^® for expansion purposes was explored. The culture plates are coated with Matrigel ^® or laminin, which are basement membrane matrix components that can be manufactured and obtained in the absence of animal products. The basement membrane matrix-like coating is preferred for culture because the foregut cells would otherwise not adhere properly to the plate for growth. The foregut cells expanded on Matrigel ^® and laminin coated plates grew properly during the 7 day culture and began to form 3D spheroids (fig. 5B). After additional culture (days 8-10), fully formed spheroids were observed, which can be further matured into liver organoids (fig. 5C). Although there was no significant difference in initial cell adhesion, spheroid formation efficiency with the laminin coating was better than that of Matrigel ^® coating.

The number of dissociated foregut cells used to inoculate the plates for expansion was also tested. 1.0X10 ⁶ or 5.0X10 ⁶ foregut cells were seeded onto laminin coated 6-well plates (9.6 cm ² surface area per well) and allowed to grow for 5 days (FIG. 5D). Spheroid formation begins earlier as the number of seeded cells increases (as seen in larger spheroids at each time point for plates seeded with 5.0 x 10 ⁶ cells). Thus, spheroid formation depends on cell density.

Since expanded foregut cells spontaneously form spheroids, they can be used as a source for manufacturing scale spheroids. Spontaneously formed 3D spheroids were collected from day 12 foregut cell culture and used for liver organoid maturation. By continuing the plate culture after spheroid collection, additional spheroids were formed during an additional 23 days (up to day 35 of culture) (fig. 5E). These newly formed spheres exhibit the same ability to mature into liver organoids.

Additional passaging of proliferating foregut cells may be performed after initial dissociation, plating and recovery of the foregut cells. Four passages (once every 10 days) of the first day 8 foregut culture differentiated from pluripotent stem cells were tested. Spheroid formation was until the third passage, but less than the fourth passage (fig. 5F). In the fourth passage, the mesenchymal cells proliferated mainly. Passage three times from the initial foregut culture can increase the number of cells by about 200-fold, providing significant advantages for scale-up. The expression of tail homeobox 2 (CDX 2), fork Box A2 (FOXA 2), AFP, vimentin (VIM), SRY-Box transcription factor 17 (SOX 17), HNF4α and ALB was quantified for the foregut cells from each passage (FIG. 5G).

Expression of CDX2 and SOX17 was maintained in passage 4. The expression of FOXA2 and hnf4α peaked at generation 2.

In general, to obtain continuous formation of human liver organoids from single cell passaging of foregut cells, dissociated foregut cells can be grown on a coating of laminin (e.g., laminin-511) in EP medium (containing FGF 4) and have a dense plating number (e.g., 5×10 ⁶ cells/6 well plate [9.6cm ² ]). The foregut cells should be passaged at most for 3 passages, as the mesenchymal cells grow mainly in the subsequent passages and spheroids cease to form. A schematic representation of liver organogenesis starting from pluripotent stem cells and including passaging of foregut cells for scale-up is illustrated in fig. 5H, where "amplifiable foregut organoids" refer to spontaneously formed foregut spheroids that are collected for maturation into a liver organoid. The "organoid reformation" step refers to the serial passage of foregut cells, and the spontaneous formation of additional foregut spheroids, which are collected and matured into liver organoids.

This process of expanding foregut cells to increase liver organoid production can be further improved by using microplates (e.g., aggresell plates (StemCell Technologies)) or other devices, such as shaping plates, designed to aggregate foregut cells to form a more uniform organoid. These methods of producing uniform organoids using the device are discussed in PCT publication WO 2021/030373, which is hereby expressly incorporated by reference in its entirety. A schematic of liver organogenesis starting from pluripotent stem cells and including passaging of foregut cells for scale-up, and aggregation of foregut cells using the device is illustrated in fig. 5I.

Example 5 scalable 3D bioreactor liver organogenesis

Liver organoids without Matrigel ^® are difficult to maintain in static culture. The use of 3D rotational culture to maintain free-floating liver organoids was explored. A schematic of this process is provided in fig. 6A. Spheroids (e.g., those formed on a basement membrane matrix or components thereof that do not contain a non-human animal component as described herein) and cultures induced from the foregut using the method described in the previous example are collected and grown in hepatocyte culture medium in a spin culture. Under this rotation culture condition, spheroids can mature into liver organoids in the course of 15 days (fig. 6B). Gene expression of AFP, HNF4α, FOXA2, ALB, CDX2 and VIM was quantified in liver organoids (3D) grown in spin culture and compared to generation 1 (P1) foregut cells (FIG. 6C). Liver organoids exhibit high expression of ALB and AFP, which is indicated as mature hepatocytes. The expression of CDX2 and hnf4α was not significantly different from that of foregut cells. Thus, this demonstrates that when grown in rotary culture, liver organoids can be cultured without the need for Matrigel ^® or other basement membrane matrix.

Example 6 additional materials and methods

Animals

All animal experiments were conducted with the approval of the institutional review board and institutional animal care and use committee. Adult Gunn (Gunn-Ugt a1 j/BluHsdRrrc) rats (breeding pair, 9-12 weeks old) were obtained from the rat resource research center (RRRC, columbia, MO). Rats were housed in standard rat cages with wood chip litter maintained at a temperature of 20-24 ℃ and 45% -55% relative humidity for 12 hours, 12 hours light, dark cycle. All animals were given a standard diet ad libitum prior to the study (CINCINNATI LAB SUPPLY, cincinnati, OH). All animals were treated according to institutional guidelines and regulations.

Maintenance of PSCs

Human iPSC line 72.3 (RRID: CVCL _A1BW) was obtained from the Xincinnati pediatric hospital medical center (CCHMC) pluripotent stem cell facility, commonly guided by CN.Mayhew and JM.Wells. The undifferentiated hiPSC was cultured in StemFit medium (Ajinomoto Company) with 100ng/mL basic fibroblast growth factor (FGF; R & D Systems) at 37℃in 5% CO2 and 95% air on laminin-511 E8 fragment (Nippi) -coated dishes.

Production of Human Liver Organoids (HLO)

Pluripotent stem cells were plated at a density of 2×10 ⁵ cells/well on 24-well plates coated with laminin iMatrix-511 Silk and maintained in StemFit medium containing Y-27632. On day 2, the medium was replaced with fresh StemFit medium. The following day, cells were treated with RPMI medium mixed with activin a and BMP4 to produce definitive endoderm. On day 4, the medium was changed to RPMI, activin a and 0.2% dFBS,0.2% dFBS was changed to 2% dFBS on day 5. From day 6-8, FGF4 and CHIR99021 were supplied to cells in advanced DMEM (supplemented with B27, N2, 10mM HEPES, 2mM L-glutamine and gentamicin-amphotericin) to induce the hindgut. On day 9, cells were dissociated into single cell suspensions using Accutase treatment. The single cell suspension was then mixed with 50% matrigel ^® and 50% EP medium (advanced DMEM/F12, B27/N2/HEPES/Glutamax, 5ng/mL FGF2, 10ng/mL VEGF, 3. Mu.M CHIR99021, 500nM A83-01 and 50. Mu.g/mL ascorbic acid) and plated in 6 well plates as 50. Mu.l drops. EP medium was supplied to these cells every 48 hours for 4 days to produce organoids. These organoids were then treated every 48 hours with advanced DMEM and Retinoic Acid (RA) for 4 days to define the liver lineage. The organoids were then fed with hepatocyte culture fluid (HCM), hepatocyte Growth Factor (HGF), oncostatin M and dexamethasone every 3-4 days to produce HLO.

HLO maturation with low doses of bilirubin

For maturation induction in HLO, the organoids on day 15 were supplied with complete HCM with supplements in addition to low doses of bilirubin (1 mg/L). The treatment was continued until day 30, at which time the organoids were harvested. Bright field images were captured on a KEYENCE BZ-X710 fluorescence microscope (KEYENCE) and analyzed using the ImageJ kit. RNA was isolated using the Rneasy Mini kit (Qiagen, hilden, germany). Reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Thermo FISHER SCIENTIFIC Inc.) according to the manufacturer's protocol. qPCR was performed on a QuantStudio 5 real-time PCR system (Thermo FISHER SCIENTIFIC inc.) using TaqMan gene expression master mix (Applied Biosystems). All primer and probe information for each target gene was obtained from the universal probe library assay design center website (available on the world wide web life science. Roche. Com/en_us/brands/universal-probe-library. Html). For whole-mount immunostaining, organoids were fixed in 4% PFA, permeabilized with 0.1% PBST, blocked with 5% normal donkey serum in 0.1% PBST, and stained with the appropriate primary and secondary antibodies. Images were captured on a Nikon A1 inverted confocal microscope.

Bilirubin and drug treatment

On day 27, mature organoids will be treated with bilirubin (1 mg/L-10 mg/L), doxycycline (100 ng/mL; added 3 days prior to activating gene expression) and/or additional drugs such as hydrocortisone, dexamethasone, ketoconazole and mifepristone (1. Mu.M-2. Mu.M each) for 5 days before harvesting for downstream assays. Bilirubin assays were performed using a colorimetric kit (ab 235627 from Abcam). RNA was isolated using the Rneasy Mini kit (Qiagen, hilden, germany). Reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Thermo FISHER SCIENTIFIC Inc.) according to the manufacturer's protocol. qPCR was performed on a QuantStudio 5 real-time PCR system (Thermo FISHER SCIENTIFIC inc.) using TaqMan gene expression master mix (Applied Biosystems). All primer and probe information for each target gene was obtained from the universal probe library assay design center (available on the world wide web life science. Roche. Com/en_us/brands/universal-probe-library. Html). Images were captured on a KEYENCE BZ-X710 fluorescence microscope (Keyence).

Organoid graft to portal vein

HLOs were harvested on day 27 and dissociated into organoid fragments by repeated pipetting, washed with PBS and resuspended with HCM containing 2% FBS and CEPT mixture (50 nmchroman 1,5 μm emtriclosan, 1:1000 polyamine and 7 μm trans-ISRIb) to increase viability. Recipient rats were treated with a single dose of cepharanthine (retrorsine) (5 mg/kg) and tacrolimus (tacrolimus) (0.8 mg/kg) 4 days prior to implantation. A midline incision was made, the intestine was pushed to one side, and 200 μl of 3 x 10 ³ organoids (approximately 5x 10 ⁵ cells) in an infusion night were injected into the portal vein by pinching the portal vein back to control bleeding using a 32g 1 inch needle. Excess blood loss is prevented by application SURGICEL SnoW of an absorbable hemostatic agent (Ethicon). The animals were then sutured with 5-0 vicryl coated surgical sutures (Ethicon) and GLUture (Zoetis inc.) and buprenorphine (0.1 mg/kg) was administered as an analgesic. The animals were then maintained on doxycycline (2 mg/kg) and tacrolimus injections every 3-4 days until the day of harvest. Blood was collected by retroorbital legal period as needed.

Live cell imaging and functional assays

For in vivo imaging of organoids, celldiscoverer (Zeiss) was used for 7 days once every 30 minutes. Visualization of bilirubin conjugation was achieved with 5 μm fluorescence UnaG, fluorescence UnaG was incubated with HLO medium and imaged for 2 days.

RNA sequencing and analysis

RNA was isolated using Rneasy mini kit (Qiagen). Reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) for RT-PCR according to the manufacturer's protocol. qPCR was performed on a QuantStudio 5 real-time PCR system (Applied Biosystems) using TaqMan gene expression master mix (Applied Biosystems). All samples were amplified using a TaqMan gene expression assay and normalized with an 18S rRNA endogenous control. For RNA sequencing, the quality of the extracted RNA was assessed using an Agilent 2100 biological analyzer (Agilent). Sequence libraries were prepared using the TruSeq STRANDED MRNA kit (Illumina) and sequenced using NovaSeq 6000 (Illumina). Reads were aligned with human genome assembly hg38 and quantified using a quasimeter Salmon (v1.8.0). Gene expression analysis was performed using the R Bioconductor package DESeq2 (v1.36.0). The read count matrix is normalized with a size factor and a Variance Stabilizing Transformation (VST) is applied to the normalized expression data. Data were visualized using clusterProfiler (v.4.4.1) and pheatmap (v 1.0.12) packages.

Alternatively, whole transcriptome RNA sequencing of HLOs (including those treated with bilirubin and those treated with mifepristone) was performed from isolated total RNA using the service Novogene co., ltd (beijin, china) on the Illumina NovaSeq platform. The RNA sequencing parameters were 150bp paired-end sequencing at 20M read depth per sample. Fastq reads for each sample were obtained and then aligned using Salmon, a quasi-mapping tool that uses RNA-seq data to align and quantify transcripts. Raw transcript counts and normalized per million Transcript (TPM) values were obtained and differential expression was analyzed using DESeq 2. For differential expression, statistical and biological significance was set to P <0.05, fdr <0.05, log fold change >1 with a minimum of 3 transcript counts in 3 of the 6 samples. For heat map visualization and hierarchical cluster analysis hclust and pheatmap were used, respectively. Finally, pathway analysis was performed in R version 4.0.3 using biomaRt and org.hs.eg.db.

MGULO edit

Murine GULO (L-gulonolactone oxidase) (mGULO) cDNA sequence was retrieved from NCBI. The 5' linker and Kozak sequence were added to the beginning of the sequence and the HA tag was added to the end of the sequence. In addition, P2A-mCherry was added to the extreme end after the HA tag and 3' linker. The custom gene was then synthesized and cloned into pAAVS-Ndi-CRISPRi (Gen 1) PCSF #117 vector using restriction sites AflII and Agel. The vector had TetON systems and then Neo ^r selection markers were inserted using Gateway technology.

MGULO iPSC production and maintenance

The PCSF #117 vector with the modified GULO sequence was then inserted into the AAVS1 locus of the 72.3 iPSC cell line using lentiviral-mediated CRISPR/Cas 9. The correct clone was then selected using G418. PCR was then used to verify correct insertion, random insertion and copy number of surviving clones, and verified by DNA sequencing. The edited ipscs were then plated onto laminin iMatrix-511 Silk coated cell culture plates and maintained with StemFit Basic complete medium containing Y-27632. Cells were passaged every 4-7 days with Accutase until passage 40 (p 40). mGULO HLO is generated according to the HLO generation scheme as described herein. mGULO protein expression was verified using GLUO ELISA kit (MBS 2890737 from MyBioSource, san Diego, CA).

MGULO HLO treatment with bilirubin and visualization of bilirubin

At day 24, mGLUO HLO was treated with doxycycline (Dox) (100 ng/ml) to induce mGULO expression. Subsequently, on day 27, mature organoids were treated with bilirubin and Dox for 5 days and then harvested for downstream assays. Bilirubin assays were performed using colorimetric kits (ab 235627) and UnaG (a green to dark light converting fluorescent protein that fluoresces only when bilirubin was bound) to measure and visualize unconjugated bilirubin and conjugated bilirubin. Images were captured on a KEYENCE BZ-X710 fluorescence microscope (Keyence).

ChIP-PCR and ChIP-qPCR

ChIP experiments were performed using the high sensitivity ChIP kit (Abcam). Briefly, organoids were fixed with PFA, and then the whole chromatin was prepared and sonicated to an optimal size of 300bp, as confirmed by gel electrophoresis. Chromatin was used for immunoprecipitation with EP300 antibodies or IgG1 isotype controls. DNA fragments were amplified using custom primers for PCR and qPCR and fold enrichment data were normalized to immunoprecipitation of IgG controls.

Protein expression assay

Albumin secretion was measured by collecting 200 μl supernatant from HLOs cultured in HCM and stored at-80 ℃ until use. Supernatants were assayed using a human albumin ELISA kit (Bethyl Laboratories) according to the manufacturer's instructions.

For the murine GULO expression assay, organoids were dissociated and washed with PBS. Cells were then lysed with RIPA lysis and extraction buffer and a mixture of hat protease and phosphatase inhibitors (Thermo Scientific) to extract total protein and assayed using the mouse GULO/L-gulonolactone oxidase ELISA kit (mybiosource.com) according to the manufacturer's instructions.

Metabolite assay

Bilirubin levels are measured by collecting supernatant from HLOs treated with bilirubin and serum from rats. Supernatants and serum were assayed using a bilirubin assay kit (Total AND DIRECT, colormetric) (abcam) and a bilirubin assay kit (Sigma-Aldrich) according to the manufacturer's instructions.

Cellular antioxidant levels were measured by harvesting HLOs, washing in PBS, and plating them into 96-well assay plates. The levels were then quantified using the cellular antioxidant assay kit (abcam) according to the manufacturer's instructions.

Activity determination

By harvesting HLOs, washing in PBS, plating them into 96-well assay plates and performing CYP3A4 and CYP1A2 assays with rifampicin and omeprazole, respectively, for 24 hours. The assays were then performed using the P450-Glo CYP3A4 and CYP1A2 assays (Promega) and normalized using the CellTiter-Glo luminescent cell viability assay according to manufacturer's instructions.

Apoptosis assays were performed by lysing HLO and assaying lysates using a caspase-3 assay kit (colorimetric) (abcam) according to the manufacturer's instructions.

Rat serum was assayed using an aspartate Aminotransferase (AST) activity assay kit and an alanine Aminotransferase (ALT) activity assay kit (Sigma-Aldrich).

Quantitative and statistical analysis

Statistical analysis was performed using R software v4.2.0 with unpaired two-tailed student t-test, dunn-Holland-Wolfe test, or Welch test. Statistical analysis of stiffness measurements was performed using the nonparametric Kruskal-Wallis and the post-Dunn-Holland-Wolfe test. For comparison between 2 unpaired groups, when the groups are independent and the variances are unequal, a non-parametric Brunner-Munzel test is performed unless otherwise noted. P values <0.05 were considered statistically significant. N values refer to biologically independent repeats. The image analysis is non-blind.

Example 7 Low doses of bilirubin promote fetal-like liver organoids maturing

The effect of bilirubin on liver development was studied using a human liver organoid model. FIG. 7A depicts an exemplary schematic of preparing a liver organoid treated with low concentrations of bilirubin (e.g., 1 mg/L) similar to human fetal physiological concentrations (about 1/10 of adult physiological concentrations). Bilirubin is added to early organoids that differentiate into the liver lineage. Exemplary methods of producing liver organoids have been previously explored in, for example, PCT publication WO 2018/085615、WO 2018/191673、WO 2018/226267、WO 2019/126626、WO 2020/023245、WO 2020/069285、WO 2020/243613、WO 2021/030373 and WO 2021/2626676, each of which is hereby expressly incorporated by reference in its entirety. Based on these exemplary methods, low concentrations of bilirubin are added to liver organoids formed after the retinoic acid-induced foregut endoderm. Liver organoids can be cultured with bilirubin in standard hepatocyte medium (HCM). For example, the hepatocyte medium may be supplemented with hepatocyte growth factor, including but not limited to Hepatocyte Growth Factor (HGF), oncostatin M, and/or dexamethasone. The liver organoids are contacted with a growth medium containing 1mg/L bilirubin for at least 5 days to 10 days to promote maturation of the liver organoids.

FIG. 7B shows that liver organoids matured with 1mg/L bilirubin exhibit luminal herniation similar to that of the bile canaliculi, a natural structure found in liver tissue. The resulting mature liver organoids exhibited lumens with smaller dimensions and reduced roundness compared to control liver organoids that were not treated with bilirubin (fig. 7C).

Quantification of gene expression by RT-qPCR on bilirubin-treated liver organoids revealed that these organoids exhibited increased expression of mature liver markers such as Albumin (ALB), solute carrier family 4 member 2 (SLC 4 A2) and heme oxygenase-1 (HO-1) and decreased expression of immature or fetal liver markers such as Alpha Fetoprotein (AFP), homeobox NANOG and tail homeobox 2 (CDX 2) relative to untreated organoids (fig. 7D).

The drug metabolizing capacity of bilirubin-treated organoids was assessed by measuring cytochrome P4503A4 (CYP 3 A4) and cytochrome P450 1A2 (CYP 1 A2) activity after treatment with rifampicin and omeprazole. Bilirubin-treated organoids exhibit increased cytochrome activity relative to untreated control organoids (fig. 7E).

Immunofluorescence microscopy revealed that bilirubin treated organoids expressed mature liver enzymes and transport proteins, including cytochrome P450E 1 (CYP 2E 1), cytochrome P450 A1 (CYP 7 A1), multi-drug resistance protein 1 (MPR 1), multi-drug resistance associated protein 3 (MRP 3), prospero homeobox 1 (PROX 1), and organic anion transport polypeptide 2 (OATP 2) (fig. 7F-7H).

Thus, low concentrations of bilirubin, similar to physiological conditions, are used to induce liver organoids maturing in culture.

Example 8 ascorbic acid promotes liver organoid viability and GULO induces portal Zhou Yang characteristics

The effect of ascorbic acid (vitamin C) on liver development was studied using a human liver organoid model. Liver organoids cultured in media lacking ascorbic acid showed loss of viability and apoptosis (fig. 8A). This result is expected because ascorbic acid is an essential nutrient and human cells cannot synthesize naturally due to nonfunctional L-gulonolactone oxidase (GULO).

Unlike humans and some other mammals such as guinea pigs, many other mammals have a functional GULO gene and are capable of enzymatic synthesis of ascorbic acid. Since ascorbic acid is important for liver development, it is hypothesized that expression of exogenous GULO in liver organoids will improve its growth and maturation in culture.

Fig. 8B depicts an exemplary schematic of the genetic engineering of human pluripotent stem cells using CRISPR/Cas9 with GULO expression constructs driven by TetOn conditional expression systems. It is contemplated that alternative methods of introducing GULO exogenous sources into cells may be used. Furthermore, although the GULO gene from mice (mGULO) is used herein, similar functional GULO genes from other mammals may also be used. FIG. 8C shows an embodiment of the GULO gene operably linked to mCherry fluorescence reporter factor for visualization and pAAVS-Ndi-CRISPRi (Gen 1) vector used.

The pluripotent stem cells were engineered with mGULO conditional expression constructs by conventional methods and these mGULO stem cells were differentiated into liver organoids by the methods described previously (fig. 8D). As provided herein, these liver organoids may be incubated with low doses of bilirubin to promote liver organoid maturation. Fig. 8E depicts bright field images and fluorescence microscopy images of liver organoids ("liver organoids") expressing mGULO constructs, where mCherry expression was observed only when doxycycline (Dox) was applied to induce TetOn expression, indicating mGULO is co-expressed in the liver organoids. mGULO organoids underwent severe apoptosis when grown in ascorbate depletion medium, but survivability was saved when doxycycline was added to induce mGULO expression, which enabled liver organoids to synthesize their own ascorbate (fig. 8F). mGULO organoids showed a dose-dependent correlation between Dox concentration and GULO expression as determined by ELISA, as well as antioxidant levels determined using the cellular antioxidant assay kit (abcam), indicating the synthesis of ascorbic acid (fig. 8G).

The expression levels of genes involved in oxidative stress and inflammation were studied by RT-qPCR in mGULO organoids cultured in ascorbic acid depleted medium with or without the addition of 100ng/mL Dox (fig. 8H). The cytoprotective protein regulated the ascorbic acid-mediated elevation of nuclear factor erythroid 2-related factor 2 (NRF 2) in mGULO organoids treated with Dox relative to control and mGULO organoids not treated with Dox. The inflammatory cytokines interleukin 1 beta (IL 1B), interleukin 6 (IL 6) and tumor necrosis factor alpha (TNFa) were elevated in ascorbic acid starved organoids, indicating the presence of significant oxidative stress, while this expression of inflammatory factors was rescued similar to controls in the mGULO organoids treated with Dox. Similarly, caspase-3 activity increased in ascorbic acid starved mGULO organoids, indicating increased apoptosis, but returned to baseline after exposure to Dox (fig. 8I).

RNA sequencing of mGULO liver organoids treated with Dox (RNA-seq) revealed increased expression of markers associated with liver maturation and/or with periportal regions of the liver (FIG. 8J). Increased expression of fumarylacetoacetate hydrolase (FAH), albumin (ALB), phenylalanine hydroxylase (PAH), cytochrome P4503A4 (CYP 3A 4), carbamyl phosphate synthase 1 (CPS 1) and homogentisate oxidase (HGD) in mGULO organoids was observed. In general, expression of genes associated with periportal access was observed in mGULO organoids treated with Dox (fig. 8K).

Microscopic examination of mGULO organoids treated with Dox and 1mg/L bilirubin showed increasingly complex and irregular lumen shapes compared to control organoids without bilirubin and bilirubin-treated organoids (not expressing mGULO L). Figure 8M shows the relative size and roundness of the mGULO organoid lumen with or without bilirubin as compared to a control. Furthermore, albumin secretion was significantly increased in mGULO organoids treated with bilirubin (fig. 8N). mGULO organoids maintained their overall morphology when treated with different concentrations of Dox (10 ng/mL, 100ng/mL, 1000 ng/mL) (FIG. 8O). mGULO organoids treated with Dox and low doses of bilirubin also showed increased CYP3A4 and CYP1A2 activity in response to rifampicin or omeprazole lesions compared to non-mGULO organoids or controls (fig. 8P).

Protein UnaG binds to unconjugated bilirubin with high specificity to form a fluorescent apoprotein. Other bilirubin-related compounds, including conjugated bilirubin, biliverdin or urobilin, do not have the same ability to fluoresce UnaG (fig. 8Q). Thus, bilirubin conjugation activity was measured in mGULO organoids with or without Dox using UnaG. A decrease in UnaG fluorescence was observed in the Dox-induced mGULO organoids compared to the uninduced mGULO organoids, indicating less bilirubin binds to UnaG due to conjugation activity (fig. 8R). Additional information regarding UnaG can be found in Kumagai et al A bilirubin-inducible fluorescent protein from eel muscle. Cell (2013) 153 (7): 1602-11, which is expressly incorporated by reference in its entirety.

Determination of the total percentage of organoids and organoid viability quantifying conjugated bilirubin using UnaG showed that conjugation activity and viability increased with mGULO expression, as represented by the concentration of Dox applied (fig. 8S). Overall, this suggests that significant maturation and morphological specialization occurs in human liver organoids when expression is enhanced with GULO. Thus, intracellular ascorbic acid promotes maturation of fetal-like liver organoids, and the introduction of exogenous GULO can be used to improve liver organoid viability, optionally along with low doses of bilirubin.

EXAMPLE 9 hyperbilirubinemia liver organoid model

Liver organoids were treated with different concentrations of bilirubin in culture to induce hyperbilirubinemia phenotypes. FIG. 9A depicts a schematic of this process, wherein bilirubin is administered at 1mg/L, 2mg/L, 5mg/L, or 10mg/L to liver organoids differentiated from pluripotent stem cells. Liver organoids showed significant morphological changes and intracellular accumulation of bilirubin when exposed to increased concentrations of bilirubin, indicating that these organoids can be used as models of hyperbilirubinemia (fig. 9B). RT-qPCR revealed that expression of UDP-glucuronyl transferase family 1 member A1 (UGT 1A 1) and NRF2 increased corresponding to bilirubin doses (FIG. 9C). UGT1A1 is an enzyme involved in the conjugation of glucuronic acid to bilirubin that occurs in the liver, and is required to render bilirubin water-soluble for excretion.

To further investigate the use of UGT1A1 in bilirubin clearance and liver organoids for a disease model associated with bilirubin dysfunction, cells were obtained from patients diagnosed with Crigler-Najjar syndrome, a genetic disorder characterized by inability to clear bilirubin from the body (fig. 9D). Genetic sequencing of patient cells revealed nonsense mutations in the UGT1A1 gene at c.858c > a (p.cys280x).

Induced pluripotent stem cells (ipscs) produced from patient cells by conventional methods were confirmed to express classical pluripotent markers Sox2 and Oct4 (fig. 9E). These ipscs successfully differentiated into definitive endoderm and further into liver organoids according to the previously described methods (fig. 9F), producing Crigler-Najjar syndrome liver organoids ("CNS organoids" or "CNS HLOs"). The functional liver phenotype of these CNS HLOs was confirmed by the expression of AFP (fig. 9G).

When treated with 10mg/L bilirubin, these treatments of CNS HLO, which were unable to conjugate bilirubin due to inactivity of UGT1A1 gene, resulted in liver organoids toxicity (FIG. 9H). However, this toxicity due to bilirubin can be temporarily saved when exogenous UGT1A1mRNA is transfected into CNS HLO (fig. 9I). Determination of unconjugated bilirubin (UCB) and Conjugated Bilirubin (CB) quantitatively showed that CNS HLO was unable to conjugate bilirubin, whereas UGT1A1 ectopic expression achieved by mRNA delivery enabled a level of bilirubin conjugation (fig. 9J). Similarly, mGULO expression in normal HLO also increased bilirubin conjugation (fig. 9K).

Thus, bilirubin is conjugated by UGT1A1, and expression of functional UGT1A1 in a liver organoid model of Crigler-Najjar syndrome restores bilirubin conjugation function and improves liver organoid survivability. This suggests that these liver organoids may be used to study bilirubin dysfunction.

Example 10 negative modulation of UGT1A1 by glucocorticoid signalling

Glucocorticoids have been implicated in the increase of human serum bilirubin levels. Thus, the role of glucocorticoid signaling regulation was studied using liver organoid models.

Conventional liver organoids (unconditional expression mGULO) were cultured in 10mg/L bilirubin to induce a hyperbilirubinemia phenotype and treated with the glucocorticoid agonist hydrocortisone or dexamethasone, which resulted in additional organoid toxicity (fig. 10A) and reduced bilirubin binding capacity (fig. 10B). In contrast, hyperbilirubinemia organoids treated with glucocorticoid antagonists ketoconazole and mifepristone showed normal organoid morphology (fig. 10C) and increased bilirubin conjugation (fig. 10D). RT-qPCR revealed that UGT1A1 expression was suppressed after treatment with hydrocortisone or dexamethasone and increased after treatment with ketoconazole or mifepristone (FIG. 10E). Ketoconazole or mifepristone treatment also improved NRF2 expression. Thus, the glucocorticoid pathway was demonstrated to play a role in bilirubin metabolism and clearance in liver organoids.

Comparison of RNA sequencing and gene expression between control organoids and those organoids treated with mifepristone showed enrichment of many genes involved in liver function (fig. 10F). These enriched genes were further classified as genes involved in oxidative stress and/or xenobiotic metabolism (fig. 10G).

ChIP-PCR and ChIP-qPCR of hyperbilirubinemia organoids treated with mifepristone or dexamethasone showed that methyl CpG binding protein 2 (MECP 2) was involved in the silencing of UGT1A1, and treatment with mifepristone reversed this silencing (fig. 10H).

EXAMPLE 11 positive displacement implantation of HLO reduces hyperbilirubinemia in rodents

The effect of administration of liver organoid compositions to a rat model of hyperbilirubinemia in vivo was studied. Gunn rats lacking all members of the UGT1A family of bilirubin conjugating enzymes exhibit lifelong high unconjugated bilirubinemia. Fig. 11A depicts an exemplary schematic of the ability to restore conjugation of bilirubin to a Gunn rat model using a human liver organoid composition (also described in example 6).

Fig. 11B shows increased albumin production in the Gunn rats transplanted with HLO, indicating increased liver function. Figure 11C shows a decrease in serum bilirubin levels in HLO transplanted rats, indicating improved bilirubin conjugation and clearance. Fig. 11D shows a decrease in serum concentrations of aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) in HLO transplanted rats, wherein elevated AST and ALT levels indicate liver injury, indicating that HLO transplanted rats exhibited healthier liver function.

EXAMPLE 12 Mass production of HLO Using 3D bioreactor

The ability to culture and amplify HLOs in a 3D bioreactor with or without Matrigel ^® was investigated. The amplified HLOs were then characterized, and reconstructed and amplified.

The 3D bioreactor is used for realizing the large-scale cultivation of HLO. FIG. 12A shows an exemplary process of subjecting HLO induced by conventional methods to amplification culture with (FIG. 12B) or without (FIG. 12C) Matrigel ^®. Dynamic methods for mass production of functional HLOs were established using 3D bioreactor cultures with 50% less Matrigel ^®% than static cultures. Fig. 12B shows that HLOs grow significantly larger after 15 days of 3D bioreactor culture and that HLOs with 3-5 fold diameters can be obtained compared to conventional static cultures. Fig. 12C shows that a large HLO is obtained even in the group to which Matrigel ^® is not added. In contrast to static cultures, 3D bioreactor cultures effectively captured high proliferation and amplification capacity.

Characterization of HLO amplified in 3D bioreactor. Immunohistochemical staining was then used to compare HLOs induced by static culture with those obtained from 3D bioreactor culture, whether Matrigel ^® was added or not. As shown in fig. 13, similar expression levels of E-cadherin, vimentin, and Prox1 were observed, indicating that the HLOs obtained from the 3D bioreactor culture had comparable properties to those obtained from the static culture.

HLO was then reconstituted and amplified in a 3D bioreactor. Fig. 14A shows an exemplary process of dissociating HLO induced by conventional methods into single cells, and passaging and reconstitution of HLO using a 3D bioreactor. Initially, HLOs induced by conventional static culture were dissociated into single cells using enzyme treatment. Single cell HLOs were then reconstituted in a 3D bioreactor using HLO formation medium containing FGF2, VEGF, EGF, A-83-01 and CHIR 99021. Figure 14B shows that, obviously, uniform HLO was induced after 6 days. This HLO reconstitution in a 3D bioreactor allows for more than 3 passages and reconstitution with the addition of Matrigel ^®.

EXAMPLE 13 treatment of Acetaminophen acute liver injury mice by transplantation of AA-treated HLO

The therapeutic effect of HLO transplantation was tested in a mouse model of acetaminophen (APAP) -induced acute liver injury.

Acetaminophen (APAP) solutions were prepared by preparing fresh batches of APAP solution one day prior to each experiment, in the form of 25mL of APAP solution, sufficient for injection into 12 adult mice. Then, 375mg APAP (25 mL) was weighed15 Mg/mL) and transferred to a50 mL tube containing 25mL of 0.9% physiological saline (NaCl). The tube was briefly vortexed to mix the APAP powder with the saline solution, and then the tube was placed in a water bath or bead bath set at 37 ℃. Steps 3 and 4 are then repeated to completely dissolve the APAP powder. The solution was sterilized with fully dissolved APAP by filtration using a 0.2 μm filter. The materials and the kit comprise pharmaceutical grade acetaminophen SIGMA ALDRICH catalog A5000-100G, DPBS Gibco catalog AT104-500, and alanine aminotransferase activity assay kit abcam catalog ab105134. APAP was diluted to 15mg/mL with 0.9% saline because it was difficult to dissolve. To dissolve the APAP, the solution was heated to 37 ℃ and vortexed frequently (about 3-4 hours required for complete dissolution).

Transplantation utilized HLO medium (HCM), HLO and aa_hlo as controls. The experimental procedure was to transplant 5,000 HLOs into one mouse with APAP-induced acute liver injury and evaluate the treatment effect 7 days after follow-up. The number of transplants for the medium-only control was 22 mice, the HLO was 29 mice, and the aa_hlo was 24 mice.

For overnight fasted NSG mice, animals were flagged and weighed one day prior to transplantation, all food removed, and only water was left available in the cage. The cage cards were labeled as indicating overnight fast according to the protocol. Overnight fast is important for the APAP overdose model, as fast allows for the depletion of liver glutathione, which is required to cause liver damage via APAP overdose. Typically, fasting begins between 4-6 pm to avoid excessive fasting periods (e.g., to avoid morning fasting).

For excess APAP in fasted NSG mice, animals were weighed and the amount of APAP solution injected per mouse was calculated. A dose of 700mg/kg has been found to produce severe Acute Liver Failure (ALF) for survival curve studies and demonstrates the therapeutic benefit of HLO transplantation. Mice were placed under anesthesia (3% isoflurane for induction, 2% for maintenance) for baseline blood drawing.

For 0 hour blood sampling, 20 μl of blood was obtained using heparinized capillaries, and 20 μl of blood was transferred into 180 μl of saline in the tube to make 200 μl of 1:10 diluted blood sample. An appropriate amount of APAP solution was withdrawn from the corresponding mice in a 3mL syringe and injected intraperitoneally into the right lower abdomen of the mice with a 27G needle. Conventional foods were then returned to the cages and hydrogels and nutritional supplements were provided to support hydration and nutrition for mice that had developed severe illness within days after APAP overdose.

For intraperitoneal HLO implantation, HLO was transplanted 6 hours after APAP injection. Mice were placed under anesthesia to draw blood at 6 hour time points. Mice are now ill, requiring less anesthesia (about 1.5% to 2%), and 1.5% -2% isoflurane is used for the rest of the experiment because the use of too high a% isoflurane can lead to death.

For pre-transplant blood sampling, 20 μl of blood was obtained using heparinized capillaries, and 20 μl of blood was transferred into 180 μl of saline in the tube to make 200 μl of 1:10 diluted blood sample. Then, 300. Mu.L of HLO suspension was withdrawn into a 1mL syringe using an 18G needle. The needle was replaced with a 23G needle and the suspension was injected into the right lower abdomen of the mouse without puncturing too deeply to damage other organs. The mice were then returned to the cages. The control was 300. Mu.L of conventional HCM without HLO.

For post-implantation follow-up, each mouse was weighed daily. For post-transplant blood sampling, 20 μl of blood was obtained using heparinized capillaries, and then 20 μl of blood was transferred into 180 μl of saline in the tube to make 200 μl of 1:10 diluted blood sample. The collected blood was then centrifuged at 1000g for 10 minutes at 4 ℃ to separate red blood cells and other cells. The supernatant fraction was then transferred to a new epothilone tube (epothilone tube) and stored at-80 ℃ until analysis. Blood samples were taken after 48 hours, 72 hours and 168 hours.

To determine liver damage and ammonia levels, APAP-induced liver damage was measured using an alanine aminotransferase activity assay kit, according to the kit instructions. A 20uL blood sample was used for ALT measurement. A dilution of 1:10 will give a value within the kit.

Dry chemical methods (mammalian liver group for ALT from Schubert RESEARCH CLINIC and our portable ammonia machine for ammonia) can be used. These should be done immediately after sample collection (samples cannot be cryopreserved and can only be stored temporarily at 4 ℃ for 1-2 hours).

The results show that the HLO transplanted group helped mice with acute liver injury survive compared to the medium alone. In particular, aa_hlo transplanted groups significantly improved survival compared to medium alone (log rank test p=0.0267).

Fig. 15A shows a schematic representation of a model for rescue of acetaminophen acute liver injury by HLO transplantation. Fig. 15B shows an image of HLO for transplantation. FIG. 15C shows Kaplan-Meier survival curves for acute liver injury rescue by HLO transplantation.

In at least some of the previously described embodiments and the following embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such substitution is technically not feasible. Those skilled in the art will appreciate that various other omissions, additions and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. For clarity, various singular/plural permutations may be explicitly set forth herein.

It will be understood by those within the art that, in general, terms used herein, and especially those used in the appended claims (e.g., bodies of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "comprising" should be interpreted as "including but not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); and so forth for the use of the indefinite articles "a" or "an" for introducing a claim recitation. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Further, where a convention analogous to "at least one of A, B, C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B and C" would include, but not be limited to, a system having a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B and C together, etc.). In the case of using a convention analogous to "A, B or at least one of C, etc." such a construction is intended in general in the sense one skilled in the art would understand the convention (e.g., "one system has at least one of A, B or C" would include but not be limited to the system having a alone a, B alone, C, A alone with B, a with C, B with C, and/or A, B and C together, etc.). Those skilled in the art will further appreciate that virtually any separating word and/or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "a or B" will be understood to encompass the possibilities of "a" or "B" or "a and B".

In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by those of skill in the art, for any and all purposes, as in providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be readily considered as fully described and achieves that the same range is broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each of the ranges discussed herein can be readily broken down into a lower third, a middle third, an upper third, and the like. As will also be understood by those skilled in the art, all language such as "up to", "at least", "greater than", "less than" and the like include the recited numbers and refer to ranges that can be subsequently broken down into sub-ranges as discussed herein. Finally, as will be appreciated by those skilled in the art, a range encompasses each individual member. Thus, for example, a group of 1 to 3 items refers to a group of 1,2, or 3 items. Similarly, a group of 1 to 5 items refers to a group of 1,2, 3, 4, or 5 items, and so on.

Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

All references cited herein, including but not limited to published and unpublished applications, patents and references, are incorporated herein by reference in their entirety and are hereby incorporated as part of this specification. In the event that publications and patents or patent applications incorporated by reference conflict with the disclosures contained in this specification, this specification intends to replace and/or take precedence over any such conflicting material.

Description of the embodiments

Exemplary embodiments of the present disclosure are provided in the following numbered embodiments:

1. An embodiment for expanding metaintestinal cells and/or metaintestinal endoderm cells, the embodiment comprising:

a) Dissociating the monolayer of metaforegut cells and/or the monolayer of metaforegut endoderm cells into metaforegut cells and/or metaforegut endoderm cells;

b) Inoculating said metaforegut cells and/or said metaforegut endoderm cells onto a tissue culture surface, and

C) Culturing the metaforegut cells and/or the metaforegut endoderm cells with a TGF-b pathway inhibitor, a FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator.

2. Embodiment 1, wherein the posterior foregut cell monolayer is dissociated into the posterior foregut cells and/or the posterior foregut endoderm cells using enzymatic dissociation and/or mechanical dissociation.

3. Any of embodiments 1 or 2, wherein the posterior foregut cells and/or the posterior foregut endoderm cells are seeded onto the tissue container surface at a cell density equal to or about 1 x 10 ⁵, 2x 10 ⁵, 3 x 10 ⁵, 4 x 10 ⁵, 5 x 10 ⁵, 6x 10 ⁵, 7 x 10 ⁵, 8 x 10 ⁵, 9 x 10 ⁵, 1 x 10 ⁶, 2x 10 ⁶, 3 x 10 ⁶, 4 x 10 ⁶, or 5 x 10 ⁶ cells/cm ² of the surface area of the tissue culture surface, or at any cell density having a range defined by any two of the cell densities described above.

4. Any of embodiments 1 to 3, wherein the tissue culture surface is coated with a basement membrane matrix or a component thereof.

5. Embodiment 4, wherein the basement membrane matrix or component thereof does not comprise a non-human animal component that renders the basement membrane matrix or component thereof xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel ^®、Cultrex^® or Geltrex ^®.

6. The any of embodiments 4 or 5, wherein the basement membrane matrix or component thereof comprises human fibronectin, collagen IV, entactin, basement membrane glycans, fibrin, and/or hydrogels.

7. Any of embodiments 1 to 6, wherein the metaforegut cells and/or the metaforegut endoderm cells are cultured until three-dimensional (3D) spheroids are spontaneously formed, optionally wherein the 3D spheroids comprise a structure with a single lumen, and/or wherein the spheroids are free of hematopoietic tissue and acquired immune cells.

8. Any of embodiments 1 to 7, wherein the metaforegut cells and/or the metaforegut endoderm cells are cultured for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days or 35 days.

9. Any one of embodiments 1 to 8, wherein the TGF-b pathway inhibitor is selected from the group consisting of A83-01, repSox, LY365947, and SB431542, optionally A83-01.

10. Any of embodiments 1 to 9, wherein the TGF-b pathway inhibitor is provided at a concentration equal to or about 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the TGF-b pathway inhibitor is provided at a concentration equal to or about 500 nM.

11. Any of embodiments 1 to 10, wherein the FGF pathway activator is selected from the group consisting of ：FGF1、FGF2、FGF3、FGF4、FGF4、FGF5、FGF6、FGF7、FGF8、FGF8、FGF9、FGF10、FGF11、FGF12、FGF13、FGF14、FGF15、FGF16、FGF17、FGF18、FGF19、FGF20、FGF21、FGF22 and FGF23, optionally FGF2.

12. Any of embodiments 1 to 11, wherein the FGF pathway activator is provided at a concentration equal to or about 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, or 10ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the FGF pathway activator is provided at a concentration equal to or about 5 ng/mL.

13. Any of embodiments 1 to 12, wherein the Wnt pathway activator is selected from the group consisting of ：Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11、Wnt16、BML 284、IQ-1、WAY 262611、CHIR99021、CHIR 98014、AZD2858、BIO、AR-A014418、SB 216763、SB 415286、 aloxin, indirubin, altbolone, kenparone, lithium chloride, TDZD 8, and TWS119, optionally CHIR99021.

14. Any of embodiments 1 to 13, wherein the Wnt pathway activator is provided at a concentration equal to or about 1 μΜ, 1.5 μΜ, 2 μΜ, 2.5 μΜ,3 μΜ, 3.5 μΜ,4 μΜ, 4.5 μΜ,5 μΜ, 5.5 μΜ,6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ or 8 μΜ, or any concentration in the range defined by any two of the foregoing concentrations, optionally wherein the Wnt pathway activator is provided at a concentration equal to or about 3 μΜ.

15. Any of embodiments 1 to 14, wherein the VEGF pathway activator is selected from the group consisting of VEGF or GS4012, optionally VEGF.

16. Any of embodiments 1 to 15, wherein the VEGF pathway activator is provided at a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the VEGF pathway activator is provided at a concentration equal to or about 10 ng/mL.

17. Any one of embodiments 1 to 16, wherein the metaintestinal cells and/or the metaintestinal endoderm cells of step c) are cultured in a medium further comprising EGF or in a medium not comprising EGF.

18. Embodiment 17, wherein the EGF is provided at a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the EGF is provided at a concentration equal to or about 20 ng/mL.

19. Any of embodiments 1 to 18, wherein the metaintestinal cells and/or the metaintestinal endoderm cells of step c) are cultured in a medium further comprising ascorbic acid, or in a medium not comprising ascorbic acid.

20. The embodiment 19, wherein the ascorbic acid is provided at a concentration equal to or about 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, or 100 μg/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ascorbic acid is provided at a concentration equal to or about 50 μg/mL.

21. Any of embodiments 1 to 20, wherein the metaintestinal cells and/or the metaintestinal endoderm cells of step c) are cultured in a medium further comprising a ROCK inhibitor, or in a medium not comprising the ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632.

22. Embodiment 21, wherein the ROCK inhibitor is provided at a concentration equal to or about 1 μΜ,2 μΜ,3 μΜ,4 μΜ,5 μΜ,6 μΜ,7 μΜ,8 μΜ,9 μΜ,10 μΜ,11 μΜ,12 μΜ,13 μΜ,14 μΜ,15 μΜ,16 μΜ,17 μΜ,18 μΜ,19 μΜ or 20 μΜ, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ROCK inhibitor is provided at a concentration equal to or about 10 μΜ.

23. Any of embodiments 1 to 22, further comprising passaging the cells of step c) one or more times.

24. Embodiment 23, wherein passage of said cells of step c) to said posterior foregut cells and/or said posterior foregut endoderm cells does not spontaneously form spheroids.

25. The method of any one of embodiments 23 or 24, wherein said cells of step c) are passaged no more than 3 times.

26. Any of embodiments 23 to 25, further comprising collecting the metaforegut cells and/or the metaforegut endoderm cells and differentiating the metaforegut cells and/or the metaforegut endoderm cells into liver organoids.

27. Embodiment 26, wherein the metaforegut cells and/or the metaforegut endoderm cells are cultured until three-dimensional (3D) spheroids are spontaneously formed, and the metaforegut cells and/or the metaforegut endoderm cells are collected from the spheroids, optionally the method further comprises dissociating the spheroids into individual masses of metaforegut cells and/or metaforegut endoderm cells prior to the differentiating step, optionally wherein the spheroids comprise a structure with a single lumen, and/or wherein the spheroids are free of hematopoietic tissue and acquired immune cells.

28. Embodiment 26, wherein the metaforegut cell monolayer is collected from the metaforegut cell monolayer prior to the differentiating step by dissociating the metaforegut cell monolayer into individual metaforegut cells and/or metaforegut endoderm cells and/or clusters of metaforegut cells and/or metaforegut endoderm cells.

29. An embodiment of a method comprising differentiating metaforegut cells and/or metaforegut endoderm cells into liver organoids, the method comprising:

i) Contacting a metaforegut cell and/or a metaforegut endoderm cell, optionally in the form of a spheroid, optionally in the form of individual cells or clusters of cells dissociated from the spheroid, and/or optionally aggregated cells in a microwell or other device described herein, optionally wherein the spheroid comprises a structure having a single lumen, and/or wherein the spheroid is free of hematopoietic tissue and acquired immune cells, and

Ii) contacting the cells of step i) with a culture medium for a period of time such that the metaforegut cells and/or the metaforegut endoderm cells differentiate into liver organoids, optionally wherein the culture medium is a hepatocyte culture medium.

30. Embodiment 29, wherein the medium is supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid.

31. Embodiment 30, wherein the cMET tyrosine kinase receptor agonist is selected from the group consisting of Hepatocyte Growth Factor (HGF), PG-001, fugondonone, tetralin Lei Walai phenanthrene, recombinant InlB321 protein, and an agonistic c-Met antibody, optionally LMH85.

32. Embodiment 30 or 31, wherein the IL-6 family cytokine is selected from the group consisting of IL-6, oncostatin M (OSM), leukemia Inhibitory Factor (LIF), cardiac dystrophin-1, ciliary neurotrophic factor (CTNF), and cardiac dystrophin-like cytokine (CLC).

33. Any of embodiments 30 to 32, wherein the corticosteroid is selected from the group consisting of dexamethasone, beclomethasone, betamethasone, flucortisone, halometasone, and mometasone.

34. Embodiment 29, wherein the medium is supplemented with HGE, OSM, and dexamethasone.

35. Embodiment 29, wherein the medium is supplemented with dexamethasone.

36. Any of embodiments 29 to 35, wherein the metaforegut cells and/or the metaforegut endoderm cells are metaforegut cells and/or the metaforegut endoderm cells produced by the method of any of embodiments 1 to 28.

37. Any of embodiments 29 to 36, wherein the metaforegut cells and/or the metaforegut endoderm cells are in the form of spheroids or metaforegut cells and/or metaforegut endoderm cells alone, and/or in the form of a pellet derived from dissociating metaforegut cells and/or metaforegut endoderm cells of the spheroids, optionally wherein the spheroids comprise a structure having a single lumen, and/or wherein the spheroids are free of hematopoietic tissue and acquired immune cells.

38. Any of embodiments 29 through 37, wherein the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally retinoic acid.

39. Any of embodiments 29 to 38, wherein the retinoic acid pathway activator is provided at a concentration of 1.0µM、1.1µM、1.2µM、1.3µM、1.4µM、1.5µM、1.6µM、1.7µM、1.8µM、1.9µM、2.0µM、2.1µM、2.2µM、2.3µM、2.4µM、2.5µM、2.6µM、2.7µM、2.8µM、2.9µM or 3.0 μm, or at any concentration within the range defined by any two of the foregoing concentrations, optionally wherein the retinoic acid pathway activator is provided at a concentration equal to or about 2.0 μm.

40. Embodiment 34, wherein said HGF is provided at a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein said HGF is provided at a concentration equal to or about 10 ng/mL.

41. Embodiment 34, wherein the OSM is provided at a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the OSM is provided at a concentration equal to or about 20 ng/mL.

42. Any of embodiments 34 or 35, wherein the dexamethasone is provided at a concentration equal to or about 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, or 200nM, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the dexamethasone is provided at a concentration equal to or about 100 nM.

43. Any one of embodiments 29 to 42, wherein the cells of step i) and/or step ii) are not contacted with EGF.

44. Any of embodiments 29 to 43, wherein the cells of step ii) are cultured in a growth medium supplemented with non-essential amino acids, and glycine.

45. Embodiment 44, wherein the growth medium after supplementation comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of nonessential amino acids by total volume, or a range defined by any two of the foregoing values, optionally wherein the growth medium after supplementation is about 4% -10%, 6% -12%, 10% -16%, 12% -15%, 13% -19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% nonessential amino acids by total volume.

46. Embodiment 44 or 45, wherein the growth medium after supplementation comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% essential amino acids by total volume, or within a range defined by any two of the foregoing values, optionally wherein the growth medium after supplementation is about 4% -10%, 6% -12%, 10% -16%, 12% -15%, 13% -19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% essential amino acids by total volume.

47. Any of embodiments 44 to 46, wherein the supplemented glycine is provided at a concentration equal to or about 5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL、21mg/mL、22mg/mL、23mg/mL、24mg/mL、25mg/mL、26mg/mL、27mg/mL、28mg/mL、29mg/mL、30mg/mL、31mg/mL、32mg/mL、33mg/mL、34mg/mL or 35mg/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the supplemented glycine is provided at a concentration equal to or about 18mg/mL-22mg/mL or 20 mg/mL.

48. Any of embodiments 29 to 47, wherein the cells of step ii) are further contacted with a low/first concentration of bilirubin, wherein the liver organoid formed is a mature liver organoid.

49. Embodiment 48, wherein the low/first concentration of bilirubin is human fetal physiological concentration of bilirubin.

50. The embodiment 48 or 49, wherein the low concentration/first concentration of bilirubin is, is about, less than, or less than about ：a) 0.1mg/L、0.2mg/L、0.3mg/L、0.4mg/L、0.5mg/L、0.6mg/L、0.7mg/L、0.8mg/L、0.9mg/L、1mg/L、1.25mg/L、1.5mg/L、1.75mg/L、2.0mg/L、2.25mg/L、2.5mg/L、2.75mg/L or 3.0mg/L, or a range defined by any two of the foregoing concentrations, e.g., 0.1mg/L to 3mg/L, 0.5mg/L to 2.0mg/L, 0.5mg/L to 1.5mg/L, 0.3mg/L to 2.5mg/L, or 0.5mg/L to 1.75mg/L, or b) 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, or 1mg/L, or a range defined by any two of the foregoing concentrations, e.g., 0.1mg/L to 1mg/L, 0.1mg/L to 0.5mg/L, or 0.5mg/L to 1.75mg/L, or b) 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L or 0.7 mg/L.

51. Any of embodiments 48 to 50, wherein the mature liver organoid exhibits a luminal protrusion resembling a bile duct, and/or has a single lumen and generally spherically shaped structure, and/or wherein the mature liver organoid is free of hematopoietic tissue and acquired immune cells.

52. Any of embodiments 48 to 51, wherein the mature liver organoid expresses reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to a liver organoid not contacted with a low/first dose of bilirubin.

53. Any of embodiments 48 to 52, wherein the mature liver organoid expresses increased levels of ALB, SLC4A2 or HO-1, or any combination thereof, relative to a liver organoid not contacted with the low/first dose of bilirubin.

54. Any one of embodiments 48 to 53, wherein the mature liver organoid expresses CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof.

55. Any of embodiments 48 to 54, wherein the mature liver organoid exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid not contacted with the low dose/first dose of bilirubin.

56. Any of embodiments 29 to 55, wherein the cells of step ii) are further contacted with a high/second concentration of bilirubin, wherein the liver organoid formed is a hyperbilirubinemia liver organoid.

57. The embodiment 56, wherein the high/second concentration of bilirubin is, is about, greater than or greater than about ：a) 2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or a range defined by any two of the foregoing concentrations, such as, for example, any of 2mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12mg/L, or b) 4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or a range defined by any two of the foregoing concentrations, such as, for example, 4mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L.

58. Embodiment 56 or 57, wherein the hyperbilirubinemia liver organoid expresses elevated levels of UGT1A1 or NRF2 or both relative to a liver organoid not treated with high/second concentrations of bilirubin.

59. Any of embodiments 29 to 58, wherein the liver organoid comprises a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA encoding the functional GULO protein or both, wherein the liver organoid is capable of synthesizing ascorbic acid.

60. Embodiment 59, wherein the functional GULO protein is murine GULO (mGluLO).

61. Embodiment 59 or 60, wherein the gene encoding the functional GULO protein is conditionally expressed, optionally using a tetracycline-inducible system.

62. Any of embodiments 59 to 61, wherein said liver organoid is engineered with said gene encoding said functional GULO protein using CRISPR.

63. Any of embodiments 59 to 62, wherein the gene or mRNA encoding the functional GULO protein, or both, is introduced to the liver organoid by transfection.

64. Any of embodiments 59 to 63, wherein said liver organoid comprising said functional GULO protein expresses increased levels of NRF2 relative to a liver organoid not comprising said functional GULO protein.

65. Any of embodiments 59 to 64, wherein the liver organoid comprising the functional GULO protein expresses reduced levels of IL1B, IL6 or tnfa or any combination thereof, relative to a liver organoid not comprising the functional GULO protein, optionally when cultured in an ascorbic acid-depleted medium or in the absence of ascorbic acid.

66. Any of embodiments 59 to 65, wherein optionally the liver organoid comprising the functional GULO protein exhibits reduced caspase-3 activity when cultured in ascorbic acid depleted medium or in the absence of ascorbic acid relative to a liver organoid not comprising the functional GULO protein.

67. Any one of embodiments 59 to 66, wherein the liver organoid comprising the functional GULO protein expresses increased levels of ALB relative to a liver organoid not comprising the functional GULO protein.

68. Any of embodiments 59 to 67, wherein the liver organoid comprising the functional GULO protein is similar to and expresses a periportal liver marker.

69. Embodiment 68, wherein said periportal liver marker comprises FAH, ALB, PAH, CPS a 1, HGD, or any combination thereof.

70. Any one of embodiments 59 to 69, wherein the liver organoid comprising the functional GULO protein exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid not comprising the functional GULO protein.

71. Any of embodiments 59 to 70, wherein the liver organoid comprising the functional GULO protein exhibits increased bilirubin conjugation activity relative to a liver organoid not comprising the functional GULO protein.

72. Any of embodiments 59 to 71, wherein the liver organoid comprising the functional GULO protein exhibits increased viability in culture relative to a liver organoid not comprising the functional GULO protein.

73. Any of embodiments 59 to 72, wherein the liver organoid has been differentiated from a pluripotent stem cell comprising a functional GULO protein and/or a gene or mRNA encoding the functional GULO protein or both, whereby the pluripotent stem cell is capable of synthesizing ascorbic acid.

74. Any of embodiments 59 to 73, wherein the liver organoid comprises an inactive UGT1A1 gene, wherein the liver organoid is a model of Crigler-Najjar syndrome.

75. Any of embodiments 29 to 74, further comprising aggregating the posterior foregut cells and/or the posterior foregut endoderm cells in a microwell or other device (e.g., aggresell) prior to step i), wherein aggregating the posterior foregut cells and/or the posterior foregut endoderm cells produces a liver organoid of more uniform size.

76. Any of embodiments 29 to 75, wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or a component thereof, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or a component thereof that is xenogeneic to humans, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or a component thereof isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the cells of step i) and/or step ii) are not in contact with Matrigel ^®、Cultrex^® or Geltrex ^®.

77. Any one of embodiments 29 to 76, wherein the cells of step i) and/or step ii) and/or the liver organoids formed therefrom are cultured in a static bioreactor or a non-static bioreactor, optionally a rotating bioreactor, optionally a 3D rotating bioreactor.

78. Embodiment 77, wherein after culturing in a static bioreactor or a non-static bioreactor, the liver organoids are dissociated into single cells and subsequently reconstituted and/or amplified via additional culturing steps in a static bioreactor or a non-static bioreactor, optionally a 3D rotating bioreactor.

79. Any of embodiments 29 to 78, further comprising cryopreserving the liver organoids.

80. Embodiment 79, wherein cryopreserving the liver organoid comprises slow cryopreservation or vitrification cryopreservation, optionally wherein the liver organoid is cryopreserved with chroman, emlicarbazen, polyamine, and trans-ISRIB (CEPT).

81. Any of embodiments 1 to 80, wherein said posterior foregut cells and/or said posterior foregut endoderm cells have been derived from pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells.

82. Any of embodiments 1 to 81, wherein said posterior foregut cells and/or said posterior foregut endoderm cells have been derived from a subject, optionally a subject suffering from a liver-related disease or disorder.

83. Any of embodiments 1 to 82, wherein the method can be used in a Good Manufacturing Practice (GMP) compliant process.

84. Including embodiments of the metaforegut cells and/or metaforegut endoderm cells or liver organoids produced by any of embodiments 1 to 83.

85. Embodiments include an in vitro composition comprising pluripotent stem cells, definitive endoderm, hindgut endoderm, and/or downstream hepatocyte type, and at least one exogenous tissue culture surface, at least one exogenous TGF-b pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator.

86. Embodiment 85, wherein the composition comprises a metaforegut cell and/or a metaforegut endoderm cell, and the metaforegut cell and/or the metaforegut endoderm cell is a dissociated metaforegut cell and/or metaforegut endoderm cell.

87. Embodiment 85 or 86, wherein the posterior foregut cells and/or the posterior foregut endoderm cells are cell densities greater than or equal to, exactly or about 1 x 10 ⁵, 2 x 10 ⁵, 3 x 10 ⁵, 4 x 10 ⁵, 5 x 10 ⁵, 6 x 10 ⁵, 7 x 10 ⁵, 8 x 10 ⁵, 9 x 10 ⁵, 1 x 10 ⁶, 2 x 10 ⁶, 3 x 10 ⁶, 4 x 10 ⁶, or 5 x 10 ⁶ cells/cm ² of the surface area of the tissue culture surface, or any cell density in the range defined by any two of the foregoing cell densities.

88. Any of embodiments 85 to 87, wherein the tissue culture surface is coated with a basement membrane matrix or a component thereof.

89. Embodiment 88, wherein the basement membrane matrix or component thereof does not comprise a non-human animal component that renders the basement membrane matrix or component thereof xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel ^®、Cultrex^® or Geltrex ^®.

90. Embodiment 88 or 89, wherein the basement membrane matrix or component thereof comprises human fibronectin, collagen IV, entactin, basement membrane glycans, fibrin, and/or hydrogels.

91. Any of embodiments 85 to 90, wherein at least a portion of the posterior foregut cells and/or the posterior foregut endoderm cells are spontaneously formed three-dimensional (3D) spheroids, optionally wherein the spheroids comprise a structure having a single lumen.

92. Any of embodiments 85 to 91, wherein said TGF-b pathway inhibitor is selected from the group consisting of A83-01, repSox, LY365947, and SB431542, optionally wherein said TGF-b pathway inhibitor comprises A83-01 or is A83-01.

93. Any of embodiments 85 to 92, wherein the TGF-b pathway inhibitor has a concentration equal to or about 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the TGF-b pathway inhibitor has a concentration equal to or about 500 nM.

94. Any of embodiments 85 to 93, wherein the FGF pathway activator is selected from the group consisting of ：FGF1、FGF2、FGF3、FGF4、FGF4、FGF5、FGF6、FGF7、FGF8、FGF8、FGF9、FGF10、FGF11、FGF12、FGF13、FGF14、FGF15、FGF16、FGF17、FGF18、FGF19、FGF20、FGF21、FGF22 and FGF23, optionally wherein the FGF pathway activator comprises FGF2 or is FGF2.

95. Any of embodiments 85 to 94, wherein the FGF pathway activator has a concentration equal to or about 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, or 10ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the FGF pathway activator has a concentration equal to or about 5 ng/mL.

96. Any of embodiments 85 to 95, wherein the Wnt pathway activator is selected from the group consisting of ：Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11、Wnt16、BML 284、IQ-1、WAY 262611、CHIR99021、CHIR 98014、AZD2858、BIO、AR-A014418、SB 216763、SB 415286、 aloxin, indirubin, altbolone, kenparone, lithium chloride, TDZD 8, and TWS119, optionally wherein the Wnt pathway activator comprises CHIR99021 or is CHIR99021.

97. Any of embodiments 85 to 96, wherein the Wnt pathway activator has a concentration equal to or about 1 μΜ, 1.5 μΜ,2 μΜ, 2.5 μΜ,3 μΜ, 3.5 μΜ,4 μΜ, 4.5 μΜ,5 μΜ, 5.5 μΜ,6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ or 8 μΜ, or any concentration in the range defined by any two of the foregoing concentrations, optionally wherein the Wnt pathway activator has a concentration equal to or about 3 μΜ.

98. Any of embodiments 85 to 97, wherein the VEGF pathway activator is selected from the group consisting of VEGF or GS4012, optionally wherein the VEGF pathway activator comprises or is VEGF.

99. Any of embodiments 85 to 98, wherein the VEGF pathway activator has a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the VEGF pathway activator has a concentration equal to or about 10 ng/mL.

100. Any of embodiments 85 to 99, wherein the composition further comprises exogenous EGF, or wherein the composition does not comprise exogenous EGF.

101. Any of embodiments 85 to 100, wherein the EGF has a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the EGF has a concentration equal to or about 20 ng/mL.

102. Any of embodiments 85 to 101, wherein the composition further comprises exogenous ascorbic acid and/or transgene-produced ascorbic acid, or wherein the composition does not comprise exogenous ascorbic acid and/or transgene-produced ascorbic acid.

103. Any of embodiments 85 to 102, wherein the ascorbic acid has a concentration equal to or about 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, or 100 μg/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ascorbic acid has a concentration equal to or about 50 μg/mL.

104. Any of embodiments 85 to 103, further comprising a ROCK inhibitor, or culturing in a medium that does not comprise the ROCK inhibitor, optionally wherein the ROCK inhibitor comprises Y-27632 or is Y-27632.

105. Any of embodiments 85 to 104, wherein the ROCK inhibitor has a concentration equal to or about 1 μΜ,2 μΜ,3 μΜ,4 μΜ,5 μΜ,6 μΜ,7 μΜ,8 μΜ,9 μΜ,10 μΜ,11 μΜ,12 μΜ,13 μΜ,14 μΜ,15 μΜ,16 μΜ,17 μΜ, 18 μΜ, 19 μΜ or 20 μΜ, or any concentration in the range defined by any two of the foregoing concentrations, optionally wherein the ROCK inhibitor has a concentration equal to or about 10 μΜ.

106. Any of embodiments 85 to 105, wherein the posterior foregut cell and/or the posterior foregut endoderm cell, definitive endoderm, posterior foregut endoderm and/or downstream liver cell is differentiated from a stem cell, optionally wherein the posterior foregut cell and/or the posterior foregut endoderm cell, definitive endoderm, posterior foregut endoderm and/or downstream liver cell is differentiated from an induced pluripotent stem cell.

107. Any of embodiments 85 to 107, wherein the posterior foregut cells and/or the posterior foregut endoderm cells, definitive endoderm, posterior foregut endoderm and/or downstream liver cells have been passaged less than 4 times.

108. Embodiment 108, wherein the cell comprises or consists essentially of a metaforegut cell and/or a metaforegut endoderm cell.

109. Any of embodiments 85 to 108, wherein the TGF-b pathway inhibitor is a83-01, the FGF pathway activator is FGF2, the Wnt pathway activator is CHIR99021, the VEGF pathway activator is VEGF, and the ROCK inhibitor is Y-27632.

110. Including embodiments of liver organoids produced by any of embodiments 29 to 83.

111. Embodiments comprising an in vitro composition comprising a) a metaintestinal cell and/or a metaintestinal endoderm cell, a liver organoid and/or a mature liver organoid, and b) a culture medium, wherein the culture medium optionally comprises a hepatocyte culture medium and is optionally supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine and a corticosteroid, and wherein the composition optionally further comprises c) a retinoic acid pathway activator.

112. Embodiment 111, wherein the cMET tyrosine kinase receptor agonist is selected from the group consisting of Hepatocyte Growth Factor (HGF), PG-001, furoneb, tetralin Lei Walai phenanthrene, recombinant InlB321 protein, and an agonistic c-Met antibody, optionally LMH85.

113. Any of embodiments 111 to 112, wherein the IL-6 family cytokine is selected from the group consisting of IL-6, oncostatin M (OSM), leukemia Inhibitory Factor (LIF), cardiac neurotrophin-1, ciliary neurotrophic factor (CTNF), and cardiac dystrophin-like cytokine (CLC).

114. Any of embodiments 111 through 113, wherein the corticosteroid is selected from the group consisting of dexamethasone, beclomethasone, betamethasone, flucortisone, halometasone, and mometasone.

115. Any of embodiments 111 to 114, wherein the medium is supplemented with HGF, OSM, and dexamethasone.

116. Any of embodiments 111 to 115, wherein the medium is supplemented with dexamethasone.

117. Any of embodiments 111 through 116, wherein the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally wherein said retinoic acid pathway activator comprises retinoic acid or is retinoic acid.

118. Any of embodiments 111 through 117, wherein the retinoic acid pathway activator has a concentration of 1.0µM、1.1µM、1.2µM、1.3µM、1.4µM、1.5µM、1.6µM、1.7µM、1.8µM、1.9µM、2.0µM、2.1µM、2.2µM、2.3µM、2.4µM、2.5µM、2.6µM、2.7µM、2.8µM、2.9µM or 3.0 μm, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the retinoic acid pathway activator has a concentration equal to or about 2.0 μm.

119. Any of embodiments 115-118, wherein said HGF has a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein said HGF has a concentration equal to or about 10 ng/mL.

120. Any of embodiments 115-119, wherein the OSM has a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the OSM has a concentration equal to or about 20 ng/mL.

121. Any of embodiments 115-120, wherein the dexamethasone has a concentration equal to or about 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, or 200nM, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the dexamethasone has a concentration equal to or about 100 nM.

122. Any of embodiments 111 to 121, wherein the composition does not comprise exogenous EGF.

123. Any of embodiments 111 to 122, further comprising a low concentration of exogenous bilirubin, optionally wherein the low concentration of bilirubin is at or near human fetal physiological bilirubin concentration.

124. Embodiment 123, wherein the bilirubin is at, is about, is less than, or is less than about ：0.1mg/L、0.2mg/L、0.3mg/L、0.4mg/L、0.5mg/L、0.6mg/L、0.7mg/L、0.8mg/L、0.9mg/L、1mg/L、1.25mg/L、1.5mg/L、1.75mg/L、2.0mg/L、2.25mg/L、2.5mg/L、2.75mg/L or is less than about 3.0mg/L, or is in a range defined by any two of the foregoing concentrations, such as 0.1mg/L to 3mg/L, 0.5mg/L to 2.0mg/L, 0.5mg/L to 1.5mg/L, 0.3mg/L to 2.5mg/L, or 0.5mg/L to 1.75mg/L, or is in a range defined by any two of the foregoing concentrations, such as 0.1mg/L to 1mg/L, 0.1mg/L to 0.5mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, or 1mg/L, or is in a range defined by any two of the foregoing concentrations, such as 0.1mg/L to 1mg/L, 0.1mg/L to 0.5mg/L, 0.5mg/L to 0.5mg/L, 0.3mg/L, 0.4mg/L, or 0.4 mg/L.

125. Embodiment 124, wherein the composition comprises a mature liver organoid, and wherein the mature liver organoid exhibits a luminal protrusion resembling a bile duct, and/or has a single lumen and generally spherically shaped structure, and/or wherein the mature liver organoid is free of hematopoietic tissue and acquired immune cells.

126. Embodiment 125, wherein the mature liver organoid expresses reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to a liver organoid not contacted with a low dose of bilirubin.

127. Embodiment 125 or 126, wherein the mature liver organoid expresses increased levels of ALB, SLC4A2 or HO-1, or any combination thereof, relative to a liver organoid not contacted with the low dose of bilirubin.

128. Any one of embodiments 125 to 127, wherein the mature liver organoid expresses CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof.

129. Any of embodiments 125-128, wherein the mature liver organoid exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid not contacted with a low dose of bilirubin.

130. Embodiments comprising an in vitro composition comprising a mature liver organoid, wherein cells of the mature liver organoid are contacted with a low dose of bilirubin, optionally wherein the low dose of bilirubin is provided exogenously, and the mature liver organoid exhibits a luminal protrusion resembling a bile duct, and/or has a single lumen and generally spherically shaped structure, and/or wherein the mature liver organoid is free of hematopoietic tissue and acquired immune cells.

131. Embodiment 130, wherein the mature liver organoid expresses reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to a liver organoid in which the cells are not contacted with low doses of bilirubin.

132. Embodiment 130 or 131, wherein the mature liver organoid expresses increased levels of ALB, SLC4A2 or HO-1, or any combination thereof, relative to a liver organoid in which the cells are not contacted with a low dose of bilirubin.

133. Any of embodiments 130-132, wherein the mature liver organoid expresses CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof.

134. Any of embodiments 130-133, wherein the mature liver organoid exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid in which the cells are not contacted with a low dose of bilirubin.

135. Any of embodiments 130 to 134, further comprising hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoid cells are contacted with a high concentration and/or a second concentration of bilirubin.

136. Embodiment 135, wherein the high/second concentration of bilirubin is, is about, is greater than or is greater than about ：2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or is greater than about 20mg/L, or is in the range defined by any two of the foregoing concentrations, such as, for example, any of 2mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12mg/L, or 4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or is in the range defined by any two of the foregoing concentrations, such as, for example, 4mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L.

137. Embodiment 135 or 136, wherein the hyperbilirubinemia liver organoid expresses elevated levels of UGT1A1 or NRF2, or both, relative to a liver organoid not treated with high/second concentrations of bilirubin.

138. Any of embodiments 111-137, wherein said posterior foregut cell and/or said posterior foregut endoderm cell, liver organoid and/or mature liver organoid comprises a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA encoding said functional GULO protein or both, wherein said posterior foregut cell and/or said posterior foregut endoderm cell, liver organoid and/or mature liver organoid is capable of synthesizing ascorbic acid.

139. Embodiment 138, wherein the functional GULO protein is murine GULO (mGULO).

140. Embodiment 138 or 139, wherein said gene encoding said functional GULO protein is conditionally expressed, optionally using a tetracycline-inducible system.

141. Any of embodiments 138 to 139, wherein the posterior foregut cells and/or the posterior foregut endoderm cells, liver organoids and/or mature liver organoids are engineered to comprise a gene encoding the functional GULO protein using CRISPR.

142. Any of embodiments 138 to 141, wherein said gene or mRNA encoding said functional GULO protein, or both, is introduced to said liver organoid by transfection.

143. Any of embodiments 138-142, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein expresses increased levels of NRF2 relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

144. Any of embodiments 138-143, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein expresses reduced levels of IL1B, IL6 or TNFa or any combination thereof relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

145. Any of embodiments 138-144, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein exhibits reduced caspase-3 activity relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

146. Any of embodiments 138-145, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein expresses increased levels of ALB relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

147. Any of embodiments 138 to 146, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein is similar to periportal liver tissue and expresses a periportal liver marker.

148. Embodiment 147, wherein the periportal liver marker comprises FAH, ALB, PAH, CPS a1, HGD, or any combination thereof.

149. Any of embodiments 138-148, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

150. Any of embodiments 138-149, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein exhibits increased bilirubin conjugation activity relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

151. Any of embodiments 138-150, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein exhibits increased viability in culture relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

152. Any of embodiments 138-151, wherein the liver organoid and/or mature liver organoid has differentiated from a pluripotent stem cell comprising a functional GULO protein and/or a gene or mRNA encoding the functional GULO protein, or both, whereby the pluripotent stem cell is capable of synthesizing ascorbic acid.

153. Embodiments comprising a method comprising administering to a subject in need thereof a liver organoid or composition according to any of embodiments 110-152.

154. Embodiments include a method for treating a liver-related disease or disorder in a subject in need thereof, the method comprising administering to the subject one or more liver organoids or compositions according to examples 110-152.

155. Embodiment 153 or 154, wherein said liver organoid has been produced by a cell derived from said subject, optionally wherein said cell derived from said subject is an induced pluripotent stem cell.

156. Any of embodiments 154 to 155, wherein administering comprises transplanting the liver organoid or composition into the subject.

157. Any of embodiments 154 to 156, wherein the liver-related disease or disorder comprises one or more types of liver dysfunction and/or failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic disease, autoimmune liver disease, wilson's disease, metabolic-related fatty liver disease, hyperammonemia, hyperbilirubinemia, crigler-Najjar syndrome, urea cycle disorder, walman's disease, liver cancer, hepatoblastoma, metabolic dysfunction-related liver disease (MASLD), metALD, metabolic dysfunction-related steatohepatitis (MASH), drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic disease, liver cyst, acetaminophen acute liver injury, and/or alcohol-related liver disease.

158. Any of embodiments 154 to 157, wherein the liver dysfunction and/or failure comprises hyperammonemia and/or hyperbilirubinemia, or wherein the metabolic disease comprises non-alcoholic fatty liver disease (NAFLD), or wherein the non-alcoholic fatty liver disease (NAFLD) comprises metabolic dysfunction-related steatohepatitis (MASH), or wherein the hepatitis comprises hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, TT hepatitis, and/or autoimmune hepatitis.

159. Any of embodiments 154 to 158, wherein the subject has a reduced serum bilirubin and/or ammonia levels, and/or increased serum protein after transplantation.

160. Any of embodiments 154 to 159, wherein the subject has improved bile duct stenosis and/or liver regeneration symptoms after transplantation.

161. Any of embodiments 154 to 160, wherein the subject has increased survival after transplantation.

162. Any of embodiments 154-161, wherein said liver organoid is implanted on said liver of said subject.

163. Any of embodiments 154 to 162, wherein said liver organoids have been treated with an Amino Acid (AA) supplement.

164. Any of embodiments 154 to 163, wherein the liver organoid has been treated with an Amino Acid (AA) supplement for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more prior to transplantation.

165. Any of embodiments 154 to 164, wherein the liver-related disease or disorder comprises acetaminophen acute liver injury.

166. Any of embodiments 154-165, wherein the method is capable of being used in a Good Manufacturing Practice (GMP) compliant process.

167. An embodiment comprising a method for screening comprising contacting a liver organoid according to any of embodiments 84-152 with a candidate compound or composition, and assessing the effect of the candidate compound or composition on the liver organoid.

168. Embodiment 167, wherein the liver organoid is a model of a liver-related disease or disorder, and assessing the effect of the candidate compound or composition on the liver organoid comprises assessing the effect of the candidate compound or composition on the liver-related disease or disorder.

169. Embodiment 167 or 168, wherein the liver organoid has been produced by a cell derived from a subject, optionally wherein the cell derived from the subject is an induced pluripotent stem cell.

170. Embodiment 169, wherein the subject has a liver-related disease or disorder.

171. Any of embodiments 167 to 170, wherein the method can be used in a Good Manufacturing Practice (GMP) compliant process.

172. Embodiments include compositions comprising liquid components of supplemental amino acids according to table 3.

173. Embodiments comprising a composition comprising a mixture of growth factors according to the embodiments of table 1 or table 2.

174. Including embodiments of compositions comprising liquid components that supplement amino acids, the liquid component of the supplemental amino acids comprises, by volume, a solution of just or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% of a non-essential amino acid (containing just or about 890mg/L alanine, 1320mg/L asparagine, 1330mg/L aspartic acid, 750mg/L glycine, 105mg/L serine, 1150mg/L proline and 1470mg/L glutamic acid), a solution of just or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% of a essential amino acid (containing just or about 6320mg/L arginine 1200mg/L cysteine, 2100mg/L histidine, 2620mg/L isoleucine, 2620mg/L leucine, 3625mg/L lysine, 755mg/L methionine, 1650mg/L phenylalanine, 2380mg/L threonine, 510mg/L tryptophan, 1800mg/L tyrosine and 2340mg/L valine) and exactly or about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% by volume of a hepatocyte medium (HCM), and is also supplemented with exactly or about 5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL、21mg/mL、22mg/mL、23mg/mL、24mg/mL、25mg/mL、26mg/mL、27mg/mL、28mg/mL、29mg/mL、30mg/mL、31mg/mL、32mg/mL、33mg/mL、34mg/mL or 35mg/mL glycine.

175. Any of embodiments 172-174, comprising a liquid component of supplemental amino acids comprising a just or about 14% non-essential amino acid solution (containing just or about 890mg/L alanine, 1320mg/L asparagine, 1330mg/L aspartic acid, 750mg/L glycine, 105mg/L serine, 1150mg/L proline, and 1470mg/L glutamic acid), a just or about 6% essential amino acid solution by volume (containing just or about 6320mg/L arginine, 1200mg/L cysteine, 2100mg/L histidine, 2620mg/L isoleucine, 2620mg/L leucine, 3625mg/L lysine, 755mg/L methionine, 1650mg/L phenylalanine, 2380mg/L threonine, 510mg/L tryptophan, 1800mg/L tyrosine, and 2340mg/L valine), and a just or about 80% hepatocyte medium (HCM) by volume, and further supplemented with just or about 20g/L glycine.

176. Any of embodiments 172 to 175, wherein the pH is between about pH6 to 8, or between pH6.5 to 7.5, or is exactly or about pH 7.0.

177. Any of embodiments 172 to 176, further comprising Hepatocyte Growth Factor (HGF), oncostatin M, dexamethasone, and/or ascorbic acid.

178. Any of embodiments 172 to 177, further comprising liver lineage committed cells differentiated from definitive endoderm cells using retinoic acid.

179. Embodiment 178, wherein said liver lineage-committed cells are characterized as liver organoids.

180. Embodiment 179, wherein the liver organoid is characterized as secreting increased levels of albumin and urea relative to a liver organoid contained in HCM without an amino acid supplement.

181. The embodiment 179 or 180, wherein the liver organoid is characterized as expressing increased levels of liver maturation-associated gene expression relative to a liver organoid comprised in HCM without the amino acid supplement.

182. Any of embodiments 179-181, wherein the liver organoid is characterized as expressing reduced levels of vimentin relative to a liver organoid comprised in HCM without the amino acid supplement.

183. Any of embodiments 172-182, wherein the composition does not comprise a non-human animal component that renders the base film matrix or component thereof xenogeneic to a human.

184. An embodiment comprising the composition of embodiment 182, wherein the composition does not comprise murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, matrigel ^®、Cultrex^®, and/or Geltrex ^®.

185. Embodiments include an in vitro hyperbilirubinemia liver organoid comprising naturally occurring and/or engineered mutations in UDP glucuronyl transferase family 1 member A1 (UGT 1 A1).

186. An embodiment comprising an in vitro hyperbilirubinemia liver organoid, wherein the hyperbilirubinemia liver organoid is produced by contacting precursor cells, precursor liver organoids, and/or precursor mature liver organoids with exogenous bilirubin for at least two cycles.

187. An embodiment of an in vitro hyperbilirubinemia liver organoid comprising embodiment 185 or 186, wherein the hyperbilirubinemia liver organoid is of clonal origin and/or derived from an iPSC.

188. Embodiments include a cryopreserved composition comprising a liver organoid, chroman a1, emlicarbazepine, a polyamine, and a trans-ISRIB (CEPT).

189. Embodiments include a cryopreserved composition comprising mature liver organoids, chroman a1, emlicarbazepine, polyamines, and trans-ISRIB (CEPT).

190. Embodiments include a cryopreserved composition comprising hyperbilirubinemia liver organoids, chroman a 1, emlicarbazin, polyamines, and trans-ISRIB (CEPT).

191. An embodiment comprising a kit comprising means for performing the method according to any one of embodiments 1 to 83 or 153 to 171.

192. An embodiment comprising a kit comprising a composition according to any of embodiments 84 to 152, 172 to 184 or 188 to 190 or means for producing the composition or means for producing a liver organoid according to any of embodiments 185 to 187.

193. Including embodiments using the method, composition or kit according to any one of embodiments 1 to 192 as a medicament, a tool for the treatment and/or prevention of a disease, a diagnostic tool and/or a medical study.

Claims (193)

1. A method for expanding metaintestinal cells and/or metaforegut endoderm cells, the method comprising:

a) Dissociating the monolayer of metaforegut cells and/or the monolayer of metaforegut endoderm cells into metaforegut cells and/or metaforegut endoderm cells;

b) Inoculating said metaforegut cells and/or said metaforegut endoderm cells onto a tissue culture surface, and

C) Culturing the metaforegut cells and/or the metaforegut endoderm cells with a TGF-b pathway inhibitor, a FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator.

2. The method of claim 1, wherein the posterior foregut cell monolayer is dissociated into the posterior foregut cells and/or the posterior foregut endoderm cells using enzymatic dissociation and/or mechanical dissociation.

3. The method of claim 1 or 2, wherein the posterior foregut cells and/or the posterior foregut endoderm cells are seeded onto the tissue container surface at a cell density equal to or about 1 x 10 ⁵, 2x 10 ⁵, 3 x 10 ⁵, 4 x 10 ⁵, 5 x 10 ⁵, 6x 10 ⁵, 7 x 10 ⁵, 8 x 10 ⁵, 9 x 10 ⁵, 1 x 10 ⁶, 2x 10 ⁶, 3 x 10 ⁶, 4 x 10 ⁶, or 5 x 10 ⁶ cells/cm ² of the surface area of the tissue culture surface, or at any cell density having a range defined by any two of the foregoing cell densities.

4. A method according to any one of claims 1 to 3, wherein the tissue culture surface is coated with a basement membrane matrix or a component thereof.

5. The method of claim 4, wherein the basement membrane matrix or component thereof does not comprise a non-human animal component that renders the basement membrane matrix or component thereof xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-swart (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel ^®、Cultrex^® or Geltrex ^®.

6. The method of claim 4 or 5, wherein the basement membrane matrix or component thereof comprises human fibronectin, collagen IV, entactin, basement membrane glycans, fibrin, and/or hydrogels.

7. The method of any one of claims 1 to 6, wherein the metaforegut cells and/or the metaforegut endoderm cells are cultured until three-dimensional (3D) spheroids are spontaneously formed, optionally wherein the 3D spheroids comprise a structure with a single lumen, and/or wherein the spheroids are free of hematopoietic tissue and acquired immune cells.

8. The method of any one of claims 1 to 7, wherein the metaforegut cells and/or the metaforegut endoderm cells are cultured for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days or 35 days.

9. The method of any one of claims 1 to 8, wherein the TGF-b pathway inhibitor is selected from the group consisting of A83-01, repSox, LY365947, and SB431542, optionally A83-01.

10. The method of any one of claims 1 to 9, wherein the TGF-b pathway inhibitor is provided at a concentration equal to or about 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the TGF-b pathway inhibitor is provided at a concentration equal to or about 500 nM.

11. The method of any one of claims 1 to 10, wherein the FGF pathway activator is selected from the group consisting of ：FGF1、FGF2、FGF3、FGF4、FGF4、FGF5、FGF6、FGF7、FGF8、FGF8、FGF9、FGF10、FGF11、FGF12、FGF13、FGF14、FGF15、FGF16、FGF17、FGF18、FGF19、FGF20、FGF21、FGF22 and FGF23, optionally FGF2.

12. The method of any one of claims 1 to 11, wherein the FGF pathway activator is provided at a concentration equal to or about 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, or 10ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the FGF pathway activator is provided at a concentration equal to or about 5 ng/mL.

13. The method of any one of claims 1 to 12, wherein the Wnt pathway activator is selected from the group consisting of ：Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11、Wnt16、BML 284、IQ-1、WAY 262611、CHIR99021、CHIR 98014、AZD2858、BIO、AR-A014418、SB 216763、SB 415286、 aloxin, indirubin, altretbolone, kenarone, lithium chloride, TDZD, and TWS119, optionally CHIR99021.

14. The method of any one of claims 1 to 13, wherein the Wnt pathway activator is provided at a concentration equal to or about 1 μΜ, 1.5 μΜ,2 μΜ, 2.5 μΜ, 3 μΜ, 3.5 μΜ, 4 μΜ, 4.5 μΜ, 5.5 μΜ,6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ, or 8 μΜ, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the Wnt pathway activator is provided at a concentration equal to or about 3 μΜ.

15. The method according to any one of claims 1 to 14, wherein the VEGF pathway activator is selected from the group consisting of VEGF or GS4012, optionally VEGF.

16. The method of any one of claims 1 to 15, wherein the VEGF pathway activator is provided at a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the VEGF pathway activator is provided at a concentration equal to or about 10 ng/mL.

17. The method of any one of claims 1 to 16, wherein the metaintestinal cells and/or the metaintestinal endoderm cells of step c) are cultured in a medium further comprising EGF or in a medium not comprising EGF.

18. The method of claim 17, wherein the EGF is provided at a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the EGF is provided at a concentration equal to or about 20 ng/mL.

19. The method of any one of claims 1 to 18, wherein the metaintestinal cells and/or the metaintestinal endoderm cells of step c) are cultured in a medium further comprising ascorbic acid, or in a medium not comprising ascorbic acid.

20. The method of claim 19, wherein the ascorbic acid is provided at a concentration equal to or about 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, or 100 μg/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ascorbic acid is provided at a concentration equal to or about 50 μg/mL.

21. The method of any one of claims 1 to 20, wherein the posterior foregut cells and/or the posterior foregut endoderm cells of step c) are cultured in a medium that further comprises a ROCK inhibitor, or in a medium that does not comprise the ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632.

22. The method of claim 21, wherein the ROCK inhibitor is provided at a concentration equal to or about 1 μΜ,2 μΜ, 3 μΜ,4 μΜ,5 μΜ,6 μΜ,7 μΜ, 8 μΜ,9 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ or 20 μΜ, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ROCK inhibitor is provided at a concentration equal to or about 10 μΜ.

23. The method of any one of claims 1 to 22, further comprising passaging the cells of step c) one or more times.

24. The method of claim 23, wherein the cells of step c) are passaged to the posterior foregut cells and/or the posterior foregut endoderm cells do not spontaneously form spheroids.

25. The method of claim 23 or 24, wherein the cells of step c) are passaged no more than 3 times.

26. The method of any one of claims 23 to 25, further comprising collecting the posterior foregut cells and/or the posterior foregut endoderm cells and differentiating the posterior foregut cells and/or the posterior foregut endoderm cells into liver organoids.

27. The method of claim 26, wherein the metaforegut cells and/or the metaforegut endoderm cells are cultured until a three-dimensional (3D) spheroid is spontaneously formed, and the metaforegut cells and/or the metaforegut endoderm cells are collected from the spheroid, optionally the method further comprises dissociating the spheroid into individual masses of metaforegut cells and/or metaforegut endoderm cells prior to the differentiating step, optionally wherein the spheroid comprises a structure with a single lumen, and/or wherein the spheroid is free of hematopoietic tissue and acquired immune cells.

28. The method of claim 26, wherein the metaforegut cell monolayer is collected from the metaforegut cell monolayer prior to the differentiating step by dissociating the metaforegut cell monolayer into individual metaforegut cells and/or metaforegut endoderm cells and/or clusters of metaforegut cells and/or metaforegut endoderm cells.

29. A method of differentiating metaforegut cells and/or metaforegut endoderm cells into liver organoids, the method comprising:

i) Contacting a metaforegut cell and/or a metaforegut endoderm cell, optionally in the form of a spheroid, optionally in the form of individual cells or clusters of cells dissociated from the spheroid, and/or optionally aggregated cells in a microwell or other device described herein, optionally wherein the spheroid comprises a structure having a single lumen, and/or wherein the spheroid is free of hematopoietic tissue and acquired immune cells, and

Ii) contacting the cells of step i) with a culture medium for a period of time such that the metaforegut cells and/or the metaforegut endoderm cells differentiate into liver organoids, optionally wherein the culture medium is a hepatocyte culture medium.

30. The method of claim 29, wherein the medium is supplemented with cMET tyrosine kinase receptor agonists, IL-6 family cytokines, and corticosteroids.

31. The method of claim 30, wherein the cMET tyrosine kinase receptor agonist is selected from the group consisting of Hepatocyte Growth Factor (HGF), PG-001, furoneb, tetralin Lei Walai phenanthrene, recombinant InlB321 protein, and an agonistic c-Met antibody, optionally LMH85.

32. The method of claim 30 or 31, wherein the IL-6 family cytokine is selected from the group consisting of IL-6, oncostatin M (OSM), leukemia Inhibitory Factor (LIF), cardiac neurotrophin-1, ciliary neurotrophic factor (CTNF), and cardiac dystrophin-like cytokine (CLC).

33. The method of any one of claims 30 to 32, wherein the corticosteroid is selected from the group consisting of dexamethasone, beclomethasone, betamethasone, flucortisone, halometasone, and mometasone.

34. The method of claim 29, wherein the medium is supplemented with HGF, OSM, and dexamethasone.

35. The method of claim 29, wherein the medium is supplemented with dexamethasone.

36. The method of any one of claims 29 to 35, wherein the metaforegut cells and/or the metaforegut endoderm cells are the metaforegut cells and/or the metaforegut endoderm cells produced by the method of any one of claims 1 to 28.

37. The method of any one of claims 29 to 36, wherein the metaforegut cells and/or the metaforegut endoderm cells are in the form of spheroids or metaforegut cells and/or metaforegut endoderm cells alone and/or in the form of a pellet derived from metaforegut cells and/or metaforegut endoderm cells dissociated from the spheroids, optionally wherein the spheroids comprise a structure having a single lumen, and/or wherein the spheroids are free of hematopoietic tissue and acquired immune cells.

38. The method of any one of claims 29-37, wherein the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally retinoic acid.

39. The method of any one of claims 29-38, wherein the retinoic acid pathway activator is provided at a concentration of 1.0µM、1.1µM、1.2µM、1.3µM、1.4µM、1.5µM、1.6µM、1.7µM、1.8µM、1.9µM、2.0µM、2.1µM、2.2µM、2.3µM、2.4µM、2.5µM、2.6µM、2.7µM、2.8µM、2.9µM or 3.0 μΜ, or at any concentration within the range defined by any two of the foregoing concentrations, optionally wherein the retinoic acid pathway activator is provided at a concentration equal to or about 2.0 μΜ.

40. The method of claim 34, wherein the HGF is provided at a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the HGF is provided at a concentration equal to or about 10 ng/mL.

41. The method of claim 34, wherein the OSM is provided at a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the OSM is provided at a concentration equal to or about 20 ng/mL.

42. The method of claim 34 or 35, wherein the dexamethasone is provided at a concentration equal to or about 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, or 200nM, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the dexamethasone is provided at a concentration equal to or about 100 nM.

43. The method of any one of claims 29 to 42, wherein the cells of step i) and/or step ii) are not contacted with EGF.

44. The method of any one of claims 29 to 43, wherein the cells of step ii) are cultured in a growth medium supplemented with non-essential amino acids, and glycine.

45. The method of claim 44, wherein the growth medium after supplementation comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of nonessential amino acids by total volume, or a range defined by any two of the foregoing values, optionally wherein the growth medium after supplementation is about 4% -10%, 6% -12%, 10% -16%, 12% -15%, 13% -19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% nonessential amino acids by total volume.

46. The method of claim 44 or 45, wherein the growth medium after supplementation comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% essential amino acids by total volume, or within a range defined by any two of the foregoing values, optionally wherein the growth medium after supplementation is about 4% -10%, 6% -12%, 10% -16%, 12% -15%, 13% -19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% essential amino acids by total volume.

47. The method as set forth in any one of claims 44 to 46 wherein the glycine supplemented is provided at a concentration equal to or about 5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL、21mg/mL、22mg/mL、23mg/mL、24mg/mL、25mg/mL、26mg/mL、27mg/mL、28mg/mL、29mg/mL、30mg/mL、31mg/mL、32mg/mL、33mg/mL、34mg/mL or 35mg/mL, or at any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the glycine supplemented is provided at a concentration equal to or about 18-22 mg/mL or 20 mg/mL.

48. The method of any one of claims 29 to 47, wherein the cells of step ii) are further contacted with a low/first concentration of bilirubin, wherein the liver organoid formed is a mature liver organoid.

49. The method of claim 48, wherein the low/first concentration of bilirubin is human fetal physiological concentration of bilirubin.

50. The method of claim 48 or 49, wherein the low concentration/first concentration of bilirubin is, is about, less than or less than about ：a) 0.1mg/L、0.2mg/L、0.3mg/L、0.4mg/L、0.5mg/L、0.6mg/L、0.7mg/L、0.8mg/L、0.9mg/L、1mg/L、1.25mg/L、1.5mg/L、1.75mg/L、2.0mg/L、2.25mg/L、2.5mg/L、2.75mg/L or 3.0mg/L, or a range defined by any two of the foregoing concentrations, such as 0.1mg/L to 3mg/L, 0.5mg/L to 2.0mg/L, 0.5mg/L to 1.5mg/L, 0.3mg/L to 2.5mg/L, or 0.5mg/L to 1.75mg/L, or b) 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, or 1mg/L, or a range defined by any two of the foregoing concentrations, such as 0.1mg/L to 1mg/L, 0.3mg/L to 2.5mg/L, or 0.5mg/L to 1.75mg/L, or b) 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, or 0.8 mg/L.

51. The method of any one of claims 48 to 50, wherein the mature liver organoid exhibits a luminal protrusion resembling a bile duct, and/or has a single lumen and generally spherically shaped structure, and/or wherein the mature liver organoid is free of hematopoietic tissue and acquired immune cells.

52. The method of any one of claims 48 to 51, wherein the mature liver organoid expresses reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to a liver organoid not contacted with a low/first dose of bilirubin.

53. The method of any one of claims 48 to 52, wherein the mature liver organoid expresses increased levels of ALB, SLC4A2 or HO-1, or any combination thereof, relative to a liver organoid not contacted with the low/first dose of bilirubin.

54. The method of any one of claims 48 to 53, wherein the mature liver organoid expresses CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof.

55. The method of any one of claims 48 to 54, wherein the mature liver organoid exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid not contacted with the low dose/first dose of bilirubin.

56. The method of any one of claims 29 to 55, wherein the cells of step ii) are further contacted with a high/second concentration of bilirubin, wherein the liver organoid formed is a hyperbilirubinemia liver organoid.

57. The method according to claim 56, wherein the high/second concentration of bilirubin is, is about, greater than or greater than about ：a) 2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or a range defined by any two of the foregoing concentrations, such as, for example, any of 2mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12mg/L, or b) 4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or a range defined by any two of the foregoing concentrations, such as, for example, 4mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L.

58. The method of claim 56 or 57, wherein the hyperbilirubinemia liver organoid expresses elevated levels of UGT1A1 or NRF2 or both relative to a liver organoid not treated with high/second concentrations of bilirubin.

59. The method of any one of claims 29-58, wherein the liver organoid comprises a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA encoding the functional GULO protein or both, wherein the liver organoid is capable of synthesizing ascorbic acid.

60. The method of claim 59, wherein the functional GULO protein is murine GULO (mGluLO).

61. A method according to claim 59 or 60, wherein the gene encoding the functional GULO protein is conditionally expressed, optionally using a tetracycline-inducible system.

62. The method of any one of claims 59 to 61, wherein the liver organoid is engineered with the gene encoding the functional GULO protein using CRISPR.

63. The method of any one of claims 59-62, wherein the gene encoding the functional GULO protein, or mRNA, or both, is introduced to the liver organoid by transfection.

64. The method of any one of claims 59-63, wherein the liver organoid comprising the functional GULO protein expresses increased levels of NRF2 relative to a liver organoid not comprising the functional GULO protein.

65. The method of any one of claims 59-64, wherein the liver organoid comprising the functional GULO protein expresses reduced levels of IL1B, IL or tnfa or any combination thereof, relative to a liver organoid not comprising the functional GULO protein, optionally when cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid.

66. The method of any one of claims 59-65, wherein the liver organoid comprising the functional GULO protein exhibits reduced caspase-3 activity relative to a liver organoid not comprising the functional GULO protein, optionally when cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid.

67. The method of any one of claims 59-66, wherein the liver organoid comprising the functional GULO protein expresses increased levels of ALB relative to a liver organoid not comprising the functional GULO protein.

68. The method of any one of claims 59-67, wherein the liver organoid comprising the functional GULO protein is similar to periportal liver tissue and expresses a periportal liver marker.

69. The method of claim 68, wherein the periportal liver marker comprises FAH, ALB, PAH, CPS a1, HGD, or any combination thereof.

70. The method of any one of claims 59-69, wherein the liver organoid comprising the functional GULO protein exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid not comprising the functional GULO protein.

71. The method of any one of claims 59-70, wherein the liver organoid comprising the functional GULO protein exhibits increased bilirubin conjugation activity relative to a liver organoid not comprising the functional GULO protein.

72. The method of any one of claims 59-71, wherein the liver organoid comprising the functional GULO protein exhibits increased viability in culture relative to a liver organoid not comprising the functional GULO protein.

73. A method according to any one of claims 59 to 72, wherein the liver organoid has been differentiated from a pluripotent stem cell comprising a functional GULO protein and/or a gene or mRNA encoding the functional GULO protein or both, whereby the pluripotent stem cell is capable of synthesizing ascorbic acid.

74. The method of any one of claims 59 to 73, wherein the liver organoid comprises an inactive UGT1A1 gene, wherein the liver organoid is a model of Crigler-Najjar syndrome.

75. The method of any one of claims 29 to 74, further comprising aggregating the posterior foregut cells and/or the posterior foregut endoderm cells in a microwell or other device (e.g., aggresell) prior to step i), wherein aggregating the posterior foregut cells and/or the posterior foregut endoderm cells produces a liver organoid of more uniform size.

76. The method of any one of claims 29 to 75, wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof that is xenogenic to humans, optionally wherein the cells of step i) and/or step ii) are not cultured with a basement membrane matrix or component thereof isolated from murine Engelbreth-Holm-swart (EHS) sarcoma cells, optionally wherein the cells of step i) and/or step ii) are not in contact with Matrigel ^®、Cultrex^® or Geltrex ^®.

77. The method of any one of claims 29 to 76, wherein the cells of step i) and/or step ii) and/or the liver organoids formed therefrom are cultured in a static bioreactor or a non-static bioreactor, optionally a rotating bioreactor, optionally a 3D rotating bioreactor.

78. The method of claim 77, wherein after culturing in a static bioreactor or a non-static bioreactor, the liver organoids are dissociated into single cells and subsequently reconstituted and/or amplified via additional culturing steps in a static bioreactor or a non-static bioreactor, optionally a 3D rotating bioreactor.

79. The method of any one of claims 29-78, further comprising cryopreserving the liver organoids.

80. The method of claim 79, wherein cryopreserving the liver organoid comprises slow freeze or vitrification cryopreservation, optionally wherein the liver organoid is cryopreserved with chroman a1, emlicarbazin, polyamines, and trans-ISRIB (CEPT).

81. The method of any one of claims 1 to 80, wherein the metaforegut cells and/or the metaforegut endoderm cells have been derived from pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells.

82. The method of any one of claims 1 to 81, wherein the metaforegut cells and/or the metaforegut endoderm cells have been derived from a subject, optionally a subject suffering from a liver-related disease or disorder.

83. The method of any one of claims 1 to 82, wherein the method can be used in a Good Manufacturing Practice (GMP) compliant process.

84. A metaforegut cell and/or a metaforegut endoderm cell or liver organoid produced by the method of any one of claims 1 to 83.

85. An in vitro composition comprising pluripotent stem cells, definitive endoderm, hindgut endoderm and/or downstream hepatocyte type, and at least one exogenous tissue culture surface, at least one exogenous TGF-b pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator.

86. The in vitro composition of claim 85, wherein the composition comprises posterior foregut cells and/or posterior foregut endoderm cells, and the posterior foregut cells and/or the posterior foregut endoderm cells are dissociated posterior foregut cells and/or posterior foregut endoderm cells.

87. The in vitro composition of claim 85 or 86, wherein the posterior foregut cells and/or the posterior foregut endoderm cells are cell densities greater than or equal to, exactly or about 1 x 10 ⁵, 2x 10 ⁵, 3 x 10 ⁵, 4 x 10 ⁵, 5 x 10 ⁵, 6x 10 ⁵, 7 x 10 ⁵, 8 x 10 ⁵, 9 x 10 ⁵, 1 x 10 ⁶, 2x 10 ⁶, 3 x 10 ⁶, 4 x 10 ⁶, or 5 x 10 ⁶ cells/cm ² of the surface area of the tissue culture surface, or any cell density of the range defined by any two of the foregoing cell densities.

88. The in vitro composition of any one of claims 85 to 87, wherein said tissue culture surface is coated with a basement membrane matrix or a component thereof.

89. The in vitro composition of claim 88, wherein the basement membrane matrix or component thereof does not comprise a non-human animal component that renders the basement membrane matrix or component thereof xenogeneic to humans, optionally wherein the basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-swart (EHS) sarcoma cells, optionally wherein the basement membrane matrix or component thereof is not Matrigel ^®、Cultrex^® or Geltrex ^®.

90. The in vitro composition of claim 88 or 89, wherein the basement membrane matrix or component thereof comprises human fibronectin, collagen IV, entactin, basement membrane glycans, fibrin, and/or hydrogels.

91. The in vitro composition of any one of claims 85 to 90, wherein at least a portion of the posterior foregut cells and/or the posterior foregut endoderm cells are spontaneously formed three-dimensional (3D) spheroids, optionally wherein the spheroids comprise a structure with a single lumen.

92. The in vitro composition of any one of claims 85 to 91, wherein the TGF-b pathway inhibitor is selected from the group consisting of a83-01, repox, LY365947, and SB431542, optionally wherein the TGF-b pathway inhibitor comprises a83-01 or a83-01.

93. The in vitro composition of any one of claims 85-92, wherein the TGF-b pathway inhibitor has a concentration equal to or about 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the TGF-b pathway inhibitor has a concentration equal to or about 500 nM.

94. The in vitro composition of any one of claims 85-93, wherein the FGF pathway activator is selected from the group ：FGF1、FGF2、FGF3、FGF4、FGF4、FGF5、FGF6、FGF7、FGF8、FGF8、FGF9、FGF10、FGF11、FGF12、FGF13、FGF14、FGF15、FGF16、FGF17、FGF18、FGF19、FGF20、FGF21、FGF22 and FGF23 consisting of, optionally wherein the FGF pathway activator comprises FGF2 or is FGF2.

95. The in vitro composition of any one of claims 85-94, wherein the FGF pathway activator has a concentration equal to or about 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, or 10ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the FGF pathway activator has a concentration equal to or about 5 ng/mL.

96. The in vitro composition of any one of claims 85-95, wherein the Wnt pathway activator is selected from the group ：Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11、Wnt16、BML 284、IQ-1、WAY 262611、CHIR99021、CHIR 98014、AZD2858、BIO、AR-A014418、SB 216763、SB 415286、 aloxin, indirubin, altbolone, kenarone, lithium chloride, TDZD, and TWS119, optionally wherein the Wnt pathway activator comprises CHIR99021 or is CHIR99021.

97. The in vitro composition of any one of claims 85 to 96, wherein the Wnt pathway activator has a concentration equal to or about 1 μΜ, 1.5 μΜ, 2 μΜ, 2.5 μΜ, 3 μΜ, 3.5 μΜ, 4 μΜ, 4.5 μΜ, 5 μΜ, 5.5 μΜ, 6 μΜ, 6.5 μΜ, 7 μΜ, 7.5 μΜ, or 8 μΜ, or any concentration in the range defined by any two of the foregoing concentrations, optionally wherein the Wnt pathway activator has a concentration equal to or about 3 μΜ.

98. The in vitro composition of any one of claims 85 to 97, wherein the VEGF pathway activator is selected from the group consisting of VEGF or GS4012, optionally wherein the VEGF pathway activator comprises VEGF or is VEGF.

99. The in vitro composition of any one of claims 85 to 98, wherein the VEGF pathway activator has a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the VEGF pathway activator has a concentration equal to or about 10 ng/mL.

100. The in vitro composition of any one of claims 85-99, wherein the composition further comprises exogenous EGF, or wherein the composition does not comprise exogenous EGF.

101. The in vitro composition of any one of claims 85 to 100, wherein the EGF has a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the EGF has a concentration equal to or about 20 ng/mL.

102. The in vitro composition of any one of claims 85 to 101, wherein the composition further comprises exogenous ascorbic acid and/or transgene-produced ascorbic acid, or wherein the composition does not comprise exogenous ascorbic acid and/or transgene-produced ascorbic acid.

103. The in vitro composition of any one of claims 85 to 102, wherein the ascorbic acid has a concentration equal to or about 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, or 100 μg/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ascorbic acid has a concentration equal to or about 50 μg/mL.

104. The in vitro composition of any one of claims 85 to 103, further comprising a ROCK inhibitor, or cultured in a medium that does not comprise the ROCK inhibitor, optionally wherein the ROCK inhibitor comprises Y-27632 or Y-27632.

105. The in vitro composition of any one of claims 85 to 104, wherein the ROCK inhibitor has a concentration equal to or about 1 μΜ,2 μΜ,3 μΜ,4 μΜ,5 μΜ,6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ or 20 μΜ, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the ROCK inhibitor has a concentration equal to or about 10 μΜ.

106. The in vitro composition of any one of claims 85 to 105, wherein said posterior foregut cells and/or said posterior foregut endoderm cells, definitive endoderm, posterior foregut endoderm and/or downstream liver cells are differentiated from stem cells, optionally wherein said posterior foregut cells and/or said posterior foregut endoderm cells, definitive endoderm, posterior foregut endoderm and/or downstream liver cells are differentiated from induced pluripotent stem cells.

107. The in vitro composition of any one of claims 85 to 107, wherein said posterior foregut cells and/or said posterior foregut endoderm cells, definitive endoderm, posterior foregut endoderm and/or downstream liver cells have been passaged less than 4 times.

108. The in vitro composition of claim 108, wherein the cells comprise or consist essentially of metaforegut cells and/or metaforegut endoderm cells.

109. The in vitro composition of any one of claims 85-108, wherein the TGF-b pathway inhibitor is a83-01, the FGF pathway activator is FGF2, the Wnt pathway activator is CHIR99021, the VEGF pathway activator is VEGF, and the ROCK inhibitor is Y-27632.

110. A liver organoid produced by the method of any of claims 29-83.

111. An in vitro composition comprising a) a metaintestinal cell and/or a metaintestinal endoderm cell, a liver organoid and/or a mature liver organoid, and b) a culture medium, wherein the culture medium optionally comprises a hepatocyte culture medium and is optionally supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine and a corticosteroid, and wherein the composition optionally further comprises c) a retinoic acid pathway activator.

112. The in vitro composition of claim 111, wherein the cMET tyrosine kinase receptor agonist is selected from the group consisting of Hepatocyte Growth Factor (HGF), PG-001, furoney, tetralin Lei Walai phenanthrene, recombinant InlB321 protein, and an agonistic c-Met antibody, optionally LMH85.

113. The in vitro composition of any one of claims 111-112, wherein the IL-6 family cytokine is selected from the group consisting of IL-6, oncostatin M (OSM), leukemia Inhibitory Factor (LIF), cardiac dystrophin-1, ciliary neurotrophic factor (CTNF), and cardiac dystrophin-like cytokine (CLC).

114. The in vitro composition of any one of claims 111-113, wherein the corticosteroid is selected from the group consisting of dexamethasone, beclomethasone, betamethasone, flucortisone, halometasone, and mometasone.

115. The in vitro composition of any one of claims 111-114, wherein the culture medium is supplemented with HGF, OSM, and dexamethasone.

116. The in vitro composition of any one of claims 111-115, wherein the culture medium is supplemented with dexamethasone.

117. The in vitro composition of any one of claims 111-116, wherein the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580, optionally wherein said retinoic acid pathway activator comprises retinoic acid or is retinoic acid.

118. The in vitro composition of any one of claims 111-117, wherein the retinoic acid pathway activator has a concentration of 1.0µM、1.1µM、1.2µM、1.3µM、1.4µM、1.5µM、1.6µM、1.7µM、1.8µM、1.9µM、2.0µM、2.1µM、2.2µM、2.3µM、2.4µM、2.5µM、2.6µM、2.7µM、2.8µM、2.9µM or 3.0 μΜ, or any concentration in the range defined by any two of the foregoing concentrations, optionally wherein the retinoic acid pathway activator has a concentration equal to or about 2.0 μΜ.

119. The in vitro composition of any one of claims 115 to 118, wherein said HGF has a concentration equal to or about 1ng/mL、2ng/mL、3ng/mL、4ng/mL、5ng/mL、6ng/mL、7ng/mL、8ng/mL、9ng/mL、10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL or 20ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein said HGF has a concentration equal to or about 10 ng/mL.

120. The in vitro composition of any one of claims 115 to 119, wherein the OSM has a concentration equal to or about 10ng/mL、11ng/mL、12ng/mL、13ng/mL、14ng/mL、15ng/mL、16ng/mL、17ng/mL、18ng/mL、19ng/mL、20ng/mL、21ng/mL、22ng/mL、23ng/mL、24ng/mL、25ng/mL、26ng/mL、27ng/mL、28ng/mL、29ng/mL or 30ng/mL, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the OSM has a concentration equal to or about 20 ng/mL.

121. The in vitro composition of any one of claims 115-120, wherein the dexamethasone has a concentration equal to or about 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, or 200nM, or any concentration within a range defined by any two of the foregoing concentrations, optionally wherein the dexamethasone has a concentration equal to or about 100 nM.

122. The in vitro composition of any one of claims 111-121, wherein the composition does not comprise exogenous EGF.

123. The in vitro composition of any one of claims 111 to 122, further comprising a low concentration of exogenous bilirubin, optionally wherein the low concentration of bilirubin is at or near human fetal physiological bilirubin concentration.

124. The in vitro composition of claim 123, wherein the bilirubin is, is about, is less than, or is less than about ：0.1mg/L、0.2mg/L、0.3mg/L、0.4mg/L、0.5mg/L、0.6mg/L、0.7mg/L、0.8mg/L、0.9mg/L、1mg/L、1.25mg/L、1.5mg/L、1.75mg/L、2.0mg/L、2.25mg/L、2.5mg/L、2.75mg/L or is less than about 3.0mg/L, or is in a range defined by any two of the foregoing concentrations, such as 0.1mg/L to 3mg/L, 0.5mg/L to 2.0mg/L, 0.5mg/L to 1.5mg/L, 0.3mg/L to 2.5mg/L, or is in any concentration within 0.5mg/L to 1.75mg/L, or is in a range defined by any two of the foregoing concentrations, such as 0.1mg/L to 1mg/L, 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, or 1mg/L, or is in a range defined by any two of the foregoing concentrations, such as 0.1mg/L to 1mg/L, 0.1mg/L to 5mg/L, 0.5mg/L or 0.5mg/L to 1.75mg/L, or is in any concentration within 0.1mg/L, 2mg/L, 0.2mg/L or 0.5mg/L or 0.3mg/L or 0.4 mg/L.

125. The in vitro composition of claim 124, wherein the composition comprises a mature liver organoid, and wherein the mature liver organoid exhibits a luminal protrusion resembling a bile duct, and/or has a single lumen and generally spherically shaped structure, and/or wherein the mature liver organoid is free of hematopoietic tissue and acquired immune cells.

126. The in vitro composition of claim 125, wherein the mature liver organoid expresses reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to a liver organoid not contacted with a low dose of bilirubin.

127. The in vitro composition of claim 125 or 126, wherein said mature liver organoid expresses increased levels of ALB, SLC4A2 or HO-1, or any combination thereof, relative to a liver organoid not contacted with said low dose of bilirubin.

128. The in vitro composition of any one of claims 125-127, wherein the mature liver organoid expresses CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof.

129. The in vitro composition of any one of claims 125-128, wherein the mature liver organoid exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid not contacted with a low dose of bilirubin.

130. An in vitro composition comprising a mature liver organoid, wherein cells of the mature liver organoid are contacted with a low dose of bilirubin, optionally wherein the low dose of bilirubin is provided exogenously and the mature liver organoid exhibits a luminal protrusion resembling a bile duct, and/or has a single lumen and generally spherical-shaped structure, and/or wherein the mature liver organoid is free of hematopoietic tissue and acquired immune cells.

131. The in vitro composition of claim 130, wherein the mature liver organoid expresses reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to a liver organoid in which the cells are not contacted with low doses of bilirubin.

132. The in vitro composition of claim 130 or 131, wherein said mature liver organoid expresses increased levels of ALB, SLC4A2 or HO-1, or any combination thereof, relative to a liver organoid in which said cells are not contacted with low doses of bilirubin.

133. The in vitro composition of any one of claims 130-132, wherein the mature liver organoid expresses CYP2E1, CYP7A1, PROX1, MRP3, or OATP2, or any combination thereof.

134. The in vitro composition of any one of claims 130-133, wherein the mature liver organoid exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid in which the cells are not contacted with low doses of bilirubin.

135. The in vitro composition of any one of claims 130 to 134, further comprising hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoid cells are contacted with a high concentration and/or a second concentration of bilirubin.

136. The in vitro composition of claim 135, wherein the high concentration/second concentration of bilirubin is, is about, greater than, or greater than about ：2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L mg/L or 20mg/L, or a range defined by any two of the foregoing concentrations, such as any concentration within 2mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12mg/L, or 4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L or 20mg/L, or a range defined by any two of the foregoing concentrations, such as 4mg/L to 20mg/L, 2mg/L to 10mg/L, 10mg/L to 20mg/L, 5mg/L to 15mg/L, or 8mg/L to 12 mg/L.

137. The in vitro composition of claim 135 or 136, wherein the hyperbilirubinemia liver organoid expresses elevated levels of UGT1A1 or NRF2, or both, relative to a liver organoid not treated with high/second concentrations of bilirubin.

138. The in vitro composition of any one of claims 111 to 137, wherein said posterior foregut cell and/or said posterior foregut endoderm cell, liver organoid and/or mature liver organoid comprises a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA or both encoding said functional GULO protein, wherein said posterior foregut cell and/or said posterior foregut endoderm cell, liver organoid and/or mature liver organoid is capable of synthesizing ascorbic acid.

139. The in vitro composition of claim 138, wherein the functional GULO protein is murine GULO (mGULO).

140. An in vitro composition according to claim 138 or 139, wherein the gene encoding the functional GULO protein is conditionally expressed, optionally using a tetracycline-inducible system.

141. The in vitro composition of any of claims 138 to 139, wherein said posterior foregut cells and/or said posterior foregut endoderm cells, liver organoids and/or mature liver organoids are engineered to comprise a gene encoding said functional GULO protein using CRISPR.

142. The in vitro composition of any one of claims 138 to 141, wherein the gene or mRNA encoding the functional GULO protein, or both, is introduced to the liver organoid by transfection.

143. The in vitro composition of any one of claims 138 to 142, wherein said liver organoid and/or mature liver organoid comprising said functional GULO protein expresses increased levels of NRF2 relative to a liver organoid and/or mature liver organoid not comprising said functional GULO protein.

144. The in vitro composition of any one of claims 138 to 143, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein expresses reduced levels of IL1B, IL6 or TNFa or any combination thereof relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

145. The in vitro composition of any one of claims 138-144, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein exhibits reduced caspase-3 activity relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

146. The in vitro composition of any one of claims 138-145, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein expresses increased levels of ALB relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

147. The in vitro composition of any one of claims 138 to 146, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein is similar to periportal liver tissue and expresses a periportal liver marker.

148. The in vitro composition of claim 147, wherein the periportal liver marker comprises FAH, ALB, PAH, CPS a1, HGD, or any combination thereof.

149. The in vitro composition of any one of claims 138-148, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein exhibits increased CYP3A4 and CYP1A2 activity relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

150. The in vitro composition of any one of claims 138 to 149, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein exhibits increased bilirubin conjugation activity relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

151. The in vitro composition of any one of claims 138 to 150, wherein the liver organoid and/or mature liver organoid comprising the functional GULO protein exhibits increased viability in culture relative to a liver organoid and/or mature liver organoid not comprising the functional GULO protein.

152. The in vitro composition of any one of claims 138 to 151, wherein said liver organoid and/or mature liver organoid has been differentiated from a pluripotent stem cell comprising a functional GULO protein and/or a gene or mRNA encoding said functional GULO protein or both, whereby said pluripotent stem cell is capable of synthesizing ascorbic acid.

153. A method comprising administering to a subject in need thereof a liver organoid or composition according to any of claims 110-152.

154. A method for treating a liver-related disease or disorder in a subject in need thereof, the method comprising administering to the subject one or more liver organoids or compositions of claims 110-152.

155. The method of claim 153 or 154, wherein the liver organoid has been produced by a cell derived from the subject, optionally wherein the cell derived from the subject is an induced pluripotent stem cell.

156. A method according to any one of claims 154 to 155, wherein administering comprises transplanting the liver organoid or composition into the subject.

157. The method of any one of claims 154-156, wherein the liver-related disease or disorder comprises one or more types of liver dysfunction and/or failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic disease, autoimmune liver disease, wilson's disease, metabolic-related fatty liver disease, hyperammonemia, hyperbilirubinemia, crigler-Najjar syndrome, urea cycle disorder, walman disease, liver cancer, hepatoblastoma, metabolic dysfunction-related liver disease (MASLD), metALD, metabolic dysfunction-related steatohepatitis (MASH), drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic disease, liver cyst, acute acetaminophen liver injury, and/or alcohol-related liver disease.

158. The method of any one of claims 154-157, wherein the liver dysfunction and/or failure comprises hyperammonemia and/or hyperbilirubinemia, or wherein the metabolic disease comprises non-alcoholic fatty liver disease (NAFLD), or wherein the non-alcoholic fatty liver disease (NAFLD) comprises metabolic dysfunction-related steatohepatitis (MASH), or wherein the hepatitis comprises hepatitis a, hepatitis b, hepatitis c, hepatitis d, hepatitis e, hepatitis G, TT and/or autoimmune hepatitis.

159. The method of any one of claims 154-158, wherein the subject has a reduced serum bilirubin and/or ammonia levels, and/or increased serum protein after transplantation.

160. The method of any one of claims 154-159, wherein the subject has improved bile duct stenosis and/or liver regeneration symptoms following transplantation.

161. The method of any one of claims 154-160, wherein the subject has increased survival after transplantation.

162. The method of any one of claims 154-161, wherein the liver organoid is implanted on the liver of the subject.

163. The method of any one of claims 154-162, wherein the liver organoid has been treated with an Amino Acid (AA) supplement.

164. The method of any one of claims 154-163, wherein the liver organoid has been treated with an Amino Acid (AA) supplement for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more prior to transplantation.

165. The method of any one of claims 154-164, wherein the liver-related disease or disorder comprises acetaminophen acute liver injury.

166. The method of any one of claims 154-165, wherein the method is capable of being used in a Good Manufacturing Practice (GMP) compliant process.

167. A method for screening, the method comprising contacting a liver organoid according to any one of claims 84 to 152 with a candidate compound or composition and assessing the effect of the candidate compound or composition on the liver organoid.

168. A method according to claim 167, wherein the liver organoid is a model of a liver-related disease or disorder, and assessing the effect of the candidate compound or composition on the liver organoid comprises assessing the effect of the candidate compound or composition on the liver-related disease or disorder.

169. The method of claim 167 or 168, wherein the liver organoid has been produced by a cell derived from a subject, optionally wherein the cell derived from the subject is an induced pluripotent stem cell.

170. The method of claim 169, wherein the subject has a liver-related disease or disorder.

171. The method of any one of claims 167 to 170, wherein the method is capable of being used in a Good Manufacturing Practice (GMP) compliant process.

172. A composition comprising a liquid component of a supplemental amino acid according to table 3.

173. A composition comprising a mixture of growth factors according to the embodiments of table 1 or table 2.

174. A composition comprising a liquid component that supplements amino acids, the liquid component of the supplemental amino acids comprises, by volume, a solution of just or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% of a non-essential amino acid (containing just or about 890mg/L alanine, 1320mg/L asparagine, 1330mg/L aspartic acid, 750mg/L glycine, 105mg/L serine, 1150mg/L proline and 1470mg/L glutamic acid), a solution of just or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% of a essential amino acid (containing just or about 6320mg/L arginine 1200mg/L cysteine, 2100mg/L histidine, 2620mg/L isoleucine, 2620mg/L leucine, 3625mg/L lysine, 755mg/L methionine, 1650mg/L phenylalanine, 2380mg/L threonine, 510mg/L tryptophan, 1800mg/L tyrosine and 2340mg/L valine) and exactly or about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% by volume of a hepatocyte medium (HCM), and is also supplemented with exactly or about 5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL、21mg/mL、22mg/mL、23mg/mL、24mg/mL、25mg/mL、26mg/mL、27mg/mL、28mg/mL、29mg/mL、30mg/mL、31mg/mL、32mg/mL、33mg/mL、34mg/mL or 35mg/mL glycine.

175. The composition of any one of claims 172-174, comprising a liquid component of a supplemental amino acid comprising a solution of a non-essential amino acid of exactly or about 14% (containing exactly or about 890mg/L alanine, 1320mg/L asparagine, 1330mg/L aspartic acid, 750mg/L glycine, 105mg/L serine, 1150mg/L proline and 1470mg/L glutamic acid), a solution of an essential amino acid of exactly or about 6% by volume (containing exactly or about 6320mg/L arginine, 1200mg/L cysteine, 2100mg/L histidine, 2620mg/L isoleucine, 2620mg/L leucine, 3625mg/L lysine, 755mg/L methionine, 1650mg/L phenylalanine, 2380mg/L threonine, 510mg/L tryptophan, 1800mg/L tyrosine and 2340mg/L valine), and a medium of hepatocytes of exactly or about 80% by volume (HCM), and further supplemented with exactly or about 20g/L glycine.

176. The composition of any of claims 172-175, wherein pH is between about pH6 and 8, or between pH6.5 and 7.5, or is exactly or about pH 7.0.

177. The composition of any one of claims 172-176, further comprising Hepatocyte Growth Factor (HGF), oncostatin M, dexamethasone, and/or ascorbic acid.

178. The composition of any one of claims 172-177, further comprising liver lineage committed cells differentiated from definitive endoderm cells using retinoic acid.

179. The composition of claim 178, wherein the liver lineage-committed cells are characterized as liver organoids.

180. The composition of claim 179, wherein the liver organoid is characterized as secreting increased levels of albumin and urea relative to a liver organoid contained in HCM without an amino acid supplement.

181. The composition of claim 179 or 180, wherein the liver organoid is characterized as having an increased level of liver maturation-related gene expression relative to a liver organoid comprised in HCM without an amino acid supplement.

182. The composition of any one of claims 179-181, wherein the liver organoid is characterized as expressing reduced levels of vimentin relative to a liver organoid contained in HCM without an amino acid supplement.

183. The composition of any one of claims 172-182, wherein the composition does not comprise a non-human animal component that renders the base film matrix or component thereof xenogeneic to humans.

184. The composition of claim 182, wherein the composition does not comprise murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, matrigel ^®、Cultrex^®, and/or Geltrex ^®.

185. An in vitro hyperbilirubinemia liver organoid comprising naturally occurring and/or engineered mutations in UDP glucuronyl transferase family 1 member A1 (UGT 1 A1).

186. An in vitro hyperbilirubinemia liver organoid, wherein the hyperbilirubinemia liver organoid is produced by contacting precursor cells, precursor liver organoids, and/or precursor mature liver organoids with exogenous bilirubin for at least two cycles.

187. The in vitro hyperbilirubinemia liver organoid of claim 185 or 186 wherein the hyperbilirubinemia liver organoid is of clonal origin and/or derived from an iPSC.

188. A cryopreserved composition comprising a liver organoid, chroman a, emlicarbazepine, a polyamine, and a trans-ISRIB (CEPT).

189. A cryopreserved composition comprising mature liver organoids, chroman a, emlicarbazepine, polyamines, and trans-ISRIB (CEPT).

190. A cryopreserved composition comprising hyperbilirubinemia liver organoids, chroman a1, emlicarbazin, polyamines, and trans-ISRIB (CEPT).

191. A kit comprising means for performing the method of any one of claims 1 to 83 or 153 to 171.

192. A kit comprising the composition of any one of claims 84 to 152, 172 to 184 or 188 to 190 or means for producing the composition or means for producing the liver organoid of any one of claims 185 to 187.

193. Use of the method, composition or kit of any one of claims 1 to 192 as a medicament, tool for the treatment and/or prophylaxis of a disease, diagnostic tool and/or medical study.