TW202505022A - Methods of producing tissue-derived epithelial organoids and uses thereof - Google Patents

Abstract

The present disclosure provides methods of preparing tissue-derived epithelial organoids. In particular, the present disclosure provides tissue-derived epithelial organoids embedded in hydrogel suspended in a medium. The present disclosure further provides methods of using such organoids.

Description

Methods for producing tissue-derived epithelial organoids and their uses

The subject matter disclosed herein relates to tissue-derived epithelial organoids and methods of generating and using such organoids.

Tissue-derived epithelial organoids are three-dimensional (3D) multicellular spheroids that recapitulate the complexity and function of in vivo tissues and have become physiologically relevant in vitro models of tissues. For example, intestinal organoids, also called "enteroids" or "colonoids," are derived from adult stem cells isolated from primary intestinal tissue and can be easily propagated and cryopreserved for long-term storage. Intestinal organoids can differentiate into a variety of intestinal cell types, perform epithelial functions such as barrier maintenance, absorption, secretion, and digestion, and recapitulate the biological characteristics and clinical responses of the patients from which they are derived (Clevers (2016) Cell 165, 1586–1597; Zachos et al. (2016) J Biol Chem 291, 3759–3766). As a result, many intestinal organoids have been widely adopted to replace traditional transformed and immortalized intestinal cell lines, thereby generating basic science discoveries and promoting translational applications in many fields including cancer biology, infectious diseases, and cystic fibrosis (Clevers (2016); Schutgens and Clevers (2019) Annu Rev Pathology Mech Dis 15, 1–24).

A challenge in the implementation of tissue-derived epithelial organoids in fields such as drug development is that existing organoid culture techniques are difficult to scale up, requiring either cumbersome manual methods or the development of advanced automation infrastructure (Louey et al. (2021) Slas Discov 26, 1138–1147). In existing techniques, tissue-derived epithelial stem cells (isolated from primary tissue or passaged from established organoid cultures) are resuspended in a cold extracellular matrix (ECM) solution, most commonly CULTREX® basement membrane extract (BME) or MATRIGEL® hydrogel. ECM is deposited onto the dish surface and then warmed to solidify the ECM cell solution into a surface-attached hydrogel dome and covered with medium. The medium in each well is changed every few days and organoids form within 1-2 weeks (Mahe et al. Curr Protoc Mouse Biology 3, 217–240; Pleguezuelos-Manzano et al. (2020) Curr Protoc Immunol 130, e106; Sato et al. (2009) Nature 459, 262–265; Sato et al. (2011) Gastroenterology 141, 1762–1772). The technique is difficult to scale up because it is limited by the surface area available for hydrogel dome formation, is time-consuming and labor-intensive, and is prone to user error, and the diffusion limitations of the hydrogel lead to heterogeneity in organoid growth and morphology (Park et al. (2022) Nat Methods 19, 1449–1460; Ringel et al. (2020) Cell Stem Cell 26, 431-440.e8; Shin et al. (2020) iScience 23, 101372). Therefore, there is a need for more efficient and high-throughput methods to generate tissue-derived epithelial organoids.

The subject matter disclosed herein relates to tissue-derived epithelial organoids and methods of generating such organoids. The present disclosure further provides methods of using tissue-derived epithelial organoids and systems for performing the methods disclosed herein.

In certain embodiments, a method of generating tissue-derived epithelial organoids comprises (a) contacting tissue-derived epithelial stem cells with hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture, (b) suspending the hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture, and (c) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to generate tissue-derived epithelial organoids. In some embodiments, a plurality of tissue-derived epithelial stem cells are contacted with the hydrogel to produce the hydrogel-tissue-derived epithelial stem cell mixture. In some embodiments, the method further comprises fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce a fragmented structure comprising the tissue-derived epithelial organoid.

In some embodiments, the hydrogel solidifies upon contact with the culture medium. In some embodiments, suspending the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium includes immersing a dispensing device containing the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium and dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the culture medium. In some embodiments, the temperature of the culture medium is about 25° C. to about 50° C. In some embodiments, the temperature of the culture medium is about 30° C. to about 50° C. In some embodiments, the temperature of the culture medium is about 30° C. to about 40° C. In some embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is less than about 20° C. In some embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is about 2° C. to about 25° C. In some embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is about 2° C. to about 20° C. In some embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is about 2° C. to about 10° C.

The present disclosure further provides a method for producing a suspended culture of tissue-derived epithelial organoids. In certain embodiments, the method comprises (a) introducing a mixture comprising a hydrogel and tissue-derived epithelial stem cells into a culture medium to produce a suspended mixture; and (b) culturing the suspended mixture in the culture medium to produce the tissue-derived epithelial organoids in a suspended state. In certain embodiments, the mixture introduced into the culture medium comprises the hydrogel and a plurality of tissue-derived epithelial stem cells. In some embodiments, the method comprises (a) introducing a mixture comprising a hydrogel and a plurality of tissue-derived epithelial stem cells into a culture medium to produce a suspended mixture, and (b) culturing the mixture in the culture medium to produce the tissue-derived epithelial organoids in a suspended state. In some embodiments, the hydrogel solidifies upon contact with the culture medium. In some embodiments, introducing the mixture into the culture medium comprises immersing a dispensing device containing the mixture in the culture medium and dispensing the mixture into the culture medium. In some embodiments, the temperature of the culture medium is about 25° C. to about 50° C. In some embodiments, the temperature of the culture medium is about 30° C. to about 50° C. In some embodiments, the temperature of the culture medium is about 25°C to about 40°C. In some embodiments, the temperature of the culture medium is about 30°C to about 40°C. In some embodiments, the temperature of the mixture is below about 20°C. In some embodiments, the temperature of the mixture is about 2°C to about 25°C. In some embodiments, the temperature of the mixture is about 2°C to about 20°C. In some embodiments, the temperature of the mixture is about 2°C to about 10°C. In some embodiments, the method further comprises fragmenting the suspended mixture to produce a fragmented structure comprising the tissue-derived epithelial organoid.

In certain embodiments, a method for generating a suspension culture of tissue-derived epithelial organoids comprises (a) contacting tissue-derived epithelial stem cells with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture; (b) depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate; (c) solidifying the hydrogel-tissue-derived epithelial stem cell mixture to generate a solidified hydrogel-tissue-derived epithelial stem cell mixture; (d) (e) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to produce tissue-derived epithelial organoids. In certain embodiments, a plurality of tissue-derived epithelial stem cells are contacted with the hydrogel to produce the hydrogel-tissue-derived epithelial stem cell mixture. For example, but not limited to, the method includes (a) contacting a plurality of tissue-derived epithelial stem cells with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture, (b) depositing the hydrogel-tissue-derived epithelial stem cell mixture on a substrate, (c) solidifying the hydrogel-tissue-derived epithelial stem cell mixture to produce a solidified hydrogel-tissue-derived epithelial stem cell mixture, (d) suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture, and (e) The suspended hydrogel-tissue-derived epithelial stem cell mixture is cultured in the culture medium to produce tissue-derived epithelial organoids. In certain embodiments, the method further comprises removing the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate before suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the culture medium.

In some embodiments, the hydrogel-tissue derived epithelial stem cell mixture is deposited onto the substrate as droplets. In some embodiments, the hydrogel-tissue derived epithelial stem cell mixture is deposited onto the substrate to have a filamentous structure. In some embodiments, the filamentous structure has a linear, serpentine, or spiral shape. In some embodiments, the method further comprises fragmenting the hydrogel-tissue derived epithelial stem cell mixture in the culture medium to produce a fragmented structure comprising the tissue derived epithelial organoid.

In certain embodiments, the geometry of the suspended hydrogel-tissue-derived epithelial stem cell mixture, the suspended mixture, or the solidified hydrogel-tissue-derived epithelial stem cell mixture comprises a length, width, and/or diameter greater than about 0.1 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue-derived epithelial stem cell mixture, the suspended mixture, or the solidified hydrogel-tissue-derived epithelial stem cell mixture comprises a length, width, and/or diameter of about 0.1 mm to about 1,000 mm ( e.g. , about 0.1 mm to about 20 mm). In some embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture, the suspended mixture, or the solidified hydrogel-tissue derived epithelial stem cell mixture is in the form of droplets. In some embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture, the suspended mixture, or the solidified hydrogel-tissue derived epithelial stem cell mixture has a filamentous structure. In some embodiments, the filamentous structure has a linear, serpentine, or spiral shape.

In some embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells are contained in tissue fragments, organoid fragments, or a combination thereof. In some embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells are isolated from native epithelial tissue. In certain embodiments, the tissue-derived epithelial stem cells are obtained from a fragment of a tissue selected from the group consisting of tear glands, tonsils, salivary glands, gastrointestinal tissue, thyroid, lung, breast, liver, bile duct, stomach, kidney, pancreas, endometrium, fallopian tube, cervix, prostate, bladder, ovary, taste buds, placenta, and combinations thereof. Alternatively or additionally, the tissue-derived epithelial stem cells are obtained from fragments of organoids selected from the group consisting of tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblastic organoids, and combinations thereof.

In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1×10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1×10 ⁷ tissue-derived epithelial stem cells/ml hydrogel.

In some embodiments, the hydrogel is selected from the group consisting of: synthetic hydrogels, natural hydrogels, and combinations thereof. In some embodiments, the natural hydrogel comprises basement membrane extract (BME) or extracellular matrix (ECM) components. In some embodiments, the hydrogel has a protein concentration greater than about 1 mg/ml. In some embodiments, the hydrogel comprises greater than about 1 w/v % of a BME component, an ECM component, or a polymer in w/v %. In some embodiments, the hydrogel has a storage modulus G' equal to or greater than a loss modulus G''.

In some embodiments, the culture medium is present in a container. In some embodiments, the container is a culture dish, a multi-well dish, a conical tube, a container, a culture bag, a bioreactor or a flask.

The present disclosure further provides a tissue-derived epithelial organoid produced by the methods disclosed herein.

In certain embodiments, the tissue-derived epithelial organoids produced by the methods of the present disclosure have a uniform morphology compared to reference tissue-derived epithelial organoids. In certain embodiments, the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. In certain embodiments, the tissue-derived epithelial organoids have a uniform size. In certain embodiments, the average diameter of the tissue-derived epithelial organoids is more uniform than the reference tissue-derived epithelial organoids.

In certain embodiments, stem cell and/or proliferation markers are expressed at higher levels in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure compared to a population of reference tissue-derived epithelial organoids. In certain embodiments, differentiation markers are expressed at lower levels in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure compared to a population of reference tissue-derived epithelial organoids. In certain embodiments, the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. In certain embodiments, the stem cell and/or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. In some embodiments, the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

The present disclosure further provides a composition comprising a tissue-derived epithelial organoid and a culture medium, wherein the tissue-derived epithelial organoid is embedded in a hydrogel suspended in the culture medium. In some embodiments, the geometry of the hydrogel comprises a length, width, and/or diameter greater than about 0.1 mm. In some embodiments, the geometry of the hydrogel comprises a length, width, and/or diameter of about 0.1 mm to about 1,000 mm ( e.g. , about 0.1 mm to about 20 mm). In some embodiments, the hydrogel is a droplet. In some embodiments, the hydrogel has a filamentous structure. In some embodiments, the filamentous structure has a linear, serpentine, or spiral shape. In certain embodiments, stem cell and/or proliferation markers are expressed at higher levels in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids. In certain embodiments, the stem cell and/or proliferation marker is selected from the group consisting of: MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. In certain embodiments, differentiation markers are expressed at lower levels in a population of epithelial organoids compared to a population of reference tissue-derived epithelial organoids. In some embodiments, the differentiation marker is selected from the group consisting of: keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. In some embodiments, the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

The present disclosure further provides a method for screening an agent ( e.g. , a therapeutic agent). In certain embodiments, the method comprises (a) contacting a tissue-derived epithelial organoid, a population of tissue-derived epithelial organoids, or a composition of tissue-derived epithelial organoids with an agent ( e.g. , a therapeutic agent), and (b) analyzing the tissue-derived epithelial organoid or a population of such tissue-derived epithelial organoids for changes indicative of the effectiveness, distribution, and/or toxicity of the agent ( e.g. , a therapeutic agent). In some embodiments, the agent ( e.g. , a therapeutic agent) is contacted with the tissue-derived epithelial organoid or a population of such tissue-derived epithelial organoids for about 1 minute to about 3 years, e.g. , about 15 minutes to about 3 years. In some embodiments, the agent is a therapeutic agent. In some embodiments, the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell-based therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof. In certain embodiments, the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redox potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, repression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof.

The present disclosure further provides a method for performing genomic screening. In some embodiments, the method includes (a) providing a tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids or a composition of tissue-derived epithelial organoids, (b) generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid, and (c) analyzing the changes associated with the mutation in the tissue-derived epithelial organoid or the group of tissue-derived epithelial organoids. In some embodiments, the mutation is generated using a gene regulation system. In some embodiments, the gene regulation system is a gene editing system. In some embodiments, the gene editing system is a CRISPR system. In certain embodiments, the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redox potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, repression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof.

The present disclosure further provides a method for generating an epithelial cell model. In some embodiments, the method includes (a) providing a tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids or a composition of tissue-derived epithelial organoids, (b) digesting the tissue-derived epithelial organoid or the group of such tissue-derived epithelial organoids into single cells, and (c) culturing the single cells in a culture medium to produce a cell monolayer. In some embodiments, the single cells are cultured on a permeable cell culture insert. In some embodiments, the culture medium is a differentiation medium. In some embodiments, the culture medium is a cell growth medium. In certain embodiments, the culture medium is a stem cell promoting medium.

The present disclosure further provides a screening method using a cell monolayer produced by the methods described herein. In some embodiments, the screening method is a method for screening an agent ( e.g. , a therapeutic agent). For example, but not limited to, the method can include (a) contacting a cell monolayer produced by the methods described herein with an agent ( e.g. , a therapeutic agent); and (b) analyzing changes in the cell monolayer that indicate the effectiveness, distribution, and/or toxicity of the agent ( e.g. , a therapeutic agent). In some embodiments, the agent ( e.g. , a therapeutic agent) is contacted with the cell monolayer for about 1 minute to about 3 years, for example , about 15 minutes to about 3 years. In some embodiments, the agent is a therapeutic agent. In some embodiments, the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell-based therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof. In some embodiments, the screening method is a genome screening. In some embodiments, the method for performing a genome screening may include (a) providing a cell monolayer produced by a method as described herein; (b) generating a mutation in a genome of one or more cells in the cell monolayer; and (c) analyzing changes in the cell monolayer associated with the mutation. In some embodiments, the mutation is generated using a gene regulation system. In some embodiments, the gene regulation system is a gene editing system. In some embodiments, the gene editing system is a CRISPR system. In some embodiments, the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redox potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, repression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof.

In some embodiments, one or more steps of the methods of the present disclosure may be performed using one or more robots and/or automated components. For example, but not limited to, one or more steps of the methods of generating tissue-derived epithelial organoids, methods of generating suspension cultures of tissue-derived epithelial organoids, methods of screening agents, methods of performing genomic screening, methods of generating epithelial cell models, and/or screening methods using cell monolayers as described herein may be performed using one or more robots and/or automated components. In some embodiments, the one or more robots and/or automated components are selected from the group consisting of: a liquid handling robot, a 3D printer, a syringe pump, and combinations thereof. In some embodiments, the one or more robots and/or automated components include a liquid handling robot.

The present disclosure further provides a system for culturing tissue-derived epithelial organoids. In some embodiments, the system includes tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium. In some embodiments, the geometry of the hydrogel comprises a length, width, and/or diameter greater than about 0.1 mm. In some embodiments, the geometry of the hydrogel comprises a length, width, and/or diameter of about 0.1 mm to about 1,000 mm ( e.g. , about 0.1 mm to about 20 mm). In some embodiments, the hydrogel is a droplet. In some embodiments, the hydrogel has a filamentous structure. In some embodiments, the filamentous structure has a linear, serpentine, or helical shape. In some embodiments, stem cell and/or proliferation markers are expressed at higher levels in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. In some embodiments, the stem cell and/or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. In certain embodiments, a differentiation marker is expressed at a lower level in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. In some embodiments, the differentiation marker is selected from the group consisting of: keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA) and combinations thereof. In some embodiments, the tissue-derived epithelial organoid is selected from the group consisting of tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblastic lining organoids, and combinations thereof. In some embodiments, the system includes one or more robots and/or automated components for generating and/or culturing the tissue-derived epithelial organoids. In some embodiments, the one or more robots and/or automated components are selected from the group consisting of: a liquid handling robot, a 3D printer, a syringe pump, and combinations thereof. In some embodiments, the one or more robots and/or automated components include a liquid handling robot.

The present disclosure further provides systems for performing the methods disclosed herein. For example, but not limited to, the present disclosure provides systems for performing the following: methods for generating tissue-derived epithelial organoids, methods for generating suspension cultures of tissue-derived epithelial organoids, methods for screening agents, methods for performing genomic screening, methods for generating epithelial cell models, and/or screening methods using cell monolayers. In some embodiments, the system includes one or more robots and/or automated components for generating and/or culturing the tissue-derived epithelial organoids. In some embodiments, the one or more robots and/or automated components are selected from the group consisting of: liquid handling robots, 3D printers, syringe pumps, and combinations thereof. In some embodiments, the one or more robots and/or automated components include a liquid handling robot.

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 63/508,132, filed on June 14, 2023, the contents of which are incorporated herein by reference in their entirety.

The present disclosure provides tissue-derived epithelial organoids in suspended cultures and methods of generating such tissue-derived epithelial organoids. The methods disclosed herein enable the generation of large-scale organoid cultures without the need for cumbersome manual manipulations, specialized equipment, or automation. By growing organoid cells in suspended hydrogels rather than using surface-attached hydrogel domes, the hydrogel volume can be significantly increased, and therefore the number of organoid cells that can be grown in a culture vessel can be significantly increased. Furthermore, since the presently disclosed method does not require the hydrogel to be deposited on a two-dimensional (2D) surface, the method is compatible with various culture containers ( eg, culture flasks and culture bags), which allows for further culture scalability, thereby achieving high throughput.

The methods disclosed herein allow the use of suspended hydrogels of various geometries, which can speed up actual culture preparation time. In addition, because organoids are in suspension, they can be sampled, split, or collected at different times during the culture process, which is difficult for conventional organoid cultures that are fixed in a dish. As shown in Example 1, organoids grew more uniformly in suspended BME hydrogels than in conventional surface-attached hydrogel domes, where limited molecular diffusion leads to nutrient gradients (Park et al. (2022); Shin et al. (2020)). The suspended hydrogel culture method disclosed herein generates epithelial organoid models of tissue origin that are more suitable for high-throughput studies, which will benefit both basic science and translational fields.

For clarity, but not limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections: I. Definitions; II. Organoids and Compositions Thereof; III. Methods of Producing Organoids; IV. Methods of Use; V. Systems; and VI. Exemplary Embodiments I. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the subject matter of this disclosure belongs. The following references provide general definitions of many of the terms used in this disclosure by those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise specified, the following terms have the meanings assigned to them below.

As used herein, when the terms "a" or "an" are used in conjunction with the term "comprising" in the claims and/or the specification, they may mean "one", but are also consistent with the meanings of "one or more", "at least one", and "one or more than one".

The term "about" or "approximately" means that a particular value is within an acceptable range of error as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e. , on the limitations of the measurement system. For example, according to practice in the art, "about" can mean within 3 times or more of a standard deviation. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term means within an order of magnitude of a value, such as within 5 times of a value, or within 2 times of a value.

The term "antibody" herein is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies ( eg , bispecific antibodies) and antibody fragments, as long as they exhibit the intended antigen-binding activity.

"Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , bifunctional antibodies, linear antibodies, single-chain antibody molecules ( e.g. , scFv), and multispecific antibodies formed from antibody fragments.

As used herein, the term "culture medium" or "medium" refers to a liquid that covers cells in a culture container (such as a culture bottle and a multiwell dish) and contains nutrients that nourish the cells. In certain embodiments, the culture medium may also include growth factors or differentiation factors to produce desired changes in the cells.

As used herein, the terms "comprise", "include", "having", "has", "may", "contain" and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude other actions or structures. The present disclosure also encompasses embodiments or elements presented herein that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

As used herein, the term "contacting" a cell with a compound , such as a therapeutic agent, refers to exposing the cell to the compound, such as placing the compound in a position that allows it to contact the cell. Contacting can be accomplished using any suitable method. For example, but not limited to, "contacting" can be accomplished by adding the compound to a container containing the cells. Contacting can also be accomplished by adding the compound to a culture medium containing the cells. In certain embodiments, "contacting" refers to exposing a cell ( e.g., a cell in an epithelial organoid or a cell monolayer of tissue origin) to an agent or compound. In certain embodiments, "contacting" refers to exposing the tissue-derived epithelial organoid or cell monolayer to a potential therapeutic agent or therapeutic agent of interest.

As used herein, the term "derived from" or "established from" or "differentiated from" when referring to any cell disclosed herein refers to a cell obtained from a cell line, tissue (such as a dissociated tissue), or a parent cell in a fluid ( e.g. , separated or purified) using any manipulation. Non-limiting examples of such manipulations include single cell isolation, in vitro culture, treatment and/or induction using, for example, proteins, chemicals, radiation, viral infection, and nucleic acid transfection. In certain embodiments, source cells can be selected from a mixed population by response to growth factors, cytokines, selected processes of cytokine treatment, adhesion, lack of adhesion, sorting procedures, or a combination thereof.

The terms "detection" or "detecting" include any means of detection, including direct and indirect detection.

As used herein, the term "droplet" when used in reference to a geometric shape refers to a spherical or spheroidal shape.

As used herein, the term "embedded" or "embedded" refers to at least partially covering or surrounding cells. In certain embodiments, as used herein, the term "embedded" or "embedded" refers to completely covering or surrounding tissue-derived epithelial organoids, such as with a hydrogel.

As used herein, the term "expression" or "express" refers to the transcription and/or translation of a nucleotide sequence.

The term "expression vector" is used to denote a linear or circular nucleic acid molecule into which another nucleic acid sequence fragment of appropriate size can be incorporated. Such nucleic acid fragments may include additional segments that provide for transcription of the gene encoded by the nucleic acid sequence fragment. Additional segments may include, but are not limited to: promoters, transcription terminators, enhancers, internal ribosomal entry sites, non-translational regions, polyadenylation signals, selectable markers, origins of replication, etc., as known in the art. Expression vectors are usually derived from plasmids, cosmids and viral vectors; vectors are usually recombinant molecules containing nucleic acid sequences from a variety of sources.

As used herein, the term "gastrointestinal" refers to the oral mucosa, pharynx (throat), esophagus, stomach, small intestine, large intestine, and rectum.

As used herein, the term "gastrointestinal stem cells" refers to stem cells of the gastrointestinal system.

As used herein, the term "subject" or "individual" refers to a vertebrate or invertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, game animals, rodents, and pets. Non-limiting examples of non-human animal individuals include rodents, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, apes, and monkeys. In certain embodiments, the subject or individual is a human.

As used herein, the term "enteral" or "intestine" refers to the rectum, small intestine, and large intestine.

As used herein, the term "intestinal stem cells" refers to the stem cells of the intestine.

As used herein, the term " in vitro " refers to an artificial environment and processes or reactions that occur within an artificial environment. An in vitro environment includes, but is not limited to, a cell culture.

As used herein, the term " in vivo " refers to the natural environment ( eg , an animal or a cell) and processes or reactions that occur in the natural environment.

As used herein, the term "linear" when used in reference to a geometric shape refers to a shape similar to a line. In some embodiments, the line can be a straight line, a curved line, or a line of any shape.

As used herein, the term "isolated" in reference to cells , such as gastrointestinal stem cells, refers to cells that have been separated from components of their natural environment.

As used herein, "label" refers to an agent that allows direct or indirect detection. Labels include, but are not limited to, fluorescent compositions, chromogenic labels, electronically precise labels, chemiluminescent labels, and radioactive labels. Non-limiting examples of markers include green fluorescent protein ("GFP"), mCherry, dtTomato or other fluorescent proteins known in the art (e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909 (2005), incorporated herein by reference), ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I, fluorophores (such as rare earth chelates or fluorescent yellow and its derivatives), rhodamine and its derivatives, dansyl, umbelliferone, luciferase (such as firefly luciferase and bacterial luciferin pure enzyme) (U.S. Patent No. 4,737,456 ), fluorescein, 2,3-dihydropyrrophenone, and enzymes that generate detectable signals, such as horseradish peroxidase (HRP), alkaline phosphatases, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase (G6PD)), and heterocyclic oxidases (such as uricase and xanthine oxidase).

The term "nucleic acid" or "polynucleotide" includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a base sequence, wherein the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually represented from 5' to 3'. The term nucleic acid includes: deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), such as messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acids described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars, phosphate backbone linkages, or chemically modified residues.

The term "operably linked", when applied to nucleic acid sequences, e.g., those in an expression vector, means that the sequences are arranged so that they function together to achieve their intended purpose, i.e. , a promoter sequence allows initiation of transcription, which proceeds through the linked coding sequence until a termination signal.

As used herein, the term "organoid" refers to a three-dimensional cell structure obtained by the expansion of stem cells ( e.g., adult stem cells) that self-organize and can differentiate into functional cell types. See Corro et al. (2020) Am. J. Physiol. Cell Physiol. 319:C151-C165.

As used herein, the term "plurality" refers to a quantity greater than one. In certain embodiments, the term "plurality of tissue-derived epithelial stem cells" refers to a quantity greater than one tissue-derived epithelial stem cell. For example, but not limited to, the plurality of tissue-derived epithelial stem cells include at least two tissue-derived epithelial stem cells. In certain non-limiting embodiments, the plurality of tissue-derived epithelial stem cells may include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 5,000, at least about 10,000, at least about 100,000, at least about 100,000, at least about 1,000,000, at least about 10,000,000, at least about 100,000,000, or at least about 1,000,000,000 tissue-derived epithelial stem cells.

As used herein, the term "a population of tissue-derived epithelial organoids" refers to a population of epithelial organoids from at least two tissues. In certain embodiments, a "population of tissue-derived epithelial organoids" refers to a population of tissue-derived epithelial organoids generated by the same method. In certain non-limiting embodiments, a population of tissue-derived epithelial organoids can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 5,000, at least about 10,000, at least about 100,000, at least about 1,000,000, at least about 10,000,000, at least about 100,000,000, or at least about 1,000,000,000 tissue-derived epithelial organoids.

As used herein, the term "proliferation" refers to an increase in the number of cells.

As used herein, the term "promoter" refers to a region within a gene to which transcription factors and/or RNA polymerases can bind to control the expression of the associated coding sequence. The promoter is usually (but not always) located in the 5' non-coding region of the gene upstream of the translation start codon. The promoter region of a gene may include one or more common sequences that serve as recognizable binding sites for sequence-specific nucleic acid binding domains of nucleic acid binding proteins. However, these binding sites may also be located in regions outside the promoter, such as in introns or in enhancer regions downstream of the coding sequence.

As used herein, the term "cure" or "solidified" refers to the hardening, thickening, polymerization and/or increase in rigidity of a substance.

As used herein, the term "snake" when used in reference to a geometric shape refers to a serpentine shape.

As used herein, the term "helix" when used in reference to a geometric shape refers to a continuous curve that spirals around a center point or around an axis.

As used herein, the term "subset" refers to a small portion of a larger body of material.

As used herein, "tissue-derived epithelial stem cells" refers to epithelial stem cells obtained from tissues. In certain embodiments, tissue-derived epithelial stem cells do not include high-potential stem cells ( eg , induced high-potential stem cells (iPSCs) and embryonic stem cells (ESCs)).

As used herein, "treatment" is a method for obtaining beneficial or desired results, including clinical results. For the purposes of the present subject matter, beneficial or desired clinical results include, but are not limited to, reduction or amelioration of one or more signs or symptoms, reduction in severity of disease, stabilization ( i.e. , not worsening) of the disease state, prevention of disease, delay or slow progression of disease, alleviation of disease ( e.g., cancer), and/or improvement or reduction of the disease state. The reduction may be at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% reduction in the severity of complications, signs, or symptoms, or the likelihood of progression to another level. In certain embodiments, "treating" may also refer to inhibiting the proliferation or progression of cancer to a higher level by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%. II. Organoids and their compositions

The present disclosure provides tissue-derived epithelial organoids. In certain embodiments, the tissue-derived epithelial organoids are embedded in a hydrogel that is not attached to a substrate ( e.g., a substrate of a culture vessel). The present disclosure further provides compositions comprising such organoids. In certain embodiments, the tissue-derived epithelial organoids are produced by a method disclosed herein, such as the method disclosed in Section III.

In certain embodiments, the present disclosure provides a composition comprising a tissue-derived epithelial organoid and a culture medium, wherein the tissue-derived epithelial organoid is embedded in a hydrogel. In certain embodiments, the hydrogel is not attached to a substrate surface, such as a cell culture dish and/or a multiwell dish. In certain embodiments, the tissue-derived epithelial organoid embedded in the hydrogel is suspended in the culture medium.

In some embodiments, the tissue-derived epithelial organoid is a tear gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a breast organoid, a liver organoid, a bile duct organoid, a stomach organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervical organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid, or a trophoblastic lining organoid. In some embodiments, the tissue-derived epithelial organoid is a tear gland organoid. In some embodiments, the tissue-derived epithelial organoid is a tonsil organoid. In some embodiments, the tissue-derived epithelial organoid is a salivary gland organoid. In some embodiments, the tissue-derived epithelial organoid is a gastrointestinal organoid. In some embodiments, the tissue-derived epithelial organoid is a thyroid organoid. In some embodiments, the tissue-derived epithelial organoid is a lung organoid. In some embodiments, the tissue-derived epithelial organoid is a mammary organoid. In some embodiments, the tissue-derived epithelial organoid is a liver organoid. In some embodiments, the tissue-derived epithelial organoid is a bile duct organoid. In some embodiments, the tissue-derived epithelial organoid is a gastric organoid. In some embodiments, the tissue-derived epithelial organoid is a kidney organoid. In some embodiments, the tissue-derived epithelial organoid is a pancreatic organoid. In some embodiments, the tissue-derived epithelial organoid is an endometrial organoid. In some embodiments, the tissue-derived epithelial organoid is a fallopian tube organoid. In some embodiments, the tissue-derived epithelial organoid is a cervical organoid. In some embodiments, the tissue-derived epithelial organoid is a prostate organoid. In some embodiments, the tissue-derived epithelial organoid is a bladder organoid. In some embodiments, the tissue-derived epithelial organoid is an ovarian organoid. In some embodiments, the tissue-derived epithelial organoid is a taste bud organoid. In some embodiments, the tissue-derived epithelial organoid is a trophoblastic lining organoid. In some embodiments, the tissue-derived epithelial organoid is selected from the group consisting of lung organoids, gastrointestinal organoids, liver organoids, pancreatic organoids, breast organoids, and combinations thereof.

In certain embodiments, the tissue-derived epithelial organoid is a lung organoid. In certain embodiments, the tissue-derived epithelial organoid is an alveolar type II (ATII) organoid.

In some embodiments, the tissue-derived epithelial organoids are gastrointestinal organoids. In some embodiments, the gastrointestinal organoids are intestinal organoids. For example, but not limited to, the tissue-derived epithelial organoids are colon or ileal organoids. In some embodiments, the tissue-derived epithelial organoids are colon organoids. In some embodiments, the tissue-derived epithelial organoids are ileal organoids. In some embodiments, the tissue-derived epithelial organoids are rectal organoids. In some embodiments, the tissue-derived epithelial organoids are esophageal organoids. In some embodiments, the tissue-derived epithelial organoids are oral maxillofacial organoids. In some embodiments, the gastrointestinal organoids comprise goblet cells and intestinal epithelial cells.

In certain embodiments, the tissue-derived epithelial organoid is a mammary organoid.

In some embodiments, the tissue-derived epithelial organoid is a pancreatic organoid. In some embodiments, the pancreatic organoid comprises vascular cells, for example , as disclosed in Example 6.

In some embodiments, the tissue-derived epithelial organoid is a liver organoid. In some embodiments, the liver organoid comprises liver cells, for example , as disclosed in Example 7.

In some embodiments, the compositions of the present disclosure may include epithelial organoids derived from one or more different types of tissues. For example, but not limited to, the compositions of the present disclosure may include colon and ileum organoids. In some embodiments, the compositions of the present disclosure may include liver and bile duct organoids.

In certain embodiments, the compositions of the present disclosure include about 1 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium, such as about 2 or more, about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, about 10,000 or more, about 50,000 or more, about 100,000 or more, about 500,000 or more, about 1,000,000 or more, about 10,000,000 or more, about 100,000,000 or more, or about 1,000,000,000 or more. In some embodiments, the compositions of the present disclosure include about 50 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In some embodiments, the compositions of the present disclosure include about 100 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In some embodiments, the compositions of the present disclosure include about 1,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the compositions of the present disclosure include about 5,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the compositions of the present disclosure include about 10,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the compositions of the present disclosure include about 100,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the compositions of the present disclosure include about 500,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the compositions of the present disclosure include about 1,000,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the compositions of the present disclosure include about 10,000,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the compositions of the present disclosure include about 100,000,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium.

In some embodiments, the hydrogel is a three-dimensional (3D) scaffold. In some embodiments, the hydrogel is composed of a material that cures at a temperature greater than about 10°C. For example, but not limited to, the hydrogel is composed of a material that cures at a temperature greater than about 15°C, greater than about 20°C, greater than about 25°C, greater than about 30°C, greater than about 35°C, greater than about 40°C, greater than about 45°C, or greater than about 50°C. In some embodiments, the hydrogel is composed of a material that cures at a temperature greater than about 25°C. In some embodiments, the hydrogel is composed of a material that cures at a temperature greater than about 30°C. In some embodiments, the hydrogel is composed of a material that cures at a temperature greater than about 35°C. In some embodiments, the hydrogel is composed of a material that cures at a temperature greater than about 40°C. In some embodiments, the hydrogel is composed of a material that cures at a temperature greater than about 45°C. In some embodiments, the hydrogel is composed of a material that cures at a temperature greater than about 50°C. In some embodiments, the hydrogel is composed of a material that cures at a temperature of about 25°C to about 50°C. In some embodiments, the hydrogel is composed of a material that cures at a temperature of about 25°C to about 40°C. In some embodiments, the hydrogel is composed of a material that cures at a temperature of about 30°C to about 50°C. In some embodiments, the hydrogel is composed of a material that cures at a temperature of about 30°C to about 40°C, such as about 37°C.

In some embodiments, the hydrogel is a synthetic hydrogel, a natural hydrogel, or a combination thereof. In some embodiments, the hydrogel is a synthetic hydrogel. In some embodiments, the hydrogel is a natural hydrogel. In some embodiments, the hydrogel may be a mixture of a synthetic hydrogel and a natural hydrogel. In some embodiments, the hydrogel does not include chemically cross-linked proteins and/or polymers.

In some embodiments, the hydrogel is a natural hydrogel. In some embodiments, the natural hydrogel includes one or more naturally occurring components. For example, but not limited to, the natural hydrogel may include one or more proteins, such as glycoproteins and/or polysaccharides. Non-limiting examples of glycoproteins include collagen ( e.g. , type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen and/or type XII collagen), fibronectin, entactin, tenascin, vitronectin, reticular protein, hyaluronic acid and laminin. In certain embodiments, the natural hydrogel may further include one or more components, such as but not limited to polysaccharides, water and/or elastin. In certain embodiments, the natural hydrogel disclosed herein includes laminin, entactin and type IV collagen. In certain embodiments, the natural hydrogel disclosed herein includes laminin, entactin, type IV collagen and heparin sulfate proteoglycans. In certain embodiments, the natural hydrogel includes extracellular matrix (ECM) secreted and/or derived from epithelial cells, endothelial cells, wall endodermal-like cells and/or connective tissue cells.

In certain embodiments, the hydrogel is a synthetic hydrogel. Non-limiting examples of synthetic hydrogels include synthetic polymers such as ProNectin (Sigma Z378666), polyethylene glycol (PEG), poly(hydroxyethyl methacrylate), poly(ethyleneimine), and polyvinyl alcohol (PVA). Additional non-limiting examples of synthetic hydrogels and polymers of such synthetic hydrogels are disclosed in Unal and West (2020) Bioconjugate Chem. 31(10):2253-2271; and Madduma-Bandarage and Madihally (2020) J. of Applied Polymer Science 138(19):e50376, each of which is incorporated herein by reference in its entirety.

In certain embodiments, the hydrogels disclosed herein do not include alginate.

In some embodiments, the hydrogel can be a commercially available ECM. Non-limiting examples of commercially available ECM include ECM proteins and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. In some embodiments, the ECM is MATRIGEL™ (BD Biosciences), which includes laminin, entactin, and collagen IV. In some embodiments, the ECM is basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of BME is CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which includes laminin, entactin, collagen IV, and heparin sulfate proteoglycans.

In some embodiments, the hydrogel has a protein concentration, such as a glycoprotein concentration, greater than about 0.7 mg/ml. In some embodiments, the hydrogel has a protein concentration, such as a glycoprotein concentration, greater than about 1 mg/ml. In some embodiments, the hydrogel has a protein concentration, such as a glycoprotein concentration, greater than about 2 mg/ml. In some embodiments, the hydrogel has a protein concentration, such as a glycoprotein concentration, greater than about 3 mg/ml. In some embodiments, the hydrogel has a protein concentration greater than about 4 mg/ml. In some embodiments, the hydrogel has a protein concentration greater than about 5 mg/ml. In some embodiments, the hydrogel has a protein concentration of greater than about 6 mg/ml, greater than about 7 mg/ml, greater than about 8 mg/ml, greater than about 9 mg/ml, or greater than about 10 mg/ml. In some embodiments, the hydrogel has a protein concentration of about 0.7 mg/ml to about 10 mg/ml. In some embodiments, the hydrogel has a protein concentration of about 1 mg/ml to about 10 mg/ml. In some embodiments, the hydrogel has a protein concentration of about 5 mg/ml to about 10 mg/ml. In some embodiments, the hydrogel has a protein concentration of about 6 mg/ml to about 10 mg/ml. In some embodiments, the hydrogel has a protein concentration of not less than 0.7 mg/ml. In some embodiments, the hydrogel has a protein concentration of not less than 1 mg/ml. In some embodiments, the hydrogel has a protein concentration of not less than 5 mg/ml. In some embodiments, the hydrogel has a protein concentration of not less than 6 mg/ml.

In certain embodiments, the hydrogel comprises greater than about 1 w/v % of a BME component, an ECM component, or a polymer. For example, but not limited to, the hydrogel comprises, in w/v%, greater than about 1 w/v%, greater than about 1.5 w/v%, greater than about 2 w/v%, greater than about 2.5 w/v%, greater than about 3 w/v%, greater than about 3.5 w/v%, greater than about 4 w/v%, greater than about 4.5 w/v%, greater than about 5 w/v%, greater than about 5.5 w/v%, greater than about 6 w/v%, greater than about 6.5 w/v%, greater than about 7 w/v%, greater than about 7.5 w/v%, greater than about 8 w/v%, greater than about 8.5 w/v%, greater than about 9 w/v%, greater than about 9.5 w/v%, greater than about 10 w/v%, greater than about 10.5 w/v%, greater than about %, greater than about 11 w/v %, greater than about 11.5 w/v %, greater than about 12 w/v %, greater than about 12.5 w/v %, greater than about 13 w/v %, greater than about 13.5 w/v %, greater than about 14 w/v %, greater than about 14.5 w/v %, greater than about 15 w/v %, greater than about 15.5 w/v %, greater than about 16 w/v %, greater than about 16.5 w/v %, greater than about 17 w/v %, greater than about 17.5 w/v %, greater than about 18 w/v %, greater than about 18.5 w/v %, greater than about 19 w/v %, greater than about 19.5 w/v %, or greater than about 20 w/v % of a BME component, an ECM component, or a polymer. In some embodiments, the hydrogel comprises about 1 w/v % to about 10 w/v % of a BME component, an ECM component, or a polymer. In some embodiments, the hydrogel comprises about 2 w/v % to about 10 w/v % of a BME component, an ECM component, or a polymer. In some embodiments, the hydrogel comprises about 5 w/v % to about 10 w/v % of a BME component, an ECM component, or a polymer.

In certain embodiments, the hydrogel has a storage modulus G' that is equal to or greater than the loss modulus G''.

In some embodiments, the hydrogel comprising the tissue-derived epithelial organoids and suspended in the culture medium has a geometry. In some embodiments, the geometry of the hydrogel has a length, width, and/or diameter greater than about 0.1 mm. In some embodiments, the geometry of the hydrogel has a length greater than about 0.1 mm. In some embodiments, the geometry of the hydrogel has a width greater than about 0.1 mm. In some embodiments, the geometry of the hydrogel has a diameter greater than about 0.1 mm. For example, but not limited to, the geometric shape of the hydrogel has a diameter greater than about 0.5 mm, greater than about 1 mm, greater than about 1.5 mm, greater than about 2 mm, greater than about 2.5 mm, greater than about 3 mm, greater than about 3.5 mm, greater than about 4 mm, greater than about 4.5 mm, greater than about 5 mm, greater than about 5.5 mm, greater than about 6 mm, greater than about 6.5 mm, greater than about 7.5 mm, greater than about 8 mm, greater than about 8.5 mm, greater than about 9 mm, greater than about 9.5 mm, greater than about 10 mm, greater than about 10.5 mm, greater than about 11 mm, greater than about 11.5 mm, greater than about 12 mm, greater than about 12.5 mm, greater than about 13 mm, greater than about 13.5 mm, greater than about 14 mm, greater than about 15 mm, greater than about 16 mm, greater than about 17 mm, greater than about 18 mm, greater than about 19 mm, greater than about 20 mm, greater than about 21 mm, greater than about 22 mm, greater than about 23 mm, greater than about 24 mm, greater than about 25 mm, greater than about 26 mm, greater than about 27 mm, greater than about 28 mm, greater than about 29 mm, greater than about 30 mm, greater than about 31 mm, greater than about 32 mm, greater than about 33 mm, greater than about 34 mm, greater than about 35 mm, greater than about 36 mm, greater than about 37 mm, greater than about 38 mm, greater than about 39 mm, 14.5 mm, greater than about 15 mm, greater than about 15.5 mm, greater than about 16 mm, greater than about 16.5 mm, greater than about 17.5 mm, greater than about 18 mm, greater than about 18.5 mm, greater than about 19 mm, greater than about 19.5 mm, greater than about 20 mm, greater than about 50 mm, greater than about 100 mm, greater than about 150 mm, greater than about 200 mm, greater than about 250 mm, greater than about 300 mm, greater than about 350 mm, greater than about 400 mm, greater than about 450 mm, greater than about 500 mm, greater than about 550 mm, greater than about 600 mm, greater than about 650 mm, greater than about 700 mm, greater than about 750 mm, greater than about 800 mm mm, greater than about 850 mm, greater than about 900 mm, greater than about 950 mm, or greater than about 1,000 mm in length, width, and/or diameter.

In certain embodiments, the geometric shape of the hydrogel has a length, width, and/or diameter of about 0.1 mm to about 1000 mm, e.g. , about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm, or about 1 mm to about 50 mm. In some embodiments, the geometric shapes of the hydrogel have a length, width, and/or diameter of about 0.1 mm to about 20.0 mm. In some embodiments, the geometric shapes of the hydrogel have a length of about 0.1 mm to about 20.0 mm. In some embodiments, the geometric shapes of the hydrogel have a width of about 0.1 mm to about 20.0 mm. In some embodiments, the geometric shapes of the hydrogel have a diameter of about 0.1 mm to about 20.0 mm. For example, but not limited to, the geometric shape of the hydrogel has a diameter of about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.5 mm to about 20 mm, about 1 mm to about 20 mm, about 2 mm to about 20 mm, about 3 mm to about 20 mm, about 4 mm to about 20 mm, about 5 mm to about 20 mm, about 6 mm to about 20 mm, about 7 mm to about 20 mm, about 8 mm to about 20 mm, about 9 mm to about 20 mm, about 10 mm to about 20 mm, about 11 mm to about 20 mm, about 12 mm to about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm In some embodiments, the geometric shape of the hydrogel has a length, width, and/or diameter of about 0.1 mm to about 1 mm. In some embodiments, the geometric shape of the hydrogel has a length, width, and/or diameter of about 0.1 mm to about 4 mm. In some embodiments, the geometric shape of the hydrogel has a length, width, and/or diameter of about 0.1 mm to about 5 mm. In some embodiments, the geometric shape of the hydrogel has a length, width, and/or diameter of about 1 mm to about 20 mm. In some embodiments, the geometric shape of the hydrogel has a length, width, and/or diameter of about 1 mm to about 10 mm. In some embodiments, the geometric shape of the hydrogel has a length, width, and/or diameter of about 1 mm to about 5 mm. In some embodiments, the geometric shape of the hydrogel has a length, width, and/or diameter of about 1 mm to about 4 mm.

In some embodiments, the geometry of the hydrogel suspended in the culture medium is a droplet, for example , as shown in FIG. 6 . In certain embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 20 mm, e.g. , about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 20 mm, about 1 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, or about 1 mm to about 15 mm. In some embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 1 mm. In some embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 4 mm. In some embodiments, the hydrogel droplets have a diameter of about 1 mm to about 20 mm. In some embodiments, the hydrogel droplets have a diameter of about 1 mm to about 10 mm. In some embodiments, the hydrogel droplets have a diameter of about 1 mm to about 4 mm. In some embodiments, each hydrogel droplet comprises about 1 or more tissue-derived epithelial organoids, e.g. , about 2 or more, about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, or about 10,000 or more tissue-derived epithelial organoids.

In some embodiments, the hydrogel has a filamentary structure, for example , as shown in FIG6 . In some embodiments, the filamentary structure has a linear, serpentine, or spiral shape. In some embodiments, the filamentary structure has a linear shape. In some embodiments, the filamentary structure has a serpentine shape. In some embodiments, the filamentary structure has a spiral shape. In certain embodiments, the filamentous structure has a length and/or width of about 0.1 mm to about 1000 mm, e.g. , about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm, or about 1 mm to about 50 mm. In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 20 mm, for example , about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 20 mm, about 1 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, or about 1 mm to about 15 mm. In some embodiments, the filamentous structure has a length and/or width of about 0.1 mm to about 4 mm. In some embodiments, the filamentous structure has a length of about 0.1 mm to about 1 mm. In some embodiments, the filamentous structure has a length of about 0.1 mm to about 4 mm. In some embodiments, the filamentous structure has a length of about 1 mm to about 20 mm. In some embodiments, the filamentous structure has a length of about 1 mm to about 10 mm. In some embodiments, the filamentous structure has a length of about 1 mm to about 4 mm. In some embodiments, the filamentous structure has a width of about 0.1 mm to about 1 mm. In some embodiments, the filamentous structure has a width of about 0.1 mm to about 4 mm. In some embodiments, the filamentary structure has a width of about 1 mm to about 20 mm. In some embodiments, the filamentary structure has a width of about 1 mm to about 10 mm. In some embodiments, the filamentary structure has a width of about 1 mm to about 4 mm.

In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 20 mm, for example , about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 20 mm, about 1 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, or about 1 mm to about 15 mm. In some embodiments, the filamentary structure has a diameter of about 0.1 mm to about 20 mm. In some embodiments, the filamentary structure has a diameter of about 0.1 mm to about 10 mm. In some embodiments, the filamentary structure has a diameter of about 0.1 mm to about 5 mm. In some embodiments, the filamentary structure has a diameter of about 1 mm to about 20 mm. In some embodiments, the filamentary structure has a diameter of about 1 mm to about 10 mm. In some embodiments, the filamentary structure has a diameter of about 1 mm to about 5 mm.

In certain embodiments, each filamentous structure comprises about 1 or more tissue-derived epithelial organoids, e.g. , about 2 or more, about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, or about 10,000 or more tissue-derived epithelial organoids.

In some embodiments, the composition includes any suitable cell culture medium. In some embodiments, the cell culture medium contains components that are important for supporting the maintenance of cultured cells. In some embodiments, the cell culture medium used in the present disclosure may be a nutrient solution, which includes standard cell culture ingredients, such as but not limited to amino acids, vitamins, inorganic salts, carbon energy ( e.g. , glucose) and buffer. In some embodiments, the culture medium is a differentiation medium. In some embodiments, the culture medium is a growth medium. In some embodiments, the culture medium is a stem cell promotion medium. Non-limiting examples of cell culture mediums are provided in the examples, such as organoid growth medium.

In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:1 or greater. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:1 to about 1:100. In certain embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:5 to about 1:100, about 1:10 to about 1:100, about 1:15 to about 1:100, about 1:20 to about 1:100, about 1:25 to about 1:100, about 1:30 to about 1:100, about 1:35 to about 1:100, about 1:40 to about 1:100, about 1:45 to about 1:100, about 1:45 to about 1:100, about 1:45 to about 1:100, about 1:45 to about 1:100, about 1:50 to about 1:100, about 1:55 to about 1:100, about 1:60 to about 1:100, about 1:65 to about 1:100, about 1:70 to about 1:100, about 1:75 to about 1:100, about 1:80 to about 1:100, about 1:85 to about 1:100, about 1:90 to about 1:100, about 1:95 to about 1:100, 1:5 to about 1:50, about 1:10 to about 1:50, about 1:15 to about 1:50, about 1:20 to about 1:50, about 1:25 to about 1:50, about 1:30 to about 1:50, about 1:35 to about 1:50, about 1:40 to about 1:50, about 1:45 to about 1:50, about 1:1 to about 1:95, about 1:1 to about 1:90, about 1:1 to about 1:85, about 1:1 to about 1:80, about 1:1 to about 1:75, about 1:1 to about 1:70, about 1:1 to about 1:65, about 1:1 to about 1:60, about 1:1 to about 1:55, about 1:1 to about 1:50, about 1:1 to about 1:45, about 1:1 to about 1:40, about 1:1 to about 1:35, about 1:1 to about 1:30, about 1:1 to about 1:35, about 1:1 to about 1:30, about 1:1 to about 1:25, about 1:1 to about 1:20, about 1:1 to about 1:15, about 1:1 to about 1:10, about 1:1 to about 1:5, about 1:5 In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:1 to about 1:50. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:2 to about 1:50. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:1 to about 1:20. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:1 to about 1:15. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:1. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:2. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:5. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:10, for example , as shown in Example 8. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:20. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:30. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:40. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:50. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:60. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:70. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:80. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:90. In some embodiments, the ratio of the volume of hydrogel to the volume of the culture medium in the composition (volume ratio) is about 1:100.

In certain embodiments, the tissue-derived epithelial organoids are more uniform in size compared to a reference tissue-derived epithelial organoid ( e.g. , a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate). In certain embodiments, the tissue-derived epithelial organoids produced by the methods of the present disclosure are more uniform in size compared to reference tissue-derived epithelial organoids due to differences in nutrient utilization as described in Example 1. For example, but not limited to, tissue-derived epithelial organoids produced by the methods of the present disclosure are more uniform in size across the width of a suspended culture droplet ( e.g. , a suspended hydrogel droplet) than a reference tissue-derived epithelial organoid ( e.g. , a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate). In certain embodiments, the reference tissue-derived epithelial organoid is produced in a hydrogel dome as disclosed in Example 1.

In certain embodiments, a tissue-derived epithelial organoid ( e.g. , a population of tissue-derived epithelial organoids) of the present disclosure expresses a marker at a different ( e.g. , higher or lower) level than a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids). For example, but not limited to, a tissue-derived epithelial organoid ( e.g. , a population of tissue-derived epithelial organoids) of the present disclosure expresses a marker at a higher level than a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids). Alternatively or additionally, in some embodiments, the tissue-derived epithelial organoids ( e.g. , a population of tissue-derived epithelial organoids) of the present disclosure express a marker at a lower level compared to a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids). In some embodiments, the marker is a stem cell and/or proliferation marker, for example , a gene associated with stem cells and/or proliferation, as shown in FIG5A . For example, but not limited to, the stem cell and/or proliferation marker is MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1 and/or CD44. In some embodiments, the marker is a differentiation marker, such as a gene associated with differentiation, as shown in FIG5B . For example, but not limited to, the differentiation marker is keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and/or smooth muscle actin (SMA). In some embodiments, the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. For example, but not limited to, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid produced in a hydrogel dome as disclosed in Example 1.

In certain embodiments, the tissue-derived epithelial organoid is a gastrointestinal organoid, and the differentially expressed markers in the gastrointestinal organoids disclosed herein are MKI67, LGR5, SOX9, CD44, MUC2, MUC5B, TFF3, KRT20, FABP1, ALPI and/or CEACAM7.

In certain embodiments, the tissue-derived epithelial organoid is a mammary organoid, and the differentially expressed markers in the mammary organoids of the present disclosure are EpCAM, CD49f, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and/or smooth muscle actin (SMA).

In certain embodiments, the tissue-derived epithelial organoid is a pancreatic organoid, and the differentially expressed markers in the pancreatic organoids of the disclosure are CD133, LGR5, PDX1, SOX9, ALDH1A1, NEUROG3, NKX6.1, keratin 19 (KRT19), MUC1, INS, GCG and/or AMY.

In certain embodiments, the tissue-derived epithelial organoid is a liver organoid, and the differentially expressed markers in the liver organoids of the disclosure are LGR5, ALB, CYP3A4, HNF4A, KRT19, KRT7 and/or SOX9.

In certain embodiments, stem cell and/or proliferation markers are expressed at higher levels by a population of tissue-derived epithelial organoids of the present disclosure ( e.g. , a population of tissue-derived epithelial organoids in a composition of the present disclosure) compared to a population of reference tissue-derived epithelial organoids. Non-limiting examples of stem cell and/or proliferation markers include MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. In certain embodiments, the stem cell and/or proliferation marker is selected from the group consisting of MKI67, ASCL2, LGR5, SOX9, SMOC2, CD44, and combinations thereof. In some embodiments, the stem cell and/or proliferation marker is MKI67. In some embodiments, the stem cell and/or proliferation marker is ASCL2. In some embodiments, the stem cell and/or proliferation marker is LGR5. In some embodiments, the stem cell and/or proliferation marker is SOX9. In some embodiments, the stem cell and/or proliferation marker is SMOC2. In some embodiments, the stem cell and/or proliferation marker is CD44. In some embodiments, the stem cell and/or proliferation marker is EpCAM. In some embodiments, the stem cell and/or proliferation marker is CD49f. In some embodiments, the stem cell and/or proliferation marker is CD133. In some embodiments, the stem cell and/or proliferation marker is ALDH1A1. In some embodiments, the stem cell and/or proliferation marker is NEUROG3. In some embodiments, the stem cell and/or proliferation marker is NKX6.1. In some embodiments, the stem cell and/or proliferation marker is PDX1. In some embodiments, the stem cell and/or proliferation marker is BMI1. In certain embodiments, the expression level of a stem cell and/or proliferation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 110% higher, at least 120% higher, at least 130% higher, at least 140% higher, at least 150% higher, at least 160% higher, at least 170% higher, at least 180% higher, at least 190% higher, at least 200% higher, at least 210% higher, at least 220% higher, at least 230% higher, at least 240% higher, at least 250% higher, at least 260% higher, at least 270% higher, at least 280% higher, at least 290% higher In some embodiments, the expression level of stem cells and/or proliferation markers in a population of epithelial organoids derived from a reference tissue is at least 50% higher than the expression level of stem cells and/or proliferation markers in a population of epithelial organoids derived from a reference tissue. In some embodiments, the expression level of stem cells and/or proliferation markers in a population of epithelial organoids derived from a reference tissue is at least 100% higher than the expression level of stem cells and/or proliferation markers in a population of epithelial organoids derived from a reference tissue. In certain embodiments, the expression level of stem cells and/or proliferation markers in a population of epithelial organoids derived from a tissue of the present disclosure is at least 200% higher than the expression level of stem cells and/or proliferation markers in a population of epithelial organoids derived from a reference tissue. In certain embodiments, the expression level of stem cells and/or proliferation markers in a population of epithelial organoids derived from a tissue of the present disclosure is at least 300% higher than the expression level of stem cells and/or proliferation markers in a population of epithelial organoids derived from a reference tissue.

In certain embodiments, a differentiation marker is expressed at a lower level by a population of tissue-derived epithelial organoids of the disclosure ( eg , a population of tissue-derived epithelial organoids in a composition of the disclosure) compared to a population of reference tissue-derived epithelial organoids. Non-limiting examples of differentiation markers include keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. In certain embodiments, the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, and combinations thereof. In some embodiments, the differentiation marker is keratin 20 (KRT20). In some embodiments, the differentiation marker is FABP1. In some embodiments, the differentiation marker is MUC2. In some embodiments, the differentiation marker is MUC5B. In some embodiments, the differentiation marker is TFF3. In some embodiments, the differentiation marker is ALPI. In some embodiments, the differentiation marker is SI. In some embodiments, the differentiation marker is CEACAM7. In some embodiments, the differentiation marker is keratin 19 (KRT19). In some embodiments, the differentiation marker is keratin 7 (KRT7). In some embodiments, the differentiation marker is SOX9. In some embodiments, the differentiation marker is MUC1. In some embodiments, the differentiation marker is INS. In some embodiments, the differentiation marker is GCG. In some embodiments, the differentiation marker is AMY. In some embodiments, the differentiation marker is ALB. In some embodiments, the differentiation marker is CYP3A4. In some embodiments, the differentiation marker is HNF4A. In some embodiments, the differentiation marker is cytokeratin 8 (K8). In some embodiments, the differentiation marker is cytokeratin 18 (K18). In some embodiments, the differentiation marker is cytokeratin 5 (K5). In some embodiments, the differentiation marker is cytokeratin 14 (K14). In some embodiments, the differentiation marker is smooth muscle actin (SMA). In some embodiments, the differentiation marker is MUC5AC. In some embodiments, the differentiation marker is MUC6. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 100% lower, at least 110% lower, at least 120% lower, at least 130% lower, at least 140% lower, at least 150% lower, at least 160% lower, at least 170% lower, at least 180% lower, at least 190% lower, at least 200% lower, at least 210% lower, at least 220% lower, at least 230% lower, at least 240% lower, at least 200% lower, at least 250% lower, at least 260% lower, at least 270% lower, at least 280% lower, at least 290% lower, or at least 300% lower than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of differentiation markers in a population of epithelial organoids derived from a tissue of the present disclosure is at least 50% lower than the expression level of differentiation markers in a population of epithelial organoids derived from a reference tissue. In certain embodiments, the expression level of differentiation markers in a population of epithelial organoids derived from a tissue of the present disclosure is at least 100% lower than the expression level of differentiation markers in a population of epithelial organoids derived from a reference tissue. In certain embodiments, the expression level of differentiation markers in a population of epithelial organoids derived from a tissue of the present disclosure is at least 200% lower than the expression level of differentiation markers in a population of epithelial organoids derived from a reference tissue. In certain embodiments, the expression level of a differentiation marker in a population of epithelial organoids derived from a tissue of the present disclosure is at least 300% lower than the expression level of a differentiation marker in a population of epithelial organoids derived from a reference tissue. III. Methods of Generating Organoids

The present disclosure provides methods for generating tissue-derived epithelial organoids. In certain embodiments, the present disclosure provides methods for generating tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. The present disclosure further provides methods for generating suspension cultures of tissue-derived epithelial organoids. In certain embodiments, the methods described in Examples 1 and 4 to 8 and Figures 10 and 11 can be used to generate organoids.

In certain embodiments, the disclosure provides methods for producing tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, or trophoblastic lining organoids. In certain embodiments, the disclosure provides methods for producing tear gland organoids. In certain embodiments, the disclosure provides methods for producing tonsil organoids. In certain embodiments, the disclosure provides methods for producing salivary gland organoids. In certain embodiments, the disclosure provides methods for producing gastrointestinal organoids. In certain embodiments, the disclosure provides methods for producing thyroid organoids. In certain embodiments, the disclosure provides methods for producing lung organoids. In certain embodiments, the disclosure provides methods for producing mammary organoids. In certain embodiments, the disclosure provides methods for producing liver organoids. In certain embodiments, the disclosure provides methods for producing bile duct organoids. In certain embodiments, the disclosure provides methods for producing gastric organoids. In certain embodiments, the disclosure provides methods for producing kidney organoids. In certain embodiments, the disclosure provides methods for producing pancreatic organoids. In certain embodiments, the disclosure provides methods for producing endometrial organoids. In certain embodiments, the disclosure provides methods for producing fallopian tube organoids. In certain embodiments, the disclosure provides methods for producing cervical organoids. In certain embodiments, the disclosure provides methods for producing prostate organoids. In certain embodiments, the disclosure provides methods for producing bladder organoids. In certain embodiments, the disclosure provides methods for producing ovarian organoids. In certain embodiments, the disclosure provides methods for producing trophoblastic lining organoids. In certain embodiments, the disclosure provides methods for producing trophoblastic lining organoids.

In certain embodiments, the present disclosure provides methods for generating tissue-derived epithelial organoids selected from the group consisting of lung organoids, gastrointestinal organoids, liver organoids, pancreatic organoids, breast organoids, and combinations thereof.

In certain embodiments, the present disclosure provides methods for producing gastrointestinal organoids. In certain embodiments, the present disclosure provides methods for producing intestinal organoids. For example, but not limited to, the intestinal organoids are colon or ileal organoids. In certain embodiments, the intestinal organoids are colon organoids. In certain embodiments, the intestinal organoids are ileal organoids. In certain embodiments, the present disclosure provides methods for producing rectal organoids. In certain embodiments, the present disclosure provides methods for producing esophageal organoids. In certain embodiments, the present disclosure provides methods for producing oral maxillofacial organoids. In certain embodiments, the methods of the present disclosure can produce epithelial organoids from two or more different types of tissue sources, such as colon and ileal organoids.

In certain embodiments, the present disclosure provides methods for generating lung organoids.

In certain embodiments, the present disclosure provides methods for generating liver organoids.

In certain embodiments, the present disclosure provides methods for generating pancreatic organoids.

In certain embodiments, the present disclosure provides methods for generating mammary organoids.

In certain embodiments, a method for generating tissue-derived epithelial organoids comprises contacting tissue-derived epithelial stem cells with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells may be combined with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture.

In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises at least two or more tissue-derived epithelial stem cells. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises at least about 10 or more tissue-derived epithelial stem cells, at least about 100 or more tissue-derived epithelial stem cells, at least about 1,000 or more tissue-derived epithelial stem cells, at least about 10,000 or more tissue-derived epithelial stem cells, at least about 100,000 or more tissue-derived epithelial stem cells, at least about 100,000 or more tissue-derived epithelial stem cells, at least about 1,000,000 or more tissue-derived epithelial stem cells, at least about 10 ... The invention relates to an epithelial cell transplantation method comprising at least about 100,000,000 or more tissue-derived epithelial stem cells, or at least about 1,000,000,000 or more tissue-derived epithelial stem cells. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises at least about 10 or more tissue-derived epithelial stem cells/ml hydrogel, at least about 100 or more tissue-derived epithelial stem cells/ml hydrogel, at least about 1,000 or more tissue-derived epithelial stem cells/ml hydrogel, at least about 10 ... In some embodiments, the plurality of tissue-derived epithelial stem cells comprises at least about 10,000,000 or more tissue-derived epithelial stem cells/ml hydrogel, at least about 100,000,000 or more tissue-derived epithelial stem cells/ml hydrogel, or at least about 1,000,000,000 or more tissue-derived epithelial stem cells/ml hydrogel. In some embodiments, the plurality of tissue-derived epithelial stem cells comprises at least about 10,000 or more tissue-derived epithelial stem cells/ml hydrogel.

In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1×10 ⁴ to about 1×10 ¹⁰ tissue-derived epithelial stem cells/ml hydrogel. For example, but not limited to, the plurality of tissue-derived epithelial stem cells include about 1 × 10 ⁴ to about 1 × 10 ⁹ tissue-derived epithelial stem cells/ml hydrogel, about 1 × 10 4 to about 1 × 10 ⁸ tissue-derived epithelial stem cells/ml hydrogel, about 1 × 10 ⁴ to about 1 × 10 ⁷ tissue-derived epithelial stem cells/ml hydrogel, about 1 × 10 ⁴ to about 1 × 10 ⁶ tissue-derived epithelial stem cells/ml hydrogel, about 1 × 10 ⁴ to about 1 × 10 ⁵ tissue-derived epithelial stem cells/ml hydrogel, about 1 × 10 ⁵ to about 1 × 10 6 ... 10 ¹⁰ tissue-derived epithelial stem cells/ml hydrogel, about 1 × 10 ⁶ to about 1 × 10 ¹⁰ tissue-derived epithelial stem cells/ml hydrogel, about 1 × 10 ⁷ to about 1 × 10 ¹⁰ tissue-derived epithelial stem cells/ml hydrogel, about 1 × 10 ⁸ to about 1 × 10 ¹⁰ tissue-derived epithelial stem cells/ml hydrogel, about 1 × 10 ⁹ to about 1 × 10 ¹⁰ tissue-derived epithelial stem cells/ml hydrogel, about 1 × 10 ⁵ to about 1 × 10 ⁷ tissue-derived epithelial stem cells/ml hydrogel, or about 1 × 10 ⁵ to about 1 × 10 ⁸ tissue-derived epithelial stem cells/ml hydrogel. In certain embodiments, the plurality of tissue-derived epithelial stem cells includes about 1 × 10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1 × 10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. In certain embodiments, the plurality of tissue-derived epithelial stem cells includes about 1 × 10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1 × 10 ⁶ tissue-derived epithelial stem cells/ml hydrogel.

In some embodiments, the method may further include suspending the hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture. In some embodiments, suspending the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium includes immersing a dispensing device containing the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium and dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the culture medium. In some embodiments, the hydrogel-tissue derived epithelial stem cell mixture can be repeatedly distributed into the culture medium to produce a plurality of separated hydrogel-tissue derived epithelial stem cell mixtures suspended in the culture medium. In some embodiments, the dispensing device can be any device that allows the mixture to be transported. In some embodiments, the transport device distributes the mixture in an accurate and controlled manner. Non-limiting examples of the dispensing device include a pipette, a dropper, and a syringe. In some embodiments, the dispensing device can be manually operated or automatically operated. For example, but not limited to, the dispensing device can be automatically operated. In some embodiments, the dispensing device can be an automated liquid handler. In some embodiments, the dispensing device can be a liquid handling robot.

In certain embodiments, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be at least about 1 µl. For example, but not limited to, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be at least about 5 µl, at least about 10 µl, at least about 50 µl, at least about 100 µl, at least about 500 µl, at least about 1 ml, at least about 10 ml, at least about 50 ml, at least about 100 ml, at least about 500 ml, at least about 1 L, at least about 1.5 L, at least about 2 L, at least about 5 L, or at least about 10 L. In certain embodiments, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium may be about 1 µl to about 1 L. In certain embodiments, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium may be about 1 µl to about 1 ml. For example, but not limited to, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium may be about 1 µl to about 900 µl, about 1 µl to about 800 µl, about 1 µl to about 700 µl, about 1 µl to about 600 µl, about 1 µl to about 500 µl, about 1 µl to about 400 µl, about 1 µl to about 300 µl, about 1 µl to about 200 µl, about 1 µl to about 100 µl, about 1 µl to about 10 µl, about 1 µl to about 900 µl, about 10 µl to about 1 ml, about 100 µl to about 1 ml, about 200 µl to about 1 ml, or about 100 µl to about 1 ml. ml, about 300 µl to about 1 ml, about 400 µl to about 1 ml, about 500 µl to about 1 ml, about 600 µl to about 1 ml, about 700 µl to about 1 ml, about 800 µl to about 1 ml, about 900 µl to about 1 ml, about 1 µl to about 50 µl, about 5 µl to about 20 µl, about 10 µl to about 100 µl, about 10 µl to about 500 µl, about 100 µl to about 200 µl, or about 100 µl to about 500 µl. In certain embodiments, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium may be about 1 µl to about 100 µl.

In some embodiments, the hydrogel solidifies when in contact with the culture medium. For example, but not limited to, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 10°C. For example, but not limited to, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 15°C, greater than about 20°C, greater than about 25°C, greater than about 30°C, greater than about 35°C, greater than about 40°C, greater than about 45°C, or greater than about 50°C. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 25°C. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that cures at a temperature greater than about 30° C. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that cures at a temperature greater than about 35° C. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that cures at a temperature greater than about 40° C. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that cures at a temperature greater than about 45° C. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature of about 25°C to about 50°C. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature of about 30°C to about 50°C. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature of about 25°C to about 40°C, such as about 37°C. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature of about 30°C to about 40°C, such as about 37°C.

In some embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 25°C or less before contacting the culture medium. In some embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 20°C or less before contacting the culture medium. In some embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 15°C or less before contacting the culture medium. In some embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 10°C or less before contacting the culture medium. In some embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 5°C or less before contacting the culture medium. In some embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 25°C, about 24°C, about 23°C, about 22°C, about 21°C, about 20°C, about 19°C, about 18°C, about 17°C, about 16°C, about 15°C, about 14°C, about 13°C, about 12°C, about 11°C, about 10°C, about 9°C, about 8°C, about 7°C, about 6°C, about 5°C, about 4°C, about 3°C, or about 2°C before contacting the culture medium. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture before contacting the culture medium is about 2° C. to about 25° C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture before contacting the culture medium is about 2° C. to about 20° C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture before contacting the culture medium is about 2° C. to about 15° C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture before contacting the culture medium is about 2° C. to about 10° C.

In some embodiments, the hydrogel may include any solidified material. In some embodiments, the hydrogel is a synthetic hydrogel, a natural hydrogel, or a combination thereof. In some embodiments, the hydrogel is a synthetic hydrogel. In some embodiments, the hydrogel is a natural hydrogel. In some embodiments, the hydrogel may be a mixture of a synthetic hydrogel and a natural hydrogel. In some embodiments, the hydrogel does not include chemically cross-linked proteins and/or polymers.

In some embodiments, the hydrogel is a natural hydrogel. In some embodiments, the natural hydrogel includes one or more naturally occurring components. For example, but not limited to, the natural hydrogel may include one or more proteins, such as glycoproteins and/or polysaccharides. Non-limiting examples of glycoproteins include collagen ( e.g. , type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen and/or type XII collagen), fibronectin, entactin, tenascin, vitronectin, reticular protein, hyaluronic acid and laminin. In certain embodiments, the natural hydrogel may further include one or more components, such as but not limited to polysaccharides, water and/or elastin. In certain embodiments, the natural hydrogel disclosed herein includes laminin, entactin and type IV collagen. In certain embodiments, the natural hydrogel disclosed herein includes laminin, entactin, type IV collagen and heparin sulfate proteoglycans. In certain embodiments, the natural hydrogel includes extracellular matrix (ECM) secreted and/or derived from epithelial cells, endothelial cells, wall endodermal-like cells and/or connective tissue cells.

In certain embodiments, the hydrogel is a synthetic hydrogel. Non-limiting examples of synthetic hydrogels include synthetic polymers such as ProNectin (Sigma Z378666), polyethylene glycol (PEG), poly(hydroxyethyl methacrylate), poly(ethyleneimine), and polyvinyl alcohol (PVA). Additional non-limiting examples of synthetic hydrogels and polymers of such synthetic hydrogels are disclosed in Unal and West (2020) Bioconjugate Chem. 31(10):2253-2271; and Madduma-Bandarage and Madihally (2020) J. of Applied Polymer Science 138(19):e50376, each of which is incorporated herein by reference in its entirety.

In certain embodiments, the hydrogels disclosed herein do not include alginate.

In some embodiments, the hydrogel can be a commercially available ECM. Non-limiting examples of commercially available ECM include ECM proteins and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. In some embodiments, the ECM is MATRIGEL™ (BD Biosciences), which includes laminin, entactin, and collagen IV. In some embodiments, the ECM is basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of BME is CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which includes laminin, entactin, collagen IV, and heparin sulfate proteoglycans.

In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration, e.g. , glycoprotein concentration, greater than about 0.7 mg/ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration, e.g. , glycoprotein concentration, greater than about 1 mg/ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration, e.g., glycoprotein concentration, greater than about 2 mg/ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration, e.g. , glycoprotein concentration, greater than about 3 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration greater than about 4 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration greater than about 5 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration greater than about 6 mg/ml, greater than about 7 mg/ml, greater than about 8 mg/ml, greater than about 9 mg/ml, or greater than about 10 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of about 0.7 mg/ml to about 10 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of about 1 mg/ml to about 10 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of about 5 mg/ml to about 10 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of about 6 mg/ml to about 10 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration of not less than 0.7 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration of not less than 1 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration of not less than 5 mg/ml. In some embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration of not less than 6 mg/ml.

In certain embodiments, the hydrogel comprises greater than about 1 w/v % of a BME component, an ECM component, or a polymer. For example, but not limited to, the hydrogel comprises, in w/v%, greater than about 1 w/v%, greater than about 1.5 w/v%, greater than about 2 w/v%, greater than about 2.5 w/v%, greater than about 3 w/v%, greater than about 3.5 w/v%, greater than about 4 w/v%, greater than about 4.5 w/v%, greater than about 5 w/v%, greater than about 5.5 w/v%, greater than about 6 w/v%, greater than about 6.5 w/v%, greater than about 7 w/v%, greater than about 7.5 w/v%, greater than about 8 w/v%, greater than about 8.5 w/v%, greater than about 9 w/v%, greater than about 9.5 w/v%, or greater than about 10 w/v%, greater than about 10.5 w/v%, greater than about %, greater than about 11 w/v %, greater than about 11.5 w/v %, greater than about 12 w/v %, greater than about 12.5 w/v %, greater than about 13 w/v %, greater than about 13.5 w/v %, greater than about 14 w/v %, greater than about 14.5 w/v %, greater than about 15 w/v %, greater than about 15.5 w/v %, greater than about 16 w/v %, greater than about 16.5 w/v %, greater than about 17 w/v %, greater than about 17.5 w/v %, greater than about 18 w/v %, greater than about 18.5 w/v %, greater than about 19 w/v %, greater than about 19.5 w/v %, or greater than about 20 w/v % of a BME component, an ECM component, or a polymer. In certain embodiments, the hydrogel comprises about 1 w/v % to about 10 w/v % of a BME component, an ECM component, or a polymer. In certain embodiments, the hydrogel comprises about 2 w/v % to about 10 w/v % of a BME component, an ECM component, or a polymer. In certain embodiments, the hydrogel comprises about 5 w/v % to about 10 w/v % of a BME component, an ECM component, or a polymer.

In certain embodiments, the hydrogel has a storage modulus G' that is equal to or greater than the loss modulus G''.

In some embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium (volume ratio) is about 1:1 to about 1:50. In some embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium (volume ratio) is about 1:2 to about 1:50. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium is about 1:5 to about 1:50, about 1:10 to about 1:50, about 1:15 to about 1:50, about 1:20 to about 1:50, about 1:25 to about 1:50, about 1:30 to about 1:50, about 1:35 to about 1:50, about 1:40 to about 1:50, about 1:45 to about 1:50, about 1:2 to about 1:45, about 1:2 to about 1:40, about 1:2 to about 1:35, about 1:2 to about 1:30, about 1:2 to about 1:35, about 1:2 to about 1:30, about 1:2 to about 1:25, about 1:2 to about 1:20, about 1:2 In some embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is about 1:2 to about 1:20. In some embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is about 1:2 to about 1:15. In some embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is about 1:2. In some embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is about 1:5. In some embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is about 1:10, for example , as shown in Example 8. In some embodiments, the ratio of hydrogel volume to culture medium volume is about 1:20. In some embodiments, the ratio of hydrogel volume to culture medium volume is about 1:30. In some embodiments, the ratio of hydrogel volume to culture medium volume is about 1:40. In some embodiments, the ratio of hydrogel volume to culture medium volume is about 1:50.

In some embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture is dispensed into a culture medium having a certain geometric shape. For example, but not limited to, the geometric shape of the suspended hydrogel-tissue derived epithelial stem cell mixture comprises a length, width, and/or diameter greater than about 0.1 mm. In some embodiments, the geometric shape of the suspended hydrogel-tissue derived epithelial stem cell mixture has a length greater than about 0.1 mm. In some embodiments, the geometric shape of the suspended hydrogel-tissue derived epithelial stem cell mixture has a width greater than about 0.1 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue-derived epithelial stem cell mixture has a diameter greater than about 0.1 mm. For example, but not limited to, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is greater than about 0.5 mm, greater than about 1 mm, greater than about 1.5 mm, greater than about 2 mm, greater than about 2.5 mm, greater than about 3 mm, greater than about 3.5 mm, greater than about 4 mm, greater than about 4.5 mm, greater than about 5 mm, greater than about 5.5 mm, greater than about 6 mm, greater than about 6.5 mm, greater than about 7.5 mm, greater than about 8 mm, greater than about 8.5 mm, greater than about 9 mm, greater than about 9.5 mm, greater than about 10 mm, greater than about 10.5 mm, greater than about 11 mm, greater than about 11.5 mm, greater than about 12 mm, greater than about 12.5 mm, greater than about 13 mm, greater than about 14 mm, greater than about 15 mm, greater than about 16 mm, greater than about 17 mm, greater than about 18 mm, greater than about 19 mm, greater than about 20 mm, greater than about 21 mm, greater than about 22 mm, greater than about 23 mm, greater than about 24 mm, greater than about 25 mm, greater than about 26 mm, greater than about 27 mm, greater than about 28 mm, greater than about 29 mm, greater than about 30 mm, greater than about 31 mm, greater than about 32 mm, greater than about 33 mm, greater than about 34 mm, greater than about 35 mm, greater than about 36 mm, greater than about 37 mm, greater than about 38 mm, greater than about 39 mm, greater than about 40 mm, greater than about 41 mm, greater than about 13 mm, greater than about 13.5 mm, greater than about 14 mm, greater than about 14.5 mm, greater than about 15 mm, greater than about 15.5 mm, greater than about 16 mm, greater than about 16.5 mm, greater than about 17.5 mm, greater than about 18 mm, greater than about 18.5 mm, greater than about 19 mm, greater than about 19.5 mm, greater than about 20 mm, greater than about 50 mm, greater than about 100 mm, greater than about 150 mm, greater than about 200 mm, greater than about 250 mm, greater than about 300 mm, greater than about 350 mm, greater than about 400 mm, greater than about 450 mm, greater than about 500 mm, greater than about 550 mm, greater than about 600 mm, greater than about 650 mm, greater than about 700 mm mm, greater than about 750 mm, greater than about 800 mm, greater than about 850 mm, greater than about 900 mm, greater than about 950 mm, or greater than about 1,000 mm in length, width, and/or diameter.

In certain embodiments, the geometry of the suspended hydrogel-tissue-derived epithelial stem cell mixture comprises a length, width, and/or diameter of about 0.1 mm to about 1000 mm, e.g. , about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm, or about 1 mm to about 50 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue-derived epithelial stem cell mixture comprises about 0.1 mm to about 20 mm, for example , about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 1 ... 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 21 mm to about 20 mm, about 22 mm to about 20 mm, about 23 mm to about 20 mm, about 24 mm to about 20 mm, about 25 mm to about 20 mm, about 26 mm to about 20 mm, about 27 mm to about 20 mm, about 28 mm to about 20 mm, about 29 mm to about 20 mm, about 30 mm to about 20 mm, about 31 mm to about 20 mm, about 32 mm to about 20 mm, about 33 mm to about 20 mm, about 34 mm to about 20 mm, about 35 mm to about 20 mm, about 36 mm to about 20 mm, about 37 mm to about 20 mm, about 38 mm to about 20 mm, about 39 mm to about 20 mm mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm in length, width, and/or diameter. In certain embodiments, the geometry of the suspended hydrogel-tissue-derived epithelial stem cell mixture comprises a length, width, and/or diameter of about 0.1 mm to about 4 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue-derived epithelial stem cell mixture has a length, width, and/or diameter of about 0.1 mm to about 1 mm. In some embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture has a length, width and/or diameter of about 0.1 mm to about 4 mm. In some embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture has a length, width and/or diameter of about 1 mm to about 20 mm. In some embodiments, the geometric shape of the suspended hydrogel-tissue derived epithelial stem cell mixture has a length, width and/or diameter of about 1 mm to about 10 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue-derived epithelial stem cell mixture has a length, width, and/or diameter of about 1 mm to about 4 mm.

In some embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is spherical or spherical. In some embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is a filamentous structure. In some embodiments, the filamentous structure has a linear, serpentine or spiral shape. In some embodiments, the filamentous structure has a linear shape. In some embodiments, the filamentous structure has a serpentine shape. In some embodiments, the filamentous structure has a spiral shape.

In some embodiments, the hydrogel-tissue derived epithelial stem cell mixture is suspended in a culture medium as a droplet ( see, e.g., FIG. 10 ). In some embodiments, the hydrogel droplet is larger than about 0.1 mm in diameter. In certain embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 20 mm, e.g. , about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 20 mm, about 1 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, or about 1 mm to about 15 mm. In some embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 4 mm. In some embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 1 mm. In some embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 4 mm. In some embodiments, the hydrogel droplets have a diameter of about 1 mm to about 20 mm. In some embodiments, the hydrogel droplets have a diameter of about 1 mm to about 10 mm. In some embodiments, the hydrogel droplets have a diameter of about 1 mm to about 4 mm. In certain embodiments, each hydrogel droplet comprises about 1 or more tissue-derived epithelial stem cells, e.g. , about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, about 10,000 or more, about 100,000 or more, about 200,000 or more, about 300,000 or more, about 400,000 or more, about 500,000 or more, about 600,000 or more, about 700,000 or more, about 800,000 or more, about 900,000 or more, about 1,000,000 or more, or about 2,000,000 or more. In some embodiments, the invention relates to epithelial stem cells of at least one tissue-derived epithelial cell. The epithelial stem cells may be about 1,000,000 or more, about 2,000,000 or more, about 3,000,000 or more, about 4,000,000 or more, about 5,000,000 or more, about 6,000,000 or more, about 7,000,000 or more, about 8,000,000 or more, about 9,000,000 or more, about 10,000,000 or more, about 100,000,000 or more, or about 1,000,000,000 or more tissue-derived epithelial stem cells.

In some embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture has a filamentous structure ( see, e.g., FIG. 11 ). In some embodiments, the filamentous structure has a length and/or width greater than about 0.1 mm. In some embodiments, the filamentous structure has a length greater than about 0.1 mm. In some embodiments, the filamentous structure has a width greater than about 0.1 mm. In certain embodiments, the filamentous structure has a length and/or width of about 0.1 mm to about 1000 mm, e.g. , about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm, or about 1 mm to about 50 mm. In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 20 mm, for example , about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 20 mm, about 1 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, or about 1 mm to about 15 mm. In some embodiments, the filamentous structure has a length and/or width of about 0.1 mm to about 4 mm. In some embodiments, the filamentous structure has a length of about 0.1 mm to about 1 mm. In some embodiments, the filamentous structure has a length of about 0.1 mm to about 4 mm. In some embodiments, the filamentous structure has a length of about 1 mm to about 20 mm. In some embodiments, the filamentous structure has a length of about 1 mm to about 10 mm. In some embodiments, the filamentous structure has a length of about 1 mm to about 4 mm. In some embodiments, the filamentous structure has a width of about 0.1 mm to about 1 mm. In some embodiments, the filamentous structure has a width of about 0.1 mm to about 4 mm. In some embodiments, the filamentary structure has a width of about 1 mm to about 20 mm. In some embodiments, the filamentary structure has a width of about 1 mm to about 10 mm. In some embodiments, the filamentary structure has a width of about 1 mm to about 4 mm.

In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 20 mm, for example , about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 20 mm, about 1 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, about 1 mm to about 20 mm, or about 1 mm to about 15 mm. In some embodiments, the filamentary structure has a diameter of about 0.1 mm to about 4 mm. In some embodiments, the filamentary structure has a diameter of about 0.1 mm to about 20 mm. In some embodiments, the filamentary structure has a diameter of about 0.1 mm to about 10 mm. In some embodiments, the filamentary structure has a diameter of about 0.1 mm to about 5 mm. In some embodiments, the filamentary structure has a diameter of about 1 mm to about 20 mm. In some embodiments, the filamentary structure has a diameter of about 1 mm to about 10 mm. In some embodiments, the filamentary structure has a diameter of about 1 mm to about 5 mm, for example , as disclosed in Example 8.

In certain embodiments, each filamentous structure comprises about 1 or more tissue-derived epithelial stem cells, for example , about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, about 10,000 or more, or about 100,000 or more, about 200,000 or more, about 300,000 or more, about 400,000 or more, about 500,000 or more, about 600,000 or more, about 700,000 or more, about 800,000 or more, about 900,000 or more, or about 1,000,000 or more. In some embodiments, the invention relates to epithelial stem cells of at least one tissue-derived epithelial cell. The epithelial stem cells may be about 1,000,000 or more, about 2,000,000 or more, about 3,000,000 or more, about 4,000,000 or more, about 5,000,000 or more, about 6,000,000 or more, about 7,000,000 or more, about 8,000,000 or more, about 9,000,000 or more, about 10,000,000 or more, about 100,000,000 or more, about 1,000,000,000 or more, or about 10,000,000,000 or more tissue-derived epithelial stem cells.

In some embodiments, the method may further include culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce tissue-derived epithelial organoids. In some embodiments, the cell culture medium contains components important for supporting the maintenance of tissue-derived epithelial stem cells and/or organoids. In some embodiments, the cell culture medium used in the present disclosure may be a nutrient solution, which includes standard cell culture ingredients, such as but not limited to amino acids, vitamins, inorganic salts, carbon energy ( e.g. , glucose) and buffer. In some embodiments, the culture medium is a stem cell promotion medium. In some embodiments, the medium is a cell growth medium. In some embodiments, the medium is a differentiation medium. Mediums known to support the growth of specific tissues and cell types are known in the art and can be used in conjunction with the subject matter disclosed herein. For example, but not limited to, examples of mediums that can be used in the present disclosure are disclosed in Calà et al., Front.Bioeng.Biotechnol.11:1058970 (2023) ( e.g. , Table 1 of Calà et al.), the contents of which are hereby incorporated by reference. In examples, such as Example 8, additional non-limiting examples of mediums used in the present disclosure are provided.

In some embodiments, the medium is present in a container. Non-limiting examples of containers include culture dishes, multi-well plates, conical tubes, containers, culture bags, bioreactors, or flasks. In some embodiments, the conical tube is a 50 ml conical tube. In some embodiments, the multi-well plate is a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate. In some embodiments, the container is a custom container. In some embodiments, the flask is a 25 ml flask, a 50 ml flask, a 250 ml flask, or a 600 ml flask. In some embodiments, the flask is a single flask or a hotel flask. In some embodiments, the container is composed of a material that minimizes the attachment of tissue-derived epithelial stem cells and/or hydrogel to the container surface. Alternatively or in addition, the container may include a surface coating that minimizes the attachment of tissue-derived epithelial stem cells and/or hydrogel to the container surface.

In some embodiments, the method may further include fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture to produce fragmented structures comprising the tissue-derived epithelial organoid. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture may include shearing the suspended hydrogel-tissue derived epithelial stem cell mixture to produce the fragmented structures, such as by pipetting a culture medium containing the suspended hydrogel-tissue derived epithelial stem cell mixture up and down. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture produces structures with shorter length and/or width. For example, but not limited to, fragmenting the filamentous structure produces a structure that is shorter in length, width, or both. In certain embodiments, the filamentous structure is fragmented to produce a structure that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% shorter in length, width, or both than the hydrogel-tissue-derived epithelial stem cell mixture (e.g., filamentous structure) that was initially extruded.

The presently disclosed subject matter further provides methods for producing suspended cultures of tissue-derived epithelial organoids. In certain embodiments, the method may include introducing a mixture comprising a hydrogel and tissue-derived epithelial stem cells into a culture medium to produce a suspended mixture. In certain embodiments, the mixture introduced into the culture medium comprises a hydrogel as described herein and a plurality of tissue-derived epithelial stem cells. In certain embodiments, the method may further include culturing a plurality of tissue-derived epithelial stem cells in the mixture in a culture medium to produce a suspended tissue-derived epithelial organoid as described herein. In certain embodiments, the method may further comprise fragmenting the suspended mixture to produce a fragmented structure comprising the tissue-derived epithelial organoid.

In certain embodiments, a method for generating a suspension culture of tissue-derived epithelial organoids may include contacting gastrointestinal stem cells ( e.g. , a plurality of tissue-derived epithelial stem cells) with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture and depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate as a droplet. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate to have a filamentous structure. In some embodiments, the method may further include solidifying the hydrogel-tissue derived epithelial stem cell mixture to produce a solidified hydrogel-tissue derived epithelial stem cell mixture. In some embodiments, the method may include suspending the solidified hydrogel-tissue derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue derived epithelial stem cell mixture. In some embodiments, the method includes culturing the suspended hydrogel-tissue derived epithelial stem cell mixture in the culture medium to produce tissue derived epithelial organoids. In some embodiments, the method may further include removing the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate before suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the culture medium. In some embodiments, the method may further include fragmenting the solidified hydrogel-tissue-derived epithelial stem cell mixture to generate a fragmented structure comprising tissue-derived epithelial organoids before or after suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the culture medium.

In certain embodiments, the tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells used in the present disclosure are contained in tissue fragments, organoid fragments, or a combination thereof. For example, but not limited to, tissue fragments and/or organoid fragments including tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells can be used to generate a hydrogel-tissue-derived epithelial stem cell mixture. Alternatively or additionally, the tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells are isolated from a tissue ( e.g. , a tissue fragment), an organoid fragment ( e.g. , a tissue-derived epithelial organoid fragment), or a combination thereof. In some embodiments, the tissue-derived epithelial stem cells do not include enriched potential stem cells ( e.g. , induced enriched potential stem cells (iPSCs) and embryonic stem cells (ESCs)). In some embodiments, the tissue-derived epithelial stem cells used in the present disclosure may be obtained from an in vitro cell culture. In some embodiments, the tissue fragments may be obtained from a frozen sample or a fresh sample, such as a frozen or fresh tissue sample and/or a frozen or fresh tissue organoid fragment. In some embodiments, the tissue fragments may be primary tissue fragments. In some embodiments, for example , the tissue of an individual ( e.g., a tissue fragment) may be normal ( e.g., non-cancerous and/or non-disease). In certain embodiments, a tissue ( eg , a tissue fragment) from a subject may be abnormal ( eg, cancerous and/or diseased). In certain embodiments, tissue-derived epithelial stem cells may be isolated from a fragment of native tissue.

In some embodiments, the primary tissue fragments can be fragments of tear glands, tonsils, salivary glands, gastrointestinal tissue, thyroid, lung, breast, liver, bile duct, stomach, kidney, pancreas, endometrium, fallopian tube, cervix, prostate, bladder, ovary, taste buds or placenta. In some embodiments, tissue-derived epithelial stem cells can be isolated from tissues (or fragments thereof) selected from the following: tear glands, tonsils, salivary glands, gastrointestinal tissue, thyroid, lung, breast, liver, bile duct, stomach, kidney, pancreas, endometrium, fallopian tube, cervix, prostate, bladder, ovary, taste buds, placenta and combinations thereof. In some embodiments, the primary tissue fragments may be fragments of lacrimal glands. In some embodiments, the primary tissue fragments may be fragments of tonsils. In some embodiments, the primary tissue fragments may be fragments of salivary glands. In some embodiments, the primary tissue fragments may be fragments of gastrointestinal tissue. In some embodiments, the primary tissue fragments may be fragments of thyroid glands. In some embodiments, the primary tissue fragments may be fragments of lungs. In some embodiments, the primary tissue fragments may be fragments of breasts. In some embodiments, the primary tissue fragments may be fragments of liver. In some embodiments, the primary tissue fragments may be fragments of bile ducts. In some embodiments, the primary tissue fragments may be fragments of stomach. In some embodiments, the primary tissue fragments may be fragments of kidneys. In some embodiments, the native tissue fragments may be fragments of the pancreas. In some embodiments, the native tissue fragments may be fragments of the endometrium. In some embodiments, the native tissue fragments may be fragments of the fallopian tube. In some embodiments, the native tissue fragments may be fragments of the cervix. In some embodiments, the native tissue fragments may be fragments of the prostate. In some embodiments, the native tissue fragments may be fragments of the bladder. In some embodiments, the native tissue fragments may be fragments of the ovary. In some embodiments, the primary tissue fragments may be taste buds. In some embodiments, the native tissue fragments may be fragments of the placenta. In certain embodiments, the primary tissue fragments are selected from the group consisting of lung tissue fragments, liver tissue fragments, gastrointestinal tissue fragments, breast tissue fragments, pancreatic tissue fragments, and combinations thereof.

In certain embodiments, the primary tissue fragments may be fragments of gastrointestinal tissue. In certain embodiments, the tissue fragments may be fragments of, for example , the oral mucosa, pharynx (throat), esophagus, stomach, small intestine, large intestine and/or rectum of an individual. In certain embodiments, the tissue fragments may be fragments of, for example, the oral mucosa of an individual. In certain embodiments, the tissue fragments may be fragments of, for example, the pharynx of an individual. In certain embodiments, the tissue fragments may be fragments of, for example, the esophagus of an individual. In certain embodiments, the tissue fragments may be fragments of, for example, the stomach of an individual. In certain embodiments, the tissue fragments may be fragments of, for example, the rectum of an individual. In certain embodiments, the tissue fragments may be fragments of, for example, the small intestine of an individual. In certain embodiments, the tissue fragments may be fragments of, for example, the large intestine of an individual. In some embodiments, the tissue fragments can be fragments of colon and/or ileum tissue, for example , of an individual. For example, but not limited to, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells can be isolated from colon and/or ileum tissue, for example, of an individual. In some embodiments, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells can be isolated from colon tissue ( for example, of an individual), for example, to generate colon organoids. In certain embodiments, a tissue-derived epithelial stem cell or a plurality of tissue-derived epithelial stem cells may be isolated from ileal tissue ( e.g., of an individual), e.g., to generate ileal organoids. In certain embodiments, the tissue of , e.g., an individual may be normal ( i.e. , non-cancerous). For example, but not limited to , the colon and/or ileal tissue of, e.g., an individual may be normal ( i.e. , non-cancerous). In certain embodiments, the tissue of, e.g. , an individual may be cancerous or diseased. For example, but not limited to, the colon and/or ileal tissue of , e.g., an individual may be cancerous and/or diseased colon and/or ileal tissue. In some embodiments, for example , the esophageal tissue of an individual may be cancerous and/or diseased esophageal tissue. In some embodiments, for example , the stomach tissue of an individual may be cancerous and/or diseased stomach tissue. In some embodiments, for example , the rectal tissue of an individual may be cancerous and/or diseased rectal tissue. In some embodiments, the tissue-derived epithelial organoid may be a gastrointestinal tumor organoid derived from an individual.

In certain embodiments, the native tissue fragments are lung tissue fragments.

In certain embodiments, the native tissue fragments are liver tissue fragments.

In certain embodiments, the native tissue fragment is a breast tissue fragment.

In certain embodiments, the native tissue fragments are pancreatic tissue fragments.

In certain embodiments, the methods of the present disclosure produce tissue-derived epithelial organoids that are uniform in size compared to a reference tissue-derived epithelial organoid ( e.g. , a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate). In certain embodiments, the tissue-derived epithelial organoids produced by the methods of the present disclosure are more uniform in size compared to the reference tissue-derived epithelial organoids due to differences in nutrient utilization as described in Example 1. For example, but not limited to, tissue-derived epithelial organoids produced by the methods of the present disclosure are more uniform in size across the width of a suspended culture droplet ( e.g. , a suspended hydrogel droplet) than a reference tissue-derived epithelial organoid ( e.g. , a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate). In certain embodiments, the reference tissue-derived epithelial organoid is produced in a hydrogel dome as disclosed in Example 1.

In certain embodiments, the methods of the present disclosure generate a population of tissue-derived epithelial organoids that express a marker at a different ( e.g., higher or lower) level than a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids). For example, but not limited to, a tissue-derived epithelial organoid ( e.g. , a population of tissue-derived epithelial organoids) of the present disclosure expresses a marker at a higher level than a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids). Alternatively or additionally, in certain embodiments, the tissue-derived epithelial organoids ( e.g. , a population of tissue-derived epithelial organoids) of the present disclosure express a marker at a lower level than a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids). In certain embodiments, the marker is a stem cell and/or proliferation marker, e.g. , a gene associated with stem cells and/or proliferation. For example, but not limited to, the stem cell and/or proliferation marker is MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1 and/or CD44. In some embodiments, the marker is a differentiation marker, for example , a gene associated with differentiation. For example, but not limited to, the differentiation marker is keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and/or smooth muscle actin (SMA). In some embodiments, the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. For example, but not limited to, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid produced in a hydrogel dome as disclosed in Example 1.

In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express stem cell and/or proliferation markers at higher levels than a population of reference tissue-derived epithelial organoids. Non-limiting examples of stem cell and/or proliferation markers include MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, and/or CD44, and combinations thereof. In certain embodiments, the stem cell and/or proliferation marker is selected from the group consisting of MKI67, ASCL2, LGR5, SOX9, SMOC2, CD44, and combinations thereof. In some embodiments, the stem cell and/or proliferation marker is MKI67. In some embodiments, the stem cell and/or proliferation marker is ASCL2. In some embodiments, the stem cell and/or proliferation marker is LGR5. In some embodiments, the stem cell and/or proliferation marker is SOX9. In some embodiments, the stem cell and/or proliferation marker is SMOC2. In some embodiments, the stem cell and/or proliferation marker is CD44. In some embodiments, the stem cell and/or proliferation marker is EpCAM. In some embodiments, the stem cell and/or proliferation marker is CD49f. In some embodiments, the stem cell and/or proliferation marker is CD133. In some embodiments, the stem cell and/or proliferation marker is ALDH1A1. In some embodiments, the stem cell and/or proliferation marker is NEUROG3. In some embodiments, the stem cell and/or proliferation marker is NKX6.1. In some embodiments, the stem cell and/or proliferation marker is PDX1. In some embodiments, the stem cell and/or proliferation marker is BMI1. In certain embodiments, the expression level of a stem cell and/or proliferation marker in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 110% higher, at least 120% higher, at least 130% higher, at least 140% higher, at least 150% higher, at least 160% higher, at least 170% higher, at least 180% higher, at least 190% higher, at least 200% higher, at least 210% higher, at least 220% higher, at least 230% higher, at least 240% higher, at least 250% higher, at least 260% higher, at least 270% higher, at least 280% higher, at least 290% higher, at least 300% higher, at least 310% higher, at least 320% higher, at least 330% higher, at least 340% higher, at least 350% higher, at least 360% higher, at least 370% higher, at least 380% higher, at least 390% higher, at least 400% higher, at least 410% higher, at least 420% higher, at least 430% higher, at least 440% higher, at least 450% higher, at least 460% higher, at least 470% higher, at least 480% higher, at least 490% higher In some embodiments, the expression level of stem cells and/or proliferation markers in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 50% higher than the expression level of stem cells and/or proliferation markers in a population of epithelial organoids derived from a reference tissue. In some embodiments, the expression level of stem cells and/or proliferation markers in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 100% higher than the expression level of stem cells and/or proliferation markers in a population of epithelial organoids derived from a reference tissue. In certain embodiments, the expression level of stem cells and/or proliferation markers in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 200% higher than the expression level of stem cells and/or proliferation markers in a population of epithelial organoids from a reference tissue. In certain embodiments, the expression level of stem cells and/or proliferation markers in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 300% higher than the expression level of stem cells and/or proliferation markers in a population of epithelial organoids from a reference tissue.

In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a differentiation marker at a lower level than a population of reference tissue-derived epithelial organoids. Non-limiting examples of differentiation markers include keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. In some embodiments, the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, and combinations thereof. In some embodiments, the differentiation marker is keratin 20 (KRT20). In some embodiments, the differentiation marker is FABP1. In some embodiments, the differentiation marker is MUC2. In some embodiments, the differentiation marker is MUC5B. In some embodiments, the differentiation marker is TFF3. In some embodiments, the differentiation marker is ALPI. In some embodiments, the differentiation marker is SI. In some embodiments, the differentiation marker is CEACAM7. In some embodiments, the differentiation marker is keratin 19 (KRT19). In some embodiments, the differentiation marker is keratin 7 (KRT7). In some embodiments, the differentiation marker is SOX9. In some embodiments, the differentiation marker is SOX9. In some embodiments, the differentiation marker is MUC1. In some embodiments, the differentiation marker is INS. In some embodiments, the differentiation marker is GCG. In some embodiments, the differentiation marker is AMY. In some embodiments, the differentiation marker is ALB. In some embodiments, the differentiation marker is CYP3A4. In some embodiments, the differentiation marker is HNF4A. In some embodiments, the differentiation marker is cytokeratin 8 (K8). In some embodiments, the differentiation marker is cytokeratin 18 (K18). In some embodiments, the differentiation marker is cytokeratin 5 (K5). In some embodiments, the differentiation marker is cytokeratin 14 (K14). In some embodiments, the differentiation marker is smooth muscle actin (SMA). In some embodiments, the differentiation marker is MUC5AC. In some embodiments, the differentiation marker is MUC6. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 100% lower, at least 110% lower, at least 120% lower, at least 130% lower, at least 140% lower, at least 150% lower, at least 160% lower, at least 170% lower, at least 180% lower, at least 190% lower, at least 200% lower, at least 210% lower, at least 220% lower, at least 230% lower, at least 240% lower, at least 200% lower, at least 250% lower, at least 260% lower, at least 270% lower, at least 280% lower, at least 290% lower, at least 300% lower, at least 310% lower, at least 320% lower, at least 330% lower, at least 340% lower, at least 350% lower, at least 360% lower, at least 370% lower, at least 380% lower, at least 390% lower, at least 400% lower, at least 410% lower, at least 420% lower, at least 430% lower, at least 440% lower In some embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 50% lower than the expression level of a differentiation marker in a population of epithelial organoids from a reference tissue. In some embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 100% lower than the expression level of a differentiation marker in a population of epithelial organoids from a reference tissue. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 200% lower than the expression level of a differentiation marker in a population of epithelial organoids from a reference tissue. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 300% lower than the expression level of a differentiation marker in a population of epithelial organoids from a reference tissue.

In certain embodiments, the method for generating gastrointestinal organoids comprises contacting tissue-derived epithelial stem cells with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1×10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1×10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. In some embodiments, the method may further include suspending the hydrogel-tissue derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue derived epithelial stem cell mixture. For example, but not limited to, the volume of the hydrogel-tissue derived epithelial stem cell mixture dispensed into the culture medium may be at least about 10 μL. In some embodiments, the ratio of the volume of the hydrogel-tissue derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In some embodiments, the hydrogel solidifies when in contact with the culture medium. For example, but not limited to, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 37°C. In some embodiments, the hydrogel can be a commercially available ECM. In some embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of the basement membrane. A non-limiting example of BME is CULTREX® Basement Membrane Extract Type 2 (R&D Systems), including laminin, entactin, collagen IV, and heparin sulfate proteoglycans. In some embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture is distributed into a culture medium having a certain geometric shape. In some embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is spherical or spherical. In some embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is a filamentous structure. In some embodiments, the filamentous structure has a linear, serpentine or spiral shape. In some embodiments, the hydrogel-tissue derived epithelial stem cell mixture is suspended in a culture medium in a droplet. In certain embodiments, the method may further comprise culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in a culture medium ( e.g. , colon passage medium (Intesticult Organoid Growth Medium (OGM, StemCell Technologies Catalog No. 06010) + 10 µM Y27632) or ileum culture medium (OGM + 10 µM Y27632 + 2.5 µM CHIR99021)) to generate colon or ileum organoids, respectively. In some embodiments, the method may further include fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture to produce fragmented structures comprising the tissue-derived epithelial organoid. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture may include shearing the suspended hydrogel-tissue derived epithelial stem cell mixture to produce the fragmented structures, such as by pipetting a culture medium containing the suspended hydrogel-tissue derived epithelial stem cell mixture up and down. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture produces structures with shorter length and/or width. For example, but not limited to, fragmenting the filamentous structure produces a structure that is shorter in length, width, or both. In certain embodiments, a tissue-derived epithelial stem cell or a plurality of tissue-derived epithelial stem cells used in the present disclosure are contained in the organoid fragments. In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a marker at a different ( e.g., higher or lower) level than a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids). In certain embodiments, the differentially expressed marker is MKI67, LGR5, SOX9, CD44, MUC2, MUC5B, TFF3, KRT20, FABP1, ALPI, and/or CEACAM7.

In certain embodiments, a method for generating lung organoids comprises contacting tissue-derived epithelial stem cells with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells may be combined with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1×10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1×10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. In some embodiments, the method may further include suspending the hydrogel-tissue derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue derived epithelial stem cell mixture. For example, but not limited to, the volume of the hydrogel-tissue derived epithelial stem cell mixture dispensed into the culture medium may be at least about 10 μL. In some embodiments, the ratio of the volume of the hydrogel-tissue derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In some embodiments, the hydrogel solidifies when in contact with the culture medium. For example, but not limited to, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 37°C. In some embodiments, the hydrogel can be a commercially available ECM. A non-limiting example of an ECM is MATRIGEL®. In some embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is distributed in a culture medium having a certain geometric shape. In some embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spherical. In some embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture is a filamentous structure. In some embodiments, the filamentous structure has a linear, serpentine or spiral shape. In some embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in a culture medium in small drops. In some embodiments, the method may further include culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in a culture medium ( e.g. , SFFF culture medium containing 10 μM ROCK inhibitor). In some embodiments, the method may further include fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce a fragmented structure comprising the tissue-derived epithelial organoid. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture may include shearing the suspended hydrogel-tissue derived epithelial stem cell mixture to produce fragmented structures, such as by pipetting a culture medium containing the suspended hydrogel-tissue derived epithelial stem cell mixture up and down. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture produces structures with shorter length and/or width. For example, but not limited to, fragmenting filamentous structures produces structures with shorter length, width, or both. In certain embodiments, a tissue-derived epithelial stem cell or a plurality of tissue-derived epithelial stem cells used in the present disclosure are contained within an organoid fragment. In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a marker at a different ( e.g., higher or lower) level than a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids).

In certain embodiments, a method for generating mammary organoids comprises contacting tissue-derived epithelial stem cells with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells may be combined with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1 × 10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1 × 10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. In some embodiments, the method may further include suspending the hydrogel-tissue derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue derived epithelial stem cell mixture. For example, but not limited to, the volume of the hydrogel-tissue derived epithelial stem cell mixture dispensed into the culture medium may be at least about 10 μL. In some embodiments, the ratio of the volume of the hydrogel-tissue derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In some embodiments, the hydrogel solidifies when in contact with the culture medium. For example, but not limited to, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 37°C. In some embodiments, the hydrogel can be a commercially available ECM. In some embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of the basement membrane. A non-limiting example of BME is CULTREX® Low Growth Factor BME Type 2 (Trevigen, 3533-010-02). In some embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture is distributed into a culture medium having a certain geometric shape. In some embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is spherical or spherical. In some embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is a filamentous structure. In some embodiments, the filamentous structure has a linear, serpentine or spiral shape. In some embodiments, the hydrogel-tissue derived epithelial stem cell mixture is suspended in a culture medium in a droplet. In certain embodiments, the method may further include culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in a culture medium ( e.g. , SFFF medium containing 10 μM ROCK inhibitor). In certain embodiments, the method may further include fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate a fragmented structure comprising the tissue-derived epithelial organoid. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture may include shearing the suspended hydrogel-tissue derived epithelial stem cell mixture to produce fragmented structures, such as by pipetting a culture medium containing the suspended hydrogel-tissue derived epithelial stem cell mixture up and down. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture produces structures with shorter length and/or width. For example, but not limited to, fragmenting filamentous structures produces structures with shorter length, width, or both. In certain embodiments, a tissue-derived epithelial stem cell or a plurality of tissue-derived epithelial stem cells used in the present disclosure are contained within an organoid fragment. In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a marker at a different ( e.g., higher or lower) level than a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids). In certain embodiments, the differentially expressed marker is EpCAM, CD49f, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and/or smooth muscle actin (SMA).

In certain embodiments, a method for generating pancreatic organoids comprises contacting tissue-derived epithelial stem cells with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells may be combined with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1×10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1×10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. In some embodiments, the method may further include suspending the hydrogel-tissue derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue derived epithelial stem cell mixture. For example, but not limited to, the volume of the hydrogel-tissue derived epithelial stem cell mixture dispensed into the culture medium may be at least about 10 μL. In some embodiments, the ratio of the volume of the hydrogel-tissue derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In some embodiments, the hydrogel solidifies when in contact with the culture medium. For example, but not limited to, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 37°C. In some embodiments, the hydrogel can be a commercially available ECM. In some embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of the basement membrane. A non-limiting example of BME is low growth factor BME 2-RGF (basement membrane extract type 2 3533-010-02; AMSBIO, CULTREX®). In some embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture is distributed into a culture medium having a certain geometric shape. In some embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is spherical or spherical. In some embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is a filamentous structure. In some embodiments, the filamentous structure has a linear, serpentine or spiral shape. In some embodiments, the hydrogel-tissue derived epithelial stem cell mixture is suspended in a culture medium in a droplet. In certain embodiments, the method can further include culturing the suspended hydrogel-tissue-derived epithelial stem cells in an optimized human pancreatic organoid expansion medium ( e.g. , a serum-free medium supplemented with 1X N2 and 1X B27 (both from GIBCO), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 10% RSPO1 conditioned medium, 10 nM human [Leu ¹⁵ ]-gastrin I (Sigma-Aldrich), 50 ng/mL EGF (Peprotech), 25 ng/mL Noggin (Peprotech), 100 ng/mL FGF10 (Peprotech), 10 mM niacinamide (Sigma-Aldrich), 5 μM A83.01 (Tocris), 10 μM FSK (Tocris) and 3 μM PGE2 (Tocris) supplemented with 10 μM Rho kinase inhibitor (Y27632, Sigma-Aldrich). In certain embodiments, the method may further comprise fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate a fragmented structure comprising the tissue-derived epithelial organoid. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture may include shearing the suspended hydrogel-tissue derived epithelial stem cell mixture to produce fragmented structures, such as by pipetting a culture medium containing the suspended hydrogel-tissue derived epithelial stem cell mixture up and down. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture produces structures with shorter length and/or width. For example, but not limited to, fragmenting filamentous structures produces structures with shorter length, width, or both. In certain embodiments, a tissue-derived epithelial stem cell or a plurality of tissue-derived epithelial stem cells used in the present disclosure are contained within an organoid fragment. In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a marker at a different ( e.g., higher or lower) level than a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids). In certain embodiments, the tissue-derived epithelial organoids are pancreatic organoids, and the differentially expressed marker is CD133, LGR5, PDX1, SOX9, ALDH1A1, NEUROG3, NKX6.1, keratin 19 (KRT19), MUC1, INS, GCG, and/or AMY.

In certain embodiments, a method for generating a liver organoid comprises contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells may be combined with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 3,000 to about 10,000 tissue-derived epithelial stem cells per well in a dish ( e.g. , a 48-well dish). In some embodiments, the method may further include suspending the hydrogel-tissue derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue derived epithelial stem cell mixture. For example, but not limited to, the volume of the hydrogel-tissue derived epithelial stem cell mixture dispensed into the culture medium may be at least about 10 μL. In some embodiments, the ratio of the volume of the hydrogel-tissue derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In some embodiments, the hydrogel solidifies when in contact with the culture medium. For example, but not limited to, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 37°C. In some embodiments, the hydrogel can be a commercially available ECM. In some embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of the basement membrane. Non-limiting examples of ECM are MATRIGEL® (BD Biosciences) or low growth factor BME 2 (basement membrane extract, type 2, Pathclear). In some embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture is distributed into a culture medium having a certain geometric shape. In some embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is spherical or spherical. In some embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture is a filamentous structure. In some embodiments, the filamentous structure has a linear, serpentine or spiral shape. In some embodiments, the hydrogel-tissue derived epithelial stem cell mixture is suspended in a culture medium in a droplet. In certain embodiments, the method may further comprise culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in a culture medium, such as AdDMEM/F12 (Invitrogen) supplemented with 1% N2 (GIBCO) and 1% B27 (GIBCO), 1.25 mM N-acetylcysteine (Sigma), 10 nM gastrin (Sigma), and growth factors: 50 ng/ml EGF (Peprotech), 10% RSPO1 conditioned medium (homemade), 100 ng/ml FGF10 (Peprotech), 25 ng/ml HGF (Peprotech), 10 mM niacinamide (Sigma), 5 µM A83.01 (Tocris) and 10 µM FSK (Tocris), supplemented with 25 ng/ml Noggin (Peprotech), 30% Wnt medium (as described in Barker et al. Cell Stem Cell 6:25-36 (2010)) and 10 µM (Y27632, Sigma Aldrich) or hES cell selective recovery solution (Stemgent) during the first 3 days after isolation to establish the culture. In certain embodiments, the medium is then changed to medium that does not contain Noggin, Wnt, Y27632, and hES cell selective recovery solution. In certain embodiments, hepatic organoids are plated and cultured in the above medium supplemented with BMP7 (25 ng/ml) for 7 to 10 days. In certain embodiments, the medium is then replaced with a differentiation medium, for example , comprising AdDMEM/F12 medium supplemented with 1% N2 and 1% B27 and containing EGF (50 ng/ml), gastrin (10 nM, Sigma), HGF (25 ng/ml), FGF19 (100 ng/ml), A8301 (500 nM), DAPT (10 µM), BMP7 (25 ng/ml) and dexamethasone (30 µM) to generate hepatocyte organoids. In certain embodiments, the method may further comprise fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate a fragmented structure comprising the tissue-derived epithelial organoids. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture may include shearing the suspended hydrogel-tissue derived epithelial stem cell mixture to produce fragmented structures, such as by pipetting a culture medium containing the suspended hydrogel-tissue derived epithelial stem cell mixture up and down. In some embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture produces structures with shorter length and/or width. For example, but not limited to, fragmenting filamentous structures produces structures with shorter length, width, or both. In certain embodiments, a tissue-derived epithelial stem cell or a plurality of tissue-derived epithelial stem cells used in the present disclosure are contained within an organoid fragment. In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a marker at a different ( e.g., higher or lower) level than a reference tissue-derived epithelial organoid ( e.g. , a population of reference tissue-derived epithelial organoids). In certain embodiments, the tissue-derived epithelial organoid is a liver organoid, and the differentially expressed marker is LGR5, ALB, CYP3A4, HNF4A, KRT19, KRT7, and/or SOX9.

In certain embodiments, one or more steps of the disclosed methods may be performed using a robot and/or automated components. In certain embodiments, the methods of the present disclosure may include the use of a robot and/or automated components to produce tissue-derived epithelial organoids. In certain embodiments, one or more steps of the disclosed methods may be performed using a robot and/or automated components to produce tissue-derived epithelial organoids. Non-limiting examples of robots and/or automated components that may be used in the disclosed methods include automated liquid handlers ( e.g. , liquid handling robots), 3D printers, syringe pumps, electronic pipettes ( e.g. , with or without a pipetting robot), or combinations thereof. A non-limiting example of an electronic pipette ( e.g. , with a pipetting robot) is the Assist Plus from Integra Biosciences. In certain embodiments, one or more steps of the disclosed methods may be performed by an automated liquid handler ( eg , a liquid handling robot).

In certain embodiments, the passage of tissue-derived epithelial organoid cultures produced by the methods of the present disclosure is performed by a robot and/or automated components. In certain embodiments, an automated robot can perform the method described in "Organoid Maintenance" of Example 1 of the present disclosure, for example , performing any of the following steps: adding TrypLE Express, heating the organoid culture, grinding the organoid culture to dissociate cells, adding PBS, sedimenting cells, resuspending cells in BME, cooling the culture, flattening cells, covering the organoid with culture medium, or a combination thereof. In certain embodiments, cell dissociation can be performed using a robot and/or automated components. In certain embodiments, medium replacement, such as during organoid maintenance or during the generation of tissue-derived epithelial organoids, can be performed using robotic and/or automated components.

In certain embodiments, the production of BOBA and/or SOBA in the cell culture medium is performed by a robot and/or automated assembly. In certain embodiments, the automated robot can perform the methods described in "Suspended hydrogel BOBA culture" and/or "Suspended hydrogel SOBA fragment culture" of Example 1 of the present disclosure, for example , performing any of the following steps: heating the culture medium, dispensing the organoid cell-BME solution as droplets into the warm culture medium to produce BOBA, dispensing the organoid cell-BME solution into the warm culture medium in a linear, serpentine or spiral motion in the XY plane to produce SOBA, grinding SOBA filament culture to produce SOBA fragments, changing the medium or a combination thereof. In some embodiments, dispensing the organoid cell-BME solution into the warm culture medium to produce BOBA and/or SOBA can be performed using a robot and/or automated components, such as a liquid handling robot. In some embodiments, changing the culture medium can be performed using a robot and/or automated components, such as a liquid handling robot. In some embodiments, grinding the SOBA silk culture to produce SOBA fragments can be performed using a robot and/or automated components, such as a liquid handling robot.

In some embodiments, high throughput methods such as those described herein can be performed using robotics and/or automated components. For example, but not limited to, any of the methods of use disclosed herein, such as those described in Section IV, can be performed in part using robotics and/or automated components. In some embodiments, methods for screening agents such as therapeutic agents and methods for performing genomic screening using the disclosed tissue-derived epithelial organoids can be performed in part using robotics and/or automated components. IV. Methods of Use

The present disclosure provides methods of using the disclosed organoids or compositions comprising such organoids. In certain embodiments, the tissue-derived epithelial organoids of the present disclosure can be used in screening assays. For example, but not limited to, the present disclosure provides methods for screening agents ( e.g., therapeutic agents), and methods for performing genomic screening using the disclosed tissue-derived epithelial organoids. In certain embodiments, the tissue-derived epithelial organoids of the present disclosure can be used to generate organoid-based models.

In certain embodiments, the organoids or compositions thereof disclosed herein can be used to identify agents having therapeutic effects. In certain embodiments, the organoids or compositions thereof disclosed herein can be used to identify therapeutic agents that can effectively prevent and/or treat diseases. In certain embodiments, the organoids or compositions thereof disclosed herein can be used to identify therapeutic agents that can effectively improve disease symptoms.

In certain embodiments, the organoids or compositions thereof disclosed herein can be used to study the biology and/or pathogenesis of a disease. For example, but not limited to, the organoids or compositions thereof disclosed herein can be contacted with a drug to study the biology and/or pathogenesis of a disease.

In certain embodiments, the disclosed organoids or compositions thereof can be used to identify potentially toxic agents, such as therapeutic agents. In certain embodiments, the disclosed organoids or compositions thereof can be used to identify the concentrations at which agents ( such as therapeutic agents) may be toxic.

In certain embodiments, methods for identifying therapeutic agents that are effective in preventing and/or treating a disease, methods for identifying therapeutic agents that are effective in ameliorating symptoms of a disease, and/or methods for identifying therapeutic agents that are potentially toxic may include contacting a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids with a therapeutic agent. In certain embodiments, the method may include contacting a composition comprising a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids with a therapeutic agent. In certain embodiments, the tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids is embedded in a hydrogel suspended in a culture medium, as described herein.

In certain embodiments, methods for studying the biology and/or pathogenesis of a disease may include contacting a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids with an agent. In certain embodiments, the method may include contacting a composition including a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids with an agent. In certain embodiments, the tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids is embedded in a hydrogel suspended in a culture medium as described herein.

In certain embodiments, an agent ( e.g., a therapeutic agent) is contacted with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 3 years, e.g. , about 15 minutes to about 3 years, about 15 minutes to about 2.5 years, about 15 minutes to about 2 years, about 15 minutes to about 1.5 years, about 15 minutes to about 1 year, about 15 minutes to about 183 days, about 15 minutes to about 150 days, about 15 minutes to about 100 days, about 15 minutes to about 50 days, about 1 day to about 3 years, about 10 days to about 3 years, about 20 days to about 3 years, about 50 days to about 3 years, about 100 days to about 3 years, about 150 days to about 3 years, about 183 days to about 3 years, about 1 year to about 3 years years, about 1.5 to about 3 years, about 2 to about 3 years, or about 2.5 to about 3 years. In some embodiments, an agent ( e.g., a therapeutic agent) is contacted with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 100 days. In some embodiments, an agent ( e.g., a therapeutic agent) is contacted with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 15 minutes to about 100 days. In some embodiments, an agent ( e.g., a therapeutic agent) is contacted with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 150 days. In some embodiments, an agent ( e.g., a therapeutic agent) is in contact with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 15 minutes to about 150 days. In some embodiments, an agent ( e.g., a therapeutic agent) is in contact with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 1 year. In some embodiments, an agent ( e.g., a therapeutic agent) is in contact with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 15 minutes to about 1 year. In some embodiments, an agent ( e.g., a therapeutic agent) is in contact with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 2 years. In some embodiments, an agent ( e.g., a therapeutic agent) is in contact with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 15 minutes to about 2 years. In some embodiments, an agent ( e.g., a therapeutic agent) is in contact with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 10 days. In certain embodiments, an agent ( eg, a therapeutic agent) is contacted with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 15 minutes to about 10 days. In certain embodiments, an agent ( e.g., a therapeutic agent) is contacted with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 1 hour to about 10 days, about 12 hours to about 10 days, about 1 day to about 10 days, about 2 days to about 10 days, about 3 days to about 10 days, about 4 days to about 10 days, about 5 days to about 10 days, about 6 days to about 10 days, about 7 days to about 10 days, about 8 days to about 10 days, about 9 days to about 10 days, about 15 minutes to about 10 days, about 15 minutes to about 9 days, about 15 minutes to about 8 days, about 15 minutes to about 7 days, about 15 minutes to about 6 days, about 15 minutes to about 5 days, about 15 minutes to about 4 days, about 15 minutes to about 3 days, about 15 minutes to about 15 days, about 15 minutes to about 10 days, about 15 minutes to about 15 ... Minutes to about 2 days, about 15 minutes to about 1 day, about 1 day to about 5 days, about 1 day to about 2 days, about 2 days to about 5 days, or about 2 days to about 10 days. In certain embodiments, an agent ( e.g., a therapeutic agent) is contacted with a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) for about 2 days to about 10 days.

In certain embodiments, the method may include contacting populations of epithelial organoids (or compositions thereof) of different tissue origin with increasing concentrations of an agent ( e.g., a therapeutic agent) to allow for dose-response studies.

In some embodiments, the agent can be any agent of interest. In some embodiments, the agent is a molecule known to affect the biology and/or pathogenesis of the disease. Non-limiting examples of agents that can be used in the disclosed methods include peptides, polypeptides, small molecules, cells, gene editing systems, or nucleic acids. In some embodiments, such agents can be agents that affect cell signaling, nucleic acid expression, protein expression, cell growth, cell differentiation, and/or cell survival.

In some embodiments, the agent is a therapeutic agent. In some embodiments, the therapeutic agent can be any therapeutic agent of interest. In some embodiments, the therapeutic agent is obtained from a library of potential therapeutic agents. Non-limiting examples of therapeutic agents that can be analyzed and/or identified using the disclosed methods include peptide-based therapeutic agents, polypeptide-based therapeutic agents, small molecule therapeutic agents, cell-based therapeutic agents, gene editing systems, nucleic acid-based therapeutic agents, and combinations thereof.

In some embodiments, the therapeutic agent is a peptide-based therapeutic agent. In some embodiments, the peptide-based therapeutic agent includes a peptide having a molecular weight of about 5,000 Da or less. Non-limiting examples of peptide-based therapeutic agents include growth factors, anti-infective agents, antifungal agents, antibacterial agents, receptor ligands, and tyrosine kinase inhibitors. Other non-limiting examples of peptide therapeutic agents are disclosed in Wang et al. (2022) Signal Transduction and Targeted Therapy 7:48 ( e.g., Tables 1 and 2), the contents of which are incorporated herein by reference in their entirety.

In certain embodiments, the therapeutic agent is a polypeptide-based therapeutic agent. Non-limiting examples of polypeptide-based therapeutic agents include antibody-based therapeutic agents, such as antibodies and antibody-drug conjugates, hormones, and enzymes. In certain embodiments, the antibody may be an agonist antibody or an antagonist antibody. In certain embodiments, the antibody may be an antibody fragment. Non-limiting examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , bifunctional antibodies, linear antibodies, single-chain antibody molecules ( e.g. , scFv), and multispecific antibodies formed from antibody fragments.

In certain embodiments, the therapeutic agent is a small molecule therapeutic agent. For example, but not limited to, a small molecule therapeutic agent is a compound with a molecular weight of less than about 1,000 Da. In certain embodiments, small molecule therapeutic agents include cell cycle regulators, kinase regulators ( e.g. , kinase inhibitors or activators), enzyme inhibitors, receptor regulators ( e.g. , receptor inhibitors or activators), anti-infective agents, antifungal agents, antibacterial agents, chemotherapeutic agents, and anti-inflammatory agents.

In certain embodiments, the therapeutic agent is a cell-based therapeutic agent. Non-limiting examples of cell-based therapeutic agents include bacterial cells and immune cells. Non-limiting examples of immune cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells (NK cells) and lymphocytes, such as B cells and T cells ( e.g. , cytotoxic T cells, natural killer T cells, regulatory T cells and helper T cells). In certain embodiments, the immune cell may be a modified immune cell that has been genetically engineered to express a chimeric antigen receptor (CAR) ( eg , CAR T cells and CAR NK cells) or a T cell receptor (TCR) ( eg , a heterologous TCR).

In some embodiments, the therapeutic agent is a gene regulatory system and/or a component of a gene regulatory system. In some embodiments, the gene regulatory system and/or a component of a gene regulatory system is a gene editing system, CRISPRi, a gene expression promoting system, a gene suppression promoting system, a nucleic acid-based therapeutic agent, a transcription factor, and/or a regulator of post-transcriptional modification.

In some embodiments, the therapeutic agent is a gene editing system. Non-limiting examples of gene editing systems include nested endonucleases or meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR gene editing systems. In some embodiments, the therapeutic agent is a CRISPR gene editing system, such as a CRISPR/Cas9 gene editing system.

In certain embodiments, the therapeutic agent is a nucleic acid-based therapeutic agent. Non-limiting examples of nucleic acid-based therapeutic agents include RNA-based therapeutic agents, including siRNA, microRNA, RNA aptamers, ribozymes, RNA baits, and RNAi. In certain embodiments, nucleic acid-based therapeutic agents include DNA-based therapeutic agents, including antisense oligonucleotides (ASOs) and DNA aptamers.

In certain embodiments, the method may further comprise analyzing the tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or cells of tissue-derived epithelial organoids) for changes. In certain embodiments, the method may further comprise analyzing the tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or cells of tissue-derived epithelial organoids) for changes that occur in the presence of an agent. For example, but not limitation, the method may further comprise analyzing the tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or cells of tissue-derived epithelial organoids) for changes that indicate effectiveness and/or toxicity of a therapeutic agent. In certain embodiments, the method comprises analyzing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids for changes indicative of the effectiveness and/or toxicity of a therapeutic agent compared to a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids not treated with the therapeutic agent.

In certain embodiments, the organoids or compositions thereof disclosed herein can be used for genome screening. In certain embodiments, genome screening can be used to identify gene editing systems for generating mutations. Non-limiting examples of mutations include deletions, duplications, insertions, and nucleotide substitutions. In certain embodiments, the method can include providing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or compositions thereof) and generating mutations in the genome of one or more cells of the tissue-derived epithelial organoids. In certain embodiments, mutations are generated using a gene regulation system and/or a component of a gene regulation system. In some embodiments, the gene regulation system and/or a component of a gene regulation system is a gene editing system, CRISPRi, RNAi, a gene expression promoting system, a gene suppression promoting system, a nucleic acid-based therapeutic agent, a transcription factor, and/or a regulator of post-transcriptional modification. In some embodiments, the gene editing system is a CRISPR system, for example , a CRISPR/Cas9 gene editing system. In some embodiments, the method may further include analyzing changes associated with mutations in a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids. In certain embodiments, the method comprises analyzing changes associated with the mutation in a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids compared to a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids that does not have the mutation.

In certain embodiments, the present disclosure further provides methods for generating epithelial cell models using tissue-derived epithelial organoids of the present disclosure. In certain embodiments, the method may include providing a tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids (or a composition thereof), digesting the tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids into single cells, and culturing the single cells in a culture medium to produce a cell monolayer. In certain embodiments, the single cells are cultured on a permeable cell culture insert. In certain embodiments, the culture medium is a differentiation medium. In certain embodiments, the culture medium is a stem cell promoting medium. In certain embodiments, the culture medium is a cell growth medium.

In certain embodiments, the cell monolayer can be used in any of the methods disclosed herein, such as in the screening methods disclosed herein. For example, but not limited to, the cell monolayer can be used to screen therapeutic agents and to perform genomic screening. In certain embodiments, the monolayer of the present disclosure can be used to study the biology and/or pathogenesis of a disease.

In certain embodiments, methods for screening therapeutic agents using cell monolayers of the present disclosure may include contacting the cell monolayer and analyzing the cell monolayer for changes that indicate the effectiveness, distribution, and/or toxicity of the therapeutic agent. In certain embodiments, the method includes analyzing changes in the cell monolayer that indicate the effectiveness and/or toxicity of the therapeutic agent compared to a cell monolayer that has not been treated with the therapeutic agent.

In certain embodiments, methods for performing genomic screening using a cell monolayer of the present disclosure may include providing a cell monolayer produced by the methods described herein, generating a mutation in the genome of one or more cells in the cell monolayer, and analyzing the cell monolayer for changes associated with the mutation. In certain embodiments, the method includes analyzing the cell monolayer for changes associated with the mutation compared to a cell monolayer without the mutation.

In certain embodiments, the changes in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) can be changes in cell viability, cell proliferation, organoid size, cell morphology, organoid morphology, hydrogel invasiveness, motility, differentiation state, mutational state, karyotype, chromosomal aberration, nucleic acid expression level, protein expression level, nucleic acid modification ( e.g. , methylation), post-translational modification ( e.g. , phosphorylation, ubiquitination and/or glycosylation), activation of a cell signaling pathway, inhibition of a cell signaling pathway, enzyme activity ( e.g. , enzyme cleavage) , chromatin accessibility, histone modifications and other epigenetic changes, physical properties of organoids (including permeability of the gastrointestinal epithelial barrier, pH, oxygen tension, and concentrations of other metabolites in the lumen and basement membrane and secreted factors in the hydrogel and/or medium), concentrations of interleukins and hormones, drug sensitivity, pharmacokinetic and pharmacodynamics of drug absorption and metabolism, force measurements, measurements of biomolecular interactions within the organoid, lumen, and medium/hydrogel, and membrane potential.

In certain embodiments, the change in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) may be a change in cell viability.

In certain embodiments, the changes in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) may be changes in cell proliferation.

In certain embodiments, the variation in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) may be a variation in organoid size.

In certain embodiments, the changes in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) may be changes in a mutant state.

In certain embodiments, the changes in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) may be changes in RNA expression levels and/or protein expression levels.

In certain embodiments, the changes in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) may be changes in RNA expression levels and/or protein expression levels of stem cells and/or proliferation markers. In certain embodiments, the changes in a tissue-derived epithelial organoid, a population of tissue-derived epithelial organoids, or a cell monolayer may be changes in protein expression levels of stem cells and/or proliferation markers. In certain embodiments, the changes in a tissue-derived epithelial organoid, a population of tissue-derived epithelial organoids, or a cell monolayer may be changes in RNA expression levels of stem cells and/or proliferation markers. For example, but not limited to, the variation may be a variation in the expression of MKI67, a variation in the expression of EpCAM, a variation in the expression of CD49f, a variation in the expression of ASCL2, a variation in the expression of CD133, a variation in the expression of LGR5, a variation in the expression of SOX9, a variation in the expression of ALDH1A1, a variation in the expression of NEUROG3, a variation in the expression of NKX6.1, a variation in the expression of SMOC2, a variation in the expression of PDX1, a variation in the expression of BMI1, and/or a variation in the expression of CD44. In certain embodiments, the variation may be a variation in the expression of MKI67. In certain embodiments, the variation may be a variation in the expression of ASCL2. In certain embodiments, the variation may be a variation in the expression of LGR5. In certain embodiments, the variation may be a variation in the expression of SOX9. In some embodiments, the variation may be a variation in the expression of SMOC2. In some embodiments, the variation may be a variation in the expression of CD44. In some embodiments, the variation may be a variation in the expression of EpCAM. In some embodiments, the variation may be a variation in the expression of CD49f. In some embodiments, the variation may be a variation in the expression of CD133. In some embodiments, the variation may be a variation in the expression of ALDH1A1. In some embodiments, the variation may be a variation in the expression of NEUROG3. In some embodiments, the variation may be a variation in the expression of NKX6.1. In some embodiments, the variation may be a variation in the expression of PDX1. In some embodiments, the variation may be a variation in the expression of BMI1.

In certain embodiments, the changes in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) may be changes in RNA expression levels and/or protein expression levels of a differentiation marker. In certain embodiments, the changes in a tissue-derived epithelial organoid, a population of tissue-derived epithelial organoids, or a cell monolayer may be changes in RNA expression levels of a differentiation marker. In certain embodiments, the changes in a tissue-derived epithelial organoid, a population of tissue-derived epithelial organoids, or a cell monolayer may be changes in protein expression levels of a differentiation marker. For example, but not limited to, the changes may be changes in keratin 20 (KRT20) expression, changes in FABP1 expression, changes in MUC2 expression, changes in MUC5B expression, changes in MUC5AC expression, changes in MUC6 expression, changes in TFF3 expression, changes in ALPI expression, changes in SI expression, changes in CEACAM7 expression, changes in keratin 19 (KRT19) expression, changes in keratin 7 (KRT7) expression, changes in SOX9 expression, changes in MUC1 expression, changes in INS expression, changes in GCG expression, changes in AMY expression, changes in ALB expression, changes in CYP3A4 expression, changes in HNF4A expression, changes in cellular keratin 8 (K8) expression. In some embodiments, the changes may be changes in the expression of KRT20. In some embodiments, the changes may be changes in the expression of FABP1. In some embodiments, the changes may be changes in the expression of MUC2. In some embodiments, the changes may be changes in the expression of MUC5B. In some embodiments, the changes may be changes in the expression of TFF3. In some embodiments, the changes may be changes in the expression of ALPI. In some embodiments, the changes may be changes in the expression of SI. In certain embodiments, the variation may be a variation in the expression of CEACAM7. In certain embodiments, the variation may be a variation in the expression of keratin 19 (KRT19). In certain embodiments, the variation may be a variation in the expression of keratin 7 (KRT7). In certain embodiments, the variation may be a variation in the expression of SOX9. In certain embodiments, the variation may be a variation in the expression of MUC1. In certain embodiments, the variation may be a variation in the expression of INS. In certain embodiments, the variation may be a variation in the expression of GCG. In certain embodiments, the variation may be a variation in the expression of AMY. In certain embodiments, the variation may be a variation in the expression of ALB. In certain embodiments, the variation may be a variation in the expression of CYP3A4. In certain embodiments, the variation may be a variation in the expression of HNF4A. In certain embodiments, the changes may be changes in the expression of cytokeratin 8 (K8). In certain embodiments, the changes may be changes in the expression of cytokeratin 18 (K18). In certain embodiments, the changes may be changes in the expression of cytokeratin 5 (K5). In certain embodiments, the changes may be changes in the expression of cytokeratin 14 (K14). In certain embodiments, the changes may be changes in the expression of smooth muscle actin (SMA). In certain embodiments, the changes may be changes in the expression of MUC5AC. In certain embodiments, the changes may be changes in the expression of MUC6.

In certain embodiments, tissue-derived epithelial organoid cultures or monolayer cultures may be analyzed by flow cytometry, RNA and protein expression, interleukin and metabolite measurements in organoids and culture media, cell viability and proliferation assays, microscopy to monitor epithelial and immune cell motility, barrier function, migration, proliferation, subcellular localization of RNA, proteins and organelles, cell stiffness, and other assays in order to determine changes in tissue-derived epithelial organoids, cells and/or monolayers or monolayers of tissue-derived epithelial organoids. Tissue-derived epithelial organoids, cells of tissue-derived epithelial organoids, cell monolayers, and/or cells of cell monolayers can be labeled with dyes or transfected with nucleic acids encoding fluorescent proteins and/or luciferases to enable visualization and quantification of cells using imaging and bioluminescence assays.

In certain embodiments, the present disclosure provides methods for identifying therapeutic agents that are effective in treating a disease. In certain embodiments, the method may include (i) contacting (a) a tissue-derived epithelial organoid (or a composition thereof), (b) a population of tissue-derived epithelial organoids (or a composition thereof), (c) a cell monolayer from a tissue-derived epithelial organoid, or (d) a population of tissue-derived epithelial organoids with a therapeutic agent, and (ii) analyzing the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for changes that indicate effectiveness and/or toxicity of the therapeutic agent. In certain embodiments, the changes indicate effectiveness of the therapeutic agent. In certain embodiments, the changes indicate toxicity of the therapeutic agent. For example, but not limited to, if the viability of the tissue-derived epithelial organoid or cells of the tissue-derived epithelial organoid is reduced in the presence of the therapeutic agent as compared to the viability of tissue-derived epithelial organoid or cells of the tissue-derived epithelial organoid not treated with the therapeutic agent, then that indicates that the therapeutic agent is toxic.

In certain embodiments, the present disclosure provides methods for performing genomic screening. In certain embodiments, the method may include (i) providing (a) a tissue-derived epithelial organoid (or a composition thereof), (b) a population of tissue-derived epithelial organoids (or a composition thereof), or (c) a cell monolayer from a tissue-derived epithelial organoid, (ii) generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid or the cell monolayer, and (iii) analyzing the changes associated with the mutation in the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids. For example, but not limited to, if a change is observed in an epithelial organoid or a cell monolayer comprising one or more cells of a tissue-derived organoid having a genome mutation compared to a reference cell- or tissue-derived epithelial organoid, this indicates that the change is a result of the genome mutation. V. Systems

The disclosure further provides systems for use in the disclosure. In certain embodiments, the disclosure further provides systems for performing the methods disclosed herein. In certain embodiments, the disclosure provides systems for generating tissue-derived epithelial organoids, such as tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, or trophoblastic lining organoids. In certain embodiments, the disclosure provides systems for identifying therapeutic agents that are effective in treating diseases. In certain embodiments, the disclosure provides systems for performing genomic screening. In certain embodiments, the present disclosure provides systems for generating epithelial cell models.

In certain embodiments, the systems of the present disclosure may include robots and/or automated components capable of performing the presently disclosed methods. In certain embodiments, the systems of the present disclosure include robots and/or automated components capable of generating tissue-derived epithelial organoids. In certain embodiments, the systems of the present disclosure include one or more robots and/or automated components for performing one or more steps of the disclosed methods for generating tissue-derived epithelial organoids. Non-limiting examples of robots and/or automated components include automated liquid handlers ( e.g. , liquid handling robots), 3D printers, syringe pumps, or combinations thereof. In certain embodiments, the systems of the present disclosure include one or more automated liquid handlers.

In certain embodiments, robots and/or automated components are used to perform the presently disclosed methods and/or generate tissue-derived epithelial organoids for use in high-throughput studies. In certain embodiments, the systems of the present disclosure may include one or more robots and/or automated components for performing high-throughput methods, for example , as described herein. For example, but not limited to, the systems of the present disclosure may include one or more robots and/or automated components that can be used to perform any of the methods of use disclosed herein. In certain embodiments, the systems of the present disclosure may include one or more robots and/or automated components for screening agents ( e.g. , therapeutic agents) and for performing genomic screening using the disclosed tissue-derived epithelial organoids.

In some embodiments, the robot and/or automated component is capable of passaging tissue-derived epithelial organoid cultures. In some embodiments, the automated robot can perform the method described in "Organoid Maintenance" of Example 1 of the present disclosure, for example , performing any of the following steps: adding TrypLE Express, heating the organoid culture, grinding the organoid culture to dissociate cells, adding PBS, sedimenting cells, resuspending cells in BME, cooling the culture, flattening cells, covering the organoid with culture medium, or a combination thereof. In some embodiments, the system of the present disclosure may include one or more robots and/or automated components for dissociating cells. In certain embodiments, the systems disclosed herein may include one or more robots and/or automated components for performing medium changes, for example, during organoid maintenance or during the generation of tissue-derived epithelial organoids.

In some embodiments, the robot and/or automated component can produce BOBA and/or SOBA. In some embodiments, the automated robot can perform the method described in "suspended hydrogel BOBA culture" and/or "suspended hydrogel SOBA fragment culture" of Example 1 of the present disclosure, for example , performing any of the following steps: heating the culture medium, dispensing the organoid cell-BME solution as droplets into the warm culture medium to produce BOBA, dispensing the organoid cell-BME solution into the warm culture medium in a linear, serpentine or spiral motion in the XY plane to produce SOBA, grinding the SOBA filament culture to produce SOBA fragments, changing the medium or a combination thereof. In certain embodiments, the systems of the present disclosure may include one or more robots and/or automated components for dispensing the organoid cell-BME solution into the warm culture medium to produce BOBA and/or SOBA. In certain embodiments, the systems of the present disclosure may include one or more liquid handling robots for dispensing the organoid cell-BME solution into the warm culture medium to produce BOBA and/or SOBA. In certain embodiments, the systems of the present disclosure may include one or more robots and/or automated components for performing medium replacement.

In certain embodiments, the disclosed systems may include tissue-derived epithelial organoids, groups of tissue-derived epithelial organoids, and/or compositions comprising tissue-derived epithelial organoids, such as tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, or trophoblastic lining organoids. In certain embodiments, the tissue-derived epithelial organoids are selected from the group consisting of lung organoids, gastrointestinal organoids, liver organoids, pancreatic organoids, breast organoids, and combinations thereof. In some embodiments, the system includes tissue-derived epithelial organoids produced from one or more tissue-derived epithelial stem cells. In some embodiments, the tissue-derived epithelial organoids are provided in the form of a hydrogel. In some embodiments, the tissue-derived epithelial organoids may be embedded in a hydrogel and suspended in a culture medium. Non-limiting examples of tissue-derived epithelial organoids (or components thereof) or methods of producing such tissue-derived epithelial organoids are disclosed in Sections II and III, respectively. In some embodiments, the tissue-derived epithelial organoids may be provided in a container, such as a culture container. In some embodiments, the tissue-derived epithelial organoids may be provided in a culture container, such as a flask and/or a multiwell dish.

In some embodiments, the systems of the present disclosure may further include instructions for using tissue-derived epithelial organoids to determine whether a therapeutic agent to be tested is effective in treating a disease. In some embodiments, the systems of the present disclosure may further include instructions for using tissue-derived epithelial organoids to determine the effects of genomic mutations. In some embodiments, the systems of the present disclosure may further include instructions for generating epithelial cell models.

In certain non-limiting embodiments, the systems of the present disclosure may further include one or more agents and other components ( e.g. , dyes, antibodies, primers, probes, etc.) to determine changes, such as protein or nucleic acid expression, changes in tissue-derived epithelial organoids, cells of tissue-derived epithelial organoids, cell monolayers, or cells of cell monolayers. Non-limiting examples of such changes are disclosed in Section III. In certain embodiments, the systems of the present disclosure may include one or more robots and/or automated components for analyzing changes in tissue-derived epithelial organoids, cells of tissue-derived epithelial organoids, cell monolayers, or cells of cell monolayers. VI. Exemplary Embodiments A. The presently disclosed subject matter provides a method for producing tissue-derived epithelial organoids, the method comprising: a) contacting tissue-derived epithelial stem cells with hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture; b) suspending the hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture; and c) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to produce tissue-derived epithelial organoids. A1. The method of A, wherein a plurality of tissue-derived epithelial stem cells are contacted with the hydrogel to produce the hydrogel-tissue-derived epithelial stem cell mixture. A2. The method of A1, wherein the plurality of tissue-derived epithelial stem cells comprise about 1 × 10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1 × 10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. A3. The method of A1 or A2, wherein the plurality of tissue-derived epithelial stem cells are contained in tissue fragments, organoid fragments, or a combination thereof. A4. The method of A to A2, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells are isolated from native epithelial tissue. A5. The method of any one of A to A4, wherein the hydrogel solidifies when in contact with the culture medium. A6. The method of any one of A to A5, wherein suspending the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium comprises immersing a dispensing device containing the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium and dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the culture medium. A7. The method of any one of A to A6, wherein the temperature of the culture medium is about 25°C to about 50°C. A7-1. The method of any one of A to A7, wherein the temperature of the culture medium is about 30°C to about 50°C. A8. The method of any one of A to A7-1, wherein the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is about 2°C to about 25°C. A8-1. The method of any one of A to A8, wherein the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is about 2°C to about 20°C. A9. The method of any one of A to A8-1, wherein the hydrogel is selected from the group consisting of: synthetic hydrogels, natural hydrogels, and combinations thereof. A10. The method of A9, wherein the natural hydrogel comprises basement membrane extract (BME) or extracellular matrix (ECM) components. A11. The method of any one of A to A10, wherein the hydrogel has a storage modulus G' equal to or greater than the loss modulus G''. A12. The method of any one of A to A11, wherein the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width and/or diameter greater than about 0.1 mm. A13. The method of A12, wherein the suspended hydrogel-tissue derived epithelial stem cell mixture has a geometric shape comprising a length, width and/or diameter of about 0.1 mm to about 1,000 mm. A14. The method of any one of A to A13, wherein the hydrogel-tissue derived epithelial stem cell mixture is suspended in the culture medium in small drops. A15. The method of any one of A to A13, wherein the suspended hydrogel-tissue derived epithelial stem cell mixture has a filamentous structure. A16. The method of A15, wherein the filamentous structure has a linear, serpentine or spiral shape. A17. The method of any one of A to A16, further comprising fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce a fragmented structure comprising the tissue-derived epithelial organoid. B. A method for producing a suspended culture of tissue-derived epithelial organoids, the method comprising: a) introducing a mixture comprising hydrogel and tissue-derived epithelial stem cells into a culture medium to produce a suspended mixture; and b) culturing the suspended mixture in the culture medium to produce the tissue-derived epithelial organoids in a suspended state. B1. The method of B, wherein the mixture introduced into the culture medium comprises the hydrogel and a plurality of tissue-derived epithelial stem cells. B2. The method of B1, wherein the plurality of tissue-derived epithelial stem cells comprises about 1 × 10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1 × 10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. B3. The method of B1 or B2, wherein the plurality of tissue-derived epithelial stem cells are contained in tissue fragments, organoid fragments, or a combination thereof. B4. The method of any one of B to B2, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells are isolated from native epithelial tissue. B5. The method of any one of B to B4, wherein the hydrogel solidifies upon contact with the culture medium. B6. The method of any one of B to B5, wherein introducing the mixture into the culture medium comprises immersing a dispensing device containing the mixture in the culture medium and dispensing the mixture into the culture medium. B7. The method of any one of B to B6, wherein the temperature of the culture medium is about 25°C to about 50°C. B7-1. The method of any one of B to B7, wherein the temperature of the culture medium is about 30°C to about 50°C. B8. The method of any one of B to B7-1, wherein the temperature of the mixture is about 2°C to about 25°C. B8-1. The method of any one of B to B8, wherein the temperature of the mixture is about 2°C to about 20°C. B9. The method of any one of B to B8-1, wherein the hydrogel is selected from the group consisting of: synthetic hydrogels, natural hydrogels, and combinations thereof. B10. The method of B9, wherein the natural hydrogel comprises basement membrane extract (BME) or extracellular matrix (ECM) components. B11. The method of any one of B to B10, wherein the hydrogel has a storage modulus G' that is equal to or greater than the loss modulus G''. B12. The method of any one of B to B11, wherein the suspended mixture has a geometric shape comprising a length, width and/or diameter greater than about 0.1 mm. B13. The method of B12, wherein the suspended mixture has a geometric shape comprising a length, width and/or diameter from about 0.1 mm to about 1,000 mm. B14. The method of any one of B to B13, wherein the mixture is introduced into the culture medium as a droplet. B15. The method of any one of B to B3, wherein the mixture is introduced into the culture medium in the form of a filamentary structure. B16. The method of B15, wherein the filamentary structure has a linear, serpentine or spiral shape. B17. The method of any one of B to B16, further comprising fragmenting the suspended mixture to produce fragmented structures comprising the tissue-derived epithelial organoid. B18. The method of any one of A to B17, wherein the culture medium is present in a container selected from the group consisting of a culture dish, a multi-well dish, a conical tube, a container, a culture bag, a bioreactor or a flask. C. A method for generating a suspension culture of tissue-derived epithelial organoids, the method comprising: a) contacting tissue-derived epithelial stem cells with hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture; b) depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate; c) solidifying the hydrogel-tissue-derived epithelial stem cell mixture to generate a solidified hydrogel-tissue-derived epithelial stem cell mixture; d) suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture; and e) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to produce tissue-derived epithelial organoids. C1. The method of C, wherein a plurality of tissue-derived epithelial stem cells are contacted with the hydrogel to produce the hydrogel-tissue-derived epithelial stem cell mixture. C2. The method of C1, wherein the plurality of tissue-derived epithelial stem cells comprises about 1 × 10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1 × 10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. C3. The method of C1 or C2, wherein the plurality of tissue-derived epithelial stem cells are contained in tissue fragments, organoid fragments, or a combination thereof. C4. The method of any one of C to C2, wherein the tissue-derived epithelial stem cells or the plurality of tissue-derived epithelial stem cells are isolated from native epithelial tissue. C5. The method of any one of C to C3, further comprising removing the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate before suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the culture medium. C6. The method of any one of C to C4, wherein the hydrogel is selected from the group consisting of: synthetic hydrogels, natural hydrogels and combinations thereof. C7. The method of C6, wherein the natural hydrogel comprises basement membrane extract (BME) or extracellular matrix (ECM) components. C8. The method of any one of C to C7, wherein the hydrogel has a storage modulus G' that is equal to or greater than the loss modulus G''. C9. The method of any one of C to C8, wherein the cured hydrogel-tissue derived epithelial stem cell mixture has a geometry comprising a length, width and/or diameter greater than about 0.1 mm. C10. The method of C9, wherein the cured hydrogel-tissue derived epithelial stem cell mixture has a geometry comprising a length, width and/or diameter of about 0.1 mm to about 1,000 mm. C11. The method of any one of C to C10, wherein the hydrogel-tissue derived epithelial stem cell mixture is deposited onto the substrate as a droplet. C12. The method of any one of C to C10, wherein the hydrogel-tissue-derived epithelial stem cell mixture is deposited on the substrate to have a filamentous structure. C13. The method of C12, wherein the filamentous structure has a linear, serpentine or spiral shape. C14. The method of any one of C to C13, further comprising fragmenting the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to produce a fragmented structure comprising the tissue-derived epithelial organoid. C15. The method of any one of C to C14, wherein the tissue-derived epithelial organoid has a uniform morphology compared to a reference tissue-derived epithelial organoid, wherein the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate. C16. The method of C15, wherein the tissue-derived epithelial organoid has a uniform size. C17. The method of C16, wherein the average diameter of the tissue-derived epithelial organoid is more uniform than that of the reference tissue-derived epithelial organoid. C18. The method of any one of A to C17, wherein the stem cell and/or proliferation marker is expressed at a higher level in a population of epithelial organoids derived from tissues compared to a population of epithelial organoids derived from reference tissues, wherein the epithelial organoids derived from reference tissues are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. C19. The method of C18, wherein the stem cell and/or proliferation marker is selected from the group consisting of: MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. C20. The method of any one of A to C18, wherein the differentiation marker is expressed at a lower level in a population of epithelial organoids derived from tissues compared to a population of epithelial organoids derived from reference tissues, wherein the epithelial organoids derived from reference tissues are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. C21. The method of C20, wherein the differentiation marker is selected from the group consisting of: keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA) and combinations thereof. D. A tissue-derived epithelial organoid produced by the method of any one of A to C21. D1. The tissue-derived epithelial organoid of D, wherein the tissue-derived epithelial organoid is selected from the group consisting of tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblastic lining organoids, and combinations thereof. E. A composition comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoid is embedded in a hydrogel suspended in the culture medium. E1. The composition of E, wherein the hydrogel has a geometry comprising a length, width and/or diameter greater than about 0.1 mm. E2. The composition of E1, wherein the hydrogel has a geometry comprising a length, width and/or diameter from about 0.1 mm to about 1,000 mm. E3. The composition of any one of E to E2, wherein the hydrogel is a droplet. E4. The composition of any one of E to E2, wherein the hydrogel has a filamentous structure. E5. The composition of E4, wherein the filamentous structure has a linear, serpentine or helical shape. E6. The composition of any one of E to E5, wherein the stem cell and/or proliferation marker is expressed at a higher level in a population of epithelial organoids derived from tissues compared to a population of epithelial organoids derived from reference tissues, wherein the epithelial organoids derived from reference tissues are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. E7. The composition of E6, wherein the stem cell and/or proliferation marker is selected from the group consisting of: MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. E8. The composition of any of E to E5, wherein a differentiation marker is expressed at a lower level in a population of epithelial organoids derived from tissues compared to a population of epithelial organoids derived from reference tissues, wherein the epithelial organoids derived from reference tissues are embedded in a hydrogel attached to a substrate. E9. The composition of E8, wherein the differentiation marker is selected from the group consisting of: keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA) and a combination thereof. F. A method for screening an agent, the method comprising: a) contacting a tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids as D or D1, or a composition as any one of E to E9, with an agent; and b) analyzing the tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids for changes indicative of the effectiveness and/or toxicity of the agent. F1. The method of F, wherein the agent is contacted with the tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids for about 1 minute to about 3 years. F1-1. The method of F1, wherein the agent is contacted with the tissue-derived epithelial organoid or a population of such tissue-derived epithelial organoids for about 15 minutes to about 3 years. F2. The method of F, F1 or F1-1, wherein the agent is a therapeutic agent. F3. The method of F2, wherein the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell-based therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof. G. A method for performing genomic screening, the method comprising: a) providing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids as described in D or D1 or a composition as described in any one of E to E9; b) generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid; and c) analyzing the tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids for changes associated with the mutation. G1. The method of G, wherein the mutation is generated using a gene regulation system. G2. The method of G1, wherein the gene regulation system is a gene editing system. G3. The method of G2, wherein the gene editing system is a CRISPR system. G4. The method of any one of F to G2, wherein the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redox potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, repression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof. H. A method for generating an epithelial cell model, the method comprising: a) providing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids as in D; b) digesting the tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids into single cells; and c) culturing the single cells in a culture medium to generate a cell monolayer. H1. The method as in H, wherein the single cells are cultured on a permeable cell culture inner. H2. The method as in H or H1, wherein the culture medium is a differentiation medium. H3. The method as in H or H1, wherein the culture medium is a cell growth or stem cell promotion medium. I. A method for screening an agent, the method comprising: a) contacting a cell monolayer produced by any of the methods of H to H3 with an agent; and b) analyzing the cell monolayer for changes indicative of the effectiveness, distribution and/or toxicity of the agent. I1. The method of I, wherein the agent is contacted with the cell monolayer for about 1 minute to about 3 years. I1-1. The method of I1, wherein the agent is contacted with the cell monolayer for about 15 minutes to about 3 years. I2. The method of I, I1 or I1-1, wherein the agent is a therapeutic agent. I3. The method of I2, wherein the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof. J. A method for performing genome screening, the method comprising: a) providing a cell monolayer produced by a method as described in any one of H to H3; b) generating a mutation in the genome of one or more cells in the cell monolayer; and c) analyzing the cell monolayer for changes associated with the mutation. J1. The method of J, wherein the mutation is generated using a gene regulation system. J2. The method of J1, wherein the gene regulation system is a gene editing system. J3. The method of J2, wherein the gene editing system is a CRISPR system. J4. The method of any one of J to J3, wherein the change is a change in a property, the property being selected from the group consisting of: cell viability, cell metabolism, redox potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, repression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof. J1. The method of any one of A to C21 and F to J4 or the composition of any one of E to E9, wherein the tissue fragment is a fragment from a tissue selected from the group consisting of lacrimal glands, tonsils, salivary glands, gastrointestinal tissue, thyroid, lung, breast, liver, bile duct, stomach, kidney, pancreas, endometrium, fallopian tube, cervix, prostate, bladder, taste buds, ovaries, placenta and combinations thereof. J5. The method of any one of A to C21 and F to J4 or the composition of any one of E to E9, wherein the tissue-derived epithelial stem cells are obtained from fragments of organoids, the organoids being selected from the group consisting of tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, gastric organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblastic organoids, and combinations thereof. K. A system for culturing tissue-derived epithelial organoids, the system comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium. K1. The system of K, wherein the tissue-derived epithelial organoids are intestinal organoids. K2. The system of K or K1, wherein the hydrogel has a geometry comprising a length, width, and/or diameter greater than about 0.1 mm. K3. The system of any one of K to K2, wherein the hydrogel has a geometry comprising a length, width, and/or diameter of about 0.1 mm to about 1,000 mm. K4. The system of any of K to K3, wherein the hydrogel is a droplet. K5. The system of any of K to K3, wherein the hydrogel has a filamentous structure. K6. The system of K5, wherein the filamentous structure has a linear, serpentine, or helical shape. K7. The system of any of K to K6, wherein stem cell and/or proliferation markers are expressed at higher levels in a population of epithelial organoids derived from tissues compared to a population of epithelial organoids derived from reference tissues, wherein the epithelial organoids derived from reference tissues are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. K8. The system of K7, wherein the stem cell and/or proliferation marker is selected from the group consisting of: MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. K9. The system of any one of K to K6, wherein the differentiation marker is expressed at a lower level in a population of epithelial organoids derived from tissues compared to a population of epithelial organoids derived from reference tissues, wherein the epithelial organoids derived from reference tissues are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. K10. A system as described in K9, wherein the differentiation marker is selected from the group consisting of: keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA) and combinations thereof. K11. The system of any one of K to K10, wherein the tissue-derived epithelial organoid is selected from the group consisting of tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblastic lining organoids, and combinations thereof. K12. The system of any one of K to K11, further comprising a robot and/or automated components. K13. A system as in K12, wherein the robot and/or automated component comprises a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof. L. A method as in any one of A to C21 and F to J5, wherein one or more steps of the method are performed by a robot and/or automated component. L1. A method as in L, wherein the robot and/or automated component comprises a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof. L2. A method as in L or L1, wherein the robot and/or automated component is a liquid handling robot. M. A method for producing epithelial organoids derived from tissues as in D to D1 or compositions as in E to E9, wherein one or more steps of the method are performed by a robot and/or automated component. M1. The method of M, wherein the robot and/or automated component comprises a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof. N. The method of any one of B to B18, wherein the mixture comprising the hydrogel and the tissue-derived epithelial stem cells is introduced into the culture medium to produce a suspended mixture by a robot and/or an automated component. N1. The method of any one of B to B18, wherein the suspended mixture is cultured in the culture medium to produce the tissue-derived epithelial organoids in a suspended state by a robot and/or an automated component. N2. The method of N or N1, wherein the robot and/or automated component comprises a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof. N3. The method of N2, wherein the robot and/or automated component comprises one or more liquid handling robots.

The subject matter disclosed herein will be better understood by reference to the following examples, which are provided by way of illustration and not by way of limitation of the subject matter disclosed herein.

Examples 1 ：Formation of intestinal organoids

This example provides a method for culturing intestinal organoids in suspended basement membrane extract (BME) hydrogels in a variety of geometries. The method simplifies the protocol, increases scalability, enables kinetic sampling, and improves culture homogeneity without the need for specialized equipment or additional expertise. The method is compatible with a variety of culture formats, and organoids generated by this method can be used in downstream applications such as performing mid-throughput drug screening and generating Transwell monolayers for barrier assessment, as shown in Examples 2 and 3. The suspended BME hydrogel culture method enables the use of intestinal organoids to a wider range of uses and at higher throughputs than previously possible.

method

Human intestinal organoids source. Organoids were sourced as previously described (Pleguezuelos-Manzano et al. (2020)) with some modifications. Unidentified human colon and ileum tissue samples from deceased donors were obtained through the Western Donor Network. Tissues were washed with Advanced DMEM/F12 medium (ThermoFisher), cut into 5 cm x 5 cm segments, and the epithelium was scraped from the submucosal layer into the medium and minced with a razor blade. The solution was pelleted at 450 x g for 5 min and then incubated in 2.5 µM EDTA in PBS without Mg ²⁺ or Ca ²⁺ at 37°C for 9 min (ileum) or 12 min (colon), vortexing every 3 to 4 min until the crypts were released. The crypts were pelleted at 450 x g for 5 min, washed in PBS, filtered through sterile gauze, then filtered through a 100 µm cell filter to remove debris, and pelleted at 450 x g for 5 min. Nests were resuspended in CULTREX® Reduced Growth Factor Basement Membrane Matrix Type II (BME, R&D Systems Catalog No. 3533-010-02) on ice, placed in 50 µL domes in 24-well plates, solidified at 37°C for 15 to 30 minutes, and then overlaid with 500 µL of either colonic medium (Intesticult Organoid Growth Medium (OGM, StemCell Technologies Catalog No. 06010) + 10 µM Y27632) or ileal medium (OGM + 10 µM Y27632 + 2.5 µM CHIR99021). After the first 2 to 3 days in culture, change the medium every 2 to 3 days or when the medium turns yellow, to plain OGM for colon cultures and to ileal medium for ileal cultures.

Organoid maintenance. Organoid cultures were passaged every 1 to 2 weeks by digestion with TrypLE Express (ThermoFisher) at 37°C for 10 min, followed by trituration with a P1000 pipette. If necessary, the culture was repeated up to 2 times to obtain a single-cell suspension. TrypLE Express was quenched by diluting with PBS and the cells were pelleted at 450 x g for 3 min. Cells were resuspended at 6 x 10 ⁵ cells/mL in BME on ice, plated in 50 µL domes in a 24-well plate, and fixed at 37°C for 15 to 30 min. For the first 2 to 3 days, cover the domes with colon passage medium or ileum medium and replace the medium with plain OGM for colon or ileum medium every 2 to 3 days. For colon organoid differentiation, wash the cultures with Advanced DMEM/F12 medium and then cover with Intesticult Organoid Differentiation Medium (ODM, StemCell Technologies Catalog No. 100-0214) + 5 µM DAPT for 5 days, replacing the medium every 2 to 3 days.

Suspended hydrogel BOBA cultures. Single organoids in BME were prepared on ice as described above. Pre-warmed colon passage medium or ileal medium was added to 6-well plates (5 mL/well), 100 cm culture dishes (15-30 mL/dish), or 50 mL conical tubes (30 mL/tube) and maintained in a warm bead bath at 37°C. Ultra-low attachment (ULA) or standard tissue culture dishes produced similar results. Figure 10 provides an exemplary schematic showing the generation of BOBA. To generate suspended BME droplets or BOBAs, dispense the organoid cell-BME solution in 10 µL volumes directly into the warm medium using an electronic continuous pipette (Integra VIAFLO 300) with a wide-bore or cut pipette tip (opening approximately 2 mm) at a slow to moderate speed to avoid the formation of threads or ribbons instead of droplets. During the dispensing process, the tip is immediately dipped below the surface of the liquid and then lifted after each dispense to ensure the separation of the droplets. For larger cultures, transfer the BOBA to a flask by serological pipette or decanting. Replace the medium with normal OGM for colon or ileum medium every 2 to 3 days. In 6-well plate cultures, place a sterile 70 µm cell filter into the well, tilt the plate and gently aspirate 4 mL of medium through the filter. In flask cultures, tilt the flask at an angle, place BOBA in a corner, and replace approximately 2/3 of the volume of used medium with a serological pipette.

Suspended hydrogel SOBA and SOBA fragment cultures. Single organoid cells in BME were prepared on ice as described above. Prewarmed colon passage medium was added to 6-well plates (5 mL/well), 100 cm culture dishes (15-30 mL/dish) and kept on a warm bead bath. Figure 11 provides an exemplary schematic showing the generation of SOBA filaments. To generate suspended SOBA filaments, the cell-BME solution was gently aspirated into a syringe with a 15-gauge blunt-tip needle and then extruded directly into the warm medium while moving the submerged needle in a linear, serpentine, or spiral motion in the XY plane. Extrusion speed and motion in the XY plane can affect filament length and/or width. To generate SOBA fragments, gently triturate SOBA filament cultures twice using a 10 mL serological pipette or wide-bore P1000 pipette tip. Add additional medium to achieve a final BME to medium ratio of 1:10. Perform medium changes as described above for BOBA cultures.

Bright field microscopy and image analysis. Cultures were imaged by bright field microscopy using a THUNDER DMi8 inverted light microscope (Leica) with 2.5X, 4X, or 10X objectives and a DFC9000 GTC camera (Leica). Images were analyzed using Imaris image analysis software and Imaris Batch software suite (Oxford Instruments). For automated organoid diameter measurements, the Imaris Surfaces detection module was used with inverted bright field images. Background and out-of-focus organoids were excluded using a background subtraction step (rolling ball, diameter 19.5 µm). The detected surfaces were then filtered according to four criteria: software-specified quality metrics (>3000 AU), minor axis length (>35 µm - to exclude debris), rectangular circularity (>0.2 - to exclude shadows), and major axis length (between 35 µm and 600 µm, to exclude false detections of overlapping organoids). In the remaining surfaces (at least 40 per analyzed image), the diameter was reported as the longest side of the smallest object oriented bounding box. This process was then performed in batches on all analyzed images. The average was calculated for each image and plotted for three total experiments with n = 3 replicates.

Spatial organoid homogeneity Organoid diameter analysis was performed using FIJI (ImageJ). A rectangular (1.5 mm x 9 mm) ROI was set at the center of each image and divided into 1 mm sections in the X-axis. For each section, the diameter across the widest point of each organoid was manually measured.

Immunofluorescence Sample Preparation and Confocal Microscopy. 24-well dome cultures were fixed with 2% paraformaldehyde (PFA) in PBS. Domes were detached from the plate using a spatula and transferred to a microcentrifuge tube using a cut P1000 pipette tip. For BOBA cultures, 500 µL of culture was transferred to a microcentrifuge tube using a cut P1000 pipette tip, the medium was removed and 2% PFA in PBS was added. Samples were incubated in fixative at room temperature for 15 to 30 minutes and then washed three times in PBS. Samples were stained in microcentrifuge tubes with primary antibodies diluted in blocking/permeabilization buffer (3% BSA, 0.1% Triton X-100, 0.02% sodium azide in PBS) for at least 4 hours at room temperature and then washed three times in PBS. The primary antibodies used were as follows: ɑ-Ki67 (Invitrogen Catalog No. MA5-14520), ɑ-MUC2 (Millipore Catalog No. MABF1989), ɑ-FABP1 (Novus Catalog No. NBP-87695), and ɑ-CHGA (Novus Catalog No. NB120-15160). Samples were then incubated with secondary antibodies (donkey ɑ-rabbit Alexa Fluor 488 (ThermoFisher Catalog No. A-21206) or goat ɑ-mouse Alexa Fluor 594 (ThermoFisher Catalog No. A-11032)), DAPI, and AlexaFluor 660 agaricus diluted in blocking/permeabilization buffer at room temperature for at least 2 hours. Images were collected on a Stellaris 8 confocal microscope (Leica) using a 40X objective and 3D reconstructions were performed using Imaris image analysis software (Oxford Instruments).

Transcriptome analysis. For RNA isolation, the RNeasy Micro Plus kit (Qiagen) was used. RLT+ lysis buffer was added to BME domes or pelleted BOBA and stored at -80°C. RNA isolation was performed using QiaCube Connect (Qiagen), and RNA was quantified using Nanodrop 8000 (ThermoFisher). Bulk mRNA-seq (NovaSeq PE150) and analysis were performed by Novogene. Reads were aligned using HISAT2 (Mortazavi et al. 2008), differential gene expression analysis was performed using DESeq2 (Anders et al. 2014), and statistical significance was calculated using a negative binomial distribution model with Benjamini-Hochberg FDR correction.

Variability of BME organoids suspended in 96 -well plates. After 9 days of culture, colon organoid SOBA fragments in 225 ^cm2 flasks were collected and gently triturated twice using a serological pipette to homogenize the sample without disrupting the intact organoids. The organoid mixture was transferred to a reagent container and then plated at 100 µL/well in a 96-well plate using a P200 multichannel pipette with a wide-bore pipette tip. Dome cultures were prepared as described above and plated in a 5 µL dome in the center of each well of a 96-well plate using a continuous pipette. Cultures were grown for 7 days before viability measurements. All viability measurements were performed using the Cell Titer Glo 3D Assay kit (Promega), and luminescence was measured on an Ensight microplate reader (Perkin Elmer).

Statistical analysis. Unless otherwise stated, all statistical analyses were performed using Prism 9 software (Graphpad). Statistical tests, n, and p values are indicated in the figure legends.

result:

Suspended BME hydrogel method for human intestinal organoid culture. The conventional intestinal organoid culture method requires that a solution of organoid cells in cold ECM be deposited onto a plastic surface, solidified in an incubator to form a hydrogel dome, and then covered with growth medium (Figure 1A-1B) (Mahe et al. (2013); Pleguezuelos-Manzano et al. (2020); Sato et al. (2009); Sato et al. (2011)). To address the limitations of scaling up this technique, a method was developed in which the cold cell-ECM solution immediately solidifies into a floating hydrogel suspended in warm medium. A method has been demonstrated using suspended droplets of BME, an Engelbreth-Holm-Swarm (EHS) cell-derived ECM equivalent to MATRIGEL®, and is termed BOBA (BME Embedded Organoid Bead Assembly) culture.

Intestinal organoid growth was first assessed in a suspended BOBA culture method and compared to the conventional surface-attached BME dome method (referred to here as dome culture). For dome culture, single cells from digested organoids were suspended in cold BME and plated as 50 µL domes in a 24-well plate, solidified in a 37°C incubator for 15 to 30 minutes, and then covered with growth medium (Intesticult Organoid Growth Medium). Medium changes were performed on individual wells every 2 to 3 days. For BOBA culture, single-cell BME solutions are deposited as 10 µL drops directly into pre-warmed medium using an electronic continuous pipette with a wide-bore tip, where the hydrogel immediately solidifies into suspended BOBA. BOBA can be produced in culture dishes, plates, or conical tubes and easily transferred to larger containers, such as cell culture flasks, via serological pipette or decanting (Figure 1A-1B). Perform a medium change for the entire flask by allowing the BOBA to settle and then replacing the top 75% of the volume of spent medium with fresh medium.

Human intestinal organoid cell-BME solutions were plated in parallel domes or BOBA cultures and grown in growth medium for 9 days. Organoid growth and size were similar in both methods, as observed by bright field microscopy (Fig. 1C) and quantified by organoid diameter measurement (Fig. 1D). Organoid cell proliferation was also comparable, as determined by quantification of cell abundance expressing the proliferation marker Ki-67 (Fig. 1E-1F). The BOBA method appeared to enable the growth of more organoid cells per ^cm2 surface area, although statistical significance was only observed in small intestinal (ileal) organoids (Fig. 1G-1H). For colon organoids, dome culture yielded an average of 2.9 x 10 ⁵ viable cells/well or 1.5 x 10 ⁵ cells/cm ² in a 24-well plate, while BOBA culture yielded an average of 2.2 x 10 ⁷ viable cells/well or 2.9 x 10 ⁵ cells/cm ² in a 75 cm ² flask (Figure 1G). For ileal organoids, dome culture yielded an average of 4.8 x 10 ⁵ viable cells/well or 2.6 x 10 ⁵ cells/cm ² in a 24-well plate, while BOBA culture yielded an average of 2.9 x 10 ⁷ viable cells/well or 3.9 x 10 ⁵ cells/cm ² in a 75 cm ² flask (Figure 1H). The number of viable cells per µL of BME hydrogel was similar, indicating that the two methods were comparable in growth at a fixed seeding density (Figure 1G).

Organoid differentiation in BOBA culture. A major advantage of the intestinal organoid model is the ability to differentiate into various intestinal epithelial cell types by changing the medium composition (e.g., by removing stem cell-promoting factors) (Clevers (2016); Schutgens and Clevers (2019); Zachos et al. (2016)). Organoid differentiation in dome and BOBA culture was compared. Organoids were grown in growth medium for 7 days and then washed and transferred to differentiation medium (Intesticut Organoid Differentiation Medium containing 5 µM DAPT) for 5 days. Bright field microscopy showed that in both culture formats, proliferating organoids in growth medium exhibited a cryptic morphology with large lumens (Figure 2A) and differentiated organoids exhibited a compact spherical morphology with elongated columnar cells and small lumens (Figure 2A).

Bulk RNA-seq analysis was performed on proliferative and differentiated organoids in dome and BOBA culture. In both culture methods, differentiated organoids downregulated the expression of stem cell and proliferation markers ( MKI67 , LGR5 , SOX9 , and CD44 ) and upregulated the expression of differentiation markers for goblet cells ( MUC2 , MUC5B , TFF3 ) and intestinal epithelial cells ( KRT20 , FABP1 , ALPI , and CEACAM7 ) relative to proliferative organoids (Figure 2B). Differentiated cell types were also observed in both culture formats by immunofluorescence (IF) confocal microscopy (Figure 2C).

Characterization and Optimization of BOBA Culture Conditions. Next, we evaluated how different culture parameters would affect organoid growth in the BOBA approach. Cultures were seeded in 6-well plates (Figures 3A-3B) or 25 ^cm2 flasks (Figure 3C) at various BME volumes and a fixed cell seeding density of 6 x ¹⁰⁵ cells/mL BME. All BOBAs were produced as 10 µL droplets in 5 mL growth medium, and organoid growth was assessed by quantifying organoid diameter and number of viable cells after 9 days of culture (Figures 3B-3C). In 6-well plates, as the total BME volume per well increased from 0.5 mL to 2 mL, organoid diameter decreased and the ratio of viable cells to BME volume decreased (although not statistically significant). Although the number of cells seeded was higher under the higher BME volume conditions, the total number of viable cells and the ratio of viable cells per ^cm2 of surface area were comparable under all conditions, indicating less proliferation per seeded cell. Together, these data suggest that organoid growth is impaired when the BME volume exceeds a threshold of a fixed amount of medium in 6-well plates (Figure 3B).

Interestingly, for BOBA cultures in 25 ^cm2 flasks, organoids grew equally well under all conditions tested (Figure 3C). As the total BME volume per flask increased from 0.5 mL to 2 mL, the organoid diameter and the ratio of viable cells to BME volume were similar. The total number of viable cells and the ratio of viable cells per ^cm2 surface area appeared to increase as the BME volume in the culture increased (although not statistically significant due to inter-experimental variability), suggesting that the thresholds for BME volume and medium are different for 6-well plates and 25 ^cm2 flasks. Both the BME to medium ratio and the vessel type should be optimized for specific applications.

Organoid Homogeneity in BOBA Culture. The conventional dome approach is known to result in organoid heterogeneity (Pleguezuelos-Manzano et al. (2020); Ringel et al. (2020)). The native ECM hydrogel restricts gas and molecular diffusion, leading to nutrient gradients and heterogeneous organoid growth (Colom et al. (2014) J Biomed Mater Res A 102 , 2776–2784; Park et al. (2022); Shin et al. (2020)). Organoids were observed to grow more uniformly in BOBA culture than in dome culture (Figure 4). Colon organoid cultures were imaged by bright field microscopy at the deepest point of each format—the bottom Z plane of the dome or the center Z plane of the BOBA—and the average organoid diameter across a rectangular ROI at the center of each hydrogel was quantified (Figure 4C). Consistent with previous reports (Park et al. (2022); Shin et al. (2020)), dome-cultured organoids had larger growth at the edges of the hydrogel and smaller growth in the core (Figures 4B-4E). However, the growth size of organoids in the BOBA across the hydrogel was comparable (Figures 4B-E).

It was also observed that organoids in the core of dome cultures generally had a dense spherical morphology with less lumen, which is generally indicative of differentiation (Figure 4B). Bulk RNA-seq analysis indicated that dome-cultured organoids had lower expression of stem cell and proliferation markers and higher expression of intestinal epithelial cell markers relative to their BOBA-cultured counterparts, supporting the hypothesis that a subpopulation of differentiated organoids may exist in dome cultures (Figure 5).

Alternative geometries for suspended BME hydrogels. While the BOBA approach has significant scalability advantages over the conventional dome approach, scale-up of large-scale cultures of suspended BOBA hydrogel droplets remains labor-intensive without automated liquid handlers. To reduce the time and labor required for suspended BME hydrogel cultures, alternative hydrogel geometries, specifically hydrogel filaments, have been designed. Extruded filaments have been used in the bioprinting field to spatially control cell growth or to construct layer-by-layer assemblies of 3D hydrogel structures, but they generally rely on attachment to a surface (Kolesky et al. (2014) Adv. Mater. 26, 2966–2966; Kolesky et al. (2016) Proc National Acad Sci 113, 3179–3184). To generate hydrogel filaments containing organoid cells, called SOBAs (syringe-extruded organoid BME assemblies), cold cell-BME solutions were loaded into a syringe with a 15-gauge (1.37 mm inner diameter) blunt-tip needle and then injected directly into warm medium while moving the tip across the XY plane ( e.g. , in a linear, serpentine, or spiral pattern). Silk fragments that more closely resemble the BOBA geometry and are termed SOBA fragments were also generated by gently triturating SOBA cultures with a wide-bore P1000 tip or a 10 mL serological pipette.

Organoids grown in BOBA, SOBA, or SOBA fragment cultures for 9 days showed similar growth as measured by bright field microscopy (Figure 6A), organoid diameter measurement (Figures 6B to 6C), and viable cell counts (Figure 6D). SOBA and SOBA fragment formats produced comparable organoid growth relative to BOBA droplets while enabling faster and less laborious culture preparation. SOBA fragments were more evenly dispersed in the medium compared to SOBA cultures, making it easier to divide, sample, or aliquot a single culture.

Discuss:

This example describes the development of a suspended BME hydrogel culture method for human intestinal epithelial organoids that overcomes several challenges associated with conventional surface-attached dome hydrogel culture methods. Compared to the dome method, the BOBA, SOBA, and SOBA-filament methods simplify and expedite the protocol, enable compatibility with scalable culture vessels, allow for kinetic culture sampling, and improve organoid culture uniformity.

Several suspension culture protocols have been proposed for intestinal organoid and tumorigenesis scale-up, but in these approaches, organoids are cultured in liquid media containing low concentrations of dissolved MATRIGEL® (similar to BME) that do not form complete hydrogels (Hirokawa et al. (2021) Commun Biology 4, 1067; Price et al. (2022) Sci Rep-Uk 12, 5571). This is in contrast to the currently described method, which relies on the formation of a fully solidified insoluble hydrogel. In addition, previous studies have shown that while organoids and tumorigenesis can be grown in 5% soluble MATRIGEL® solutions, concentrations above 5% result in reduced growth. Furthermore, intestinal organoids grown in 5% MATRIGEL® exhibited reversed epithelial polarity (Hirokawa et al. (2021)), which is consistent with previous reports of organoid polarity reversal under low ECM conditions (Co et al. (2019) Cell Reports 26, 2509-2520.e4).

The BOBA method offers several advantages over the conventional dome method. First, it simplifies the protocol. Because the hydrogel cures directly in the culture medium, a separate curing incubation step is not required, saving time and labor. Second, it reduces the vessel surface area and the technical precision required to lay the dome. By reducing the reliance on available surface area, the BOBA method utilizes all 3 dimensions of the culture vessel, thereby increasing the volume of hydrogel and organoid cells per culture. The BOBA method facilitates large-scale culture because it enables compatibility with flasks, which can be larger in size and easier to handle, and allows for rapid changes of culture medium. For example, in BOBA culture, 10 mL of BME can be cultured in a single 225 ^cm2 flask, and the medium can be changed simply by replacing the supernatant with a serological pipette. However, using the dome method, the equivalent culture requires nine 24-well plates (200 wells of a 50 µL dome), and the medium needs to be changed individually for each well. This also reduces plastic consumption by more than 70% (a 225 ^cm2 flask contains 152.7 g of plastic, and replaces 562.5 g of plastic in nine 24-well plates, data not shown). Further scalability can be achieved using multi-layer flasks or "cell factories" that can accommodate multiple liters of culture volume. Table 1 provides general guidance for the setup of suspended BME hydrogel cultures for several vessel types. Culture vessel type has been reported to affect parameters such as gas transport (Allen et al. (2001) Am J Physiol-Lung C 281, L1021–L1027), and factors such as medium formulation, hydrogel composition, and organoid strain-specific growth rates can affect organoid growth. Table 1 Surface-attached BME domes Suspended BME hydrogel (BOBA, SOBA, SOBA fragments) container 24 hole plate 6 hole plate 75 cm ² Flask 75 cm ² Flask Culture medium per well (mL) 0.5-1 3-5 n/a n/a Medium volume per plate/flask (mL) 12-24 18-30 15-30 80-100 BME per well (mL) 30-50 300-700 n/a n/a BME per plate/flask (mL) 720-1200 1800-4200 1500-6000 8000-15000 Inoculation density (cells/mL) 3 x 10 ⁵ – 6.0 x 10 ⁵ 3 x 10 ⁵ – 6.0 x 10 ⁵ 3 x 10 ⁵ – 6.0 x 10 ⁵ 3 x 10 ⁵ – 6.0 x 10 ⁵ Inoculation conditions for suspended BME hydrogel cultures.

The BOBA approach also overcomes the culture heterogeneity that arises from hydrogel diffusion limitations in dome cultures (Park et al. (2022); Shin et al. (2020)). Some protocols recommend plating smaller 10-15 µL hydrogel domes (Pleguezuelos-Manzano et al. (2020); Stewart et al. (2020) Methods Mol Biology 2121, 185–198) to reduce diffusion pathways for molecular transport. However, plating smaller domes reduces the total volume of hydrogel per well, even when plating multiple domes per well. Another approach to overcome the diffusion limitations of hydrogels is to flip the dish during the hydrogel solidification step, allowing gravity to cause cells to settle near the top surface of the dome, with few or no cells in the dome core (Pleguezuelos-Manzano et al. (2020)). This results in suboptimal use of expensive hydrogel volume and ultimately fewer organoid cells can be seeded per µL of hydrogel. Complex bioengineering methods have been developed to increase hydrogel surface area to improve molecular transport (Park et al. (2022)), but these are difficult to scale and still rely on anchoring the hydrogel to a 2D surface. The BOBA approach produces more homogeneous cultures without sacrificing the amount of hydrogel per well or the number of cells per µL of hydrogel. Without being bound by a particular theory, the observed improvement in organoid culture uniformity may be explained by BOBA hydrogels having (1) smaller diameters and therefore shorter diffusion pathways, and (2) all external surfaces exposed to the culture medium, allowing for even diffusion of molecules into the hydrogel.

Another documented challenge to organoid morphological heterogeneity in hydrogel domes is that organoids located near the bottom of the dish can attach to the dish surface, unfold and flatten, and lose their 3D structure (Pleguezuelos-Manzano et al. (2020); Price et al. (2022)). Since the suspended hydrogel droplets do not come into direct contact with the dish surface, organoid unfolding and flattening do not occur.

In addition to BOBA hydrogel droplets, organoids were grown as SOBA filaments and SOBA filament fragments. Organoid growth was similar in all 3 configurations, demonstrating the robustness of the suspended BME culture method. The SOBA method facilitates and speeds up culture preparation because a large volume of cell-BME solution can be loaded into a single syringe to produce SOBA hydrogel filaments. Approximately 10 mL of SOBA can be produced in one minute, while the equivalent dome culture requires more than 15 minutes for leveling and an additional 15 to 30 minutes for solidification. Despite the much longer length of SOBAs than BOBAs, no heterogeneity in organoid growth (as observed in dome cultures) was observed, likely due to the smaller diameter of SOBAs (typically <2 mm) and the ability to efficiently transport nutrients from the culture medium. SOBA fragments more closely resemble BOBAs in size and geometry, but are generated much more rapidly. Like BOBAs, SOBA fragments are evenly dispersed throughout the culture, which may be useful for dynamic sampling or for partitioning cultures for multiple applications or readouts.

Overall, the suspended hydrogel culture method enables large-scale organoid scalability, improves organoid culture uniformity, and saves labor, time, and resources. These culture improvements make intestinal organoids more suitable for high-throughput applications such as compound or genomic screening. The method may be extended to the culture of diseased intestinal organoids and tumor-like structures, intestinal organoids from other species, and organoids from different tissue types. Therefore, BOBA, SOBA, and SOBA filaments have the potential to promote and expand the adoption of organoid technology.

Examples 2 ：The use of organoids in screening methods

This example discloses the use of intestinal organoids produced by the method of Example 1 in cytotoxicity assays.

method:

Cytotoxicity assay of suspended BME organoids in 96 -well plates. Two 96-well plates were seeded with 90 µl per well from homogenized colon organoid SOBA fragments into 225 ^cm2 flasks. Compound stocks were diluted to 10-fold final concentration in Intesticult OGM and 10 µL was added to each well. An 8-dose dilution series starting at 100 µM with 5-fold dilutions was evaluated in technical quadruplicate. After 3 days of treatment, viability was measured using the Cell Titer Glo 3D assay kit (Promega) and luminescence was measured on an Ensight ELISA reader (Perkin Elmer). 4PL fit curves were generated using Prism (GraphPad).

result:

Application of suspended BME hydrogel organoids in drug toxicity screening assays. Intestinal organoids generated as BOBA, SOBA, or SOBA fragments can be used directly in downstream assays without the need for additional organoid digestion or passaging steps. As a proof of concept, the implementation of these organoids was demonstrated in drug toxicity screening. Organoids cultured in SOBA fragments were grown in 225 ^cm2 flasks (Figures 7A-7B), homogenized by trituration with a serological pipette, and then transferred to 96-well plates (Figures 7A-7C). Inter-well variability measured by the Cell Titer Glo 3D ATP-based viability assay was comparable to SOBA filament-grown organoids and dome cultures plated in 96-well plates ( FIG. 7D ).

For drug toxicity screening, SOBA filament-grown organoids in 96-well plates were treated for 3 days with compounds known to cause enterotoxicity (diacerein, sorafenib, SN-38, or docetaxel) or DMSO vehicle control. Viability was determined as a measure of ATP and a dose-response curve was generated (Figure 7E). This is an example of how suspended BME hydrogel cultures can be used directly in downstream applications.

The utility of suspended BME hydrogel-grown cultures was demonstrated here in two applications. First, SOBA filament cultures could be homogenized to generate 96-well plate cultures with low well-to-well organoid variability. As a proof of concept, drug toxicity screening was performed; this approach can be applied to other medium- to high-throughput screens.

Examples 3 ：Generation of organoid-derived models

This example discloses the use of intestinal organoids produced by the method of Example 1 in providing an alternative organoid source model.

method:

Transwell monolayer. Collect colon organoid SOBA fragments from 225 ^cm2 flasks into 50 mL conical tubes and pellet at 800 xg for 3 min. Remove supernatant and digest organoids into single cells by incubating in TrypLE Express in a 37°C water bath for 10 min, then triturate with a P1000 pipette. Repeat incubation and trituration up to 2 times as needed. Pellet cells and wash with PBS, then resuspend in monolayer growth medium (Intesticult OGM + 10 µM Y-27632) or monolayer differentiation medium (Intesticult ODM + 10 µM Y-27632). For each well of a 96-well PET Transwell plate (pore size 0.4 µm, Corning catalog #3450), 2.0 x 10 ⁵ cells in 100 µL of medium were seeded into the apical chamber, and 200 µL of medium was added to the basal chamber. The medium in both chambers was changed every 2 to 3 days. Bright field imaging was performed using a 10X objective and a THUNDER microscope (Leica) and a DFC9000 GTC camera (Leica). Transepithelial electrical resistance (TEER) was determined by measuring the electrical resistance with a volt/ohmmeter (EVOM3, WPI) and then multiplying the resistance by the Transwell surface area (0.143 cm ² ).

result:

Suspended BME hydrogel cultures facilitate alternative organoid-derived models. In addition to using organoids directly in their suspended BME hydrogel format, these organoids can facilitate the generation of other organoid-derived models, particularly those that require large cell input, such as monolayers and microphysiological system (MPS) devices. Suspended BME hydrogel organoids were used to generate colon epithelial Transwell monolayers and assess epithelial barrier function under two conditions. SOBA filament organoids cultured in 225 ^cm2 flasks were digested into single cells, plated on 96-well Transwell inserts at confluence, and cultured for 7 days in either monolayer growth or monolayer differentiation media (Figure 8A). The two conditions produced Transwell epithelial cultures with different morphologies: in growth medium, the resulting epithelium formed 3D structures protruding from the monolayer surface, whereas in differentiation medium these structures were absent and the typical "cobblestone" cell morphology of differentiated epithelial cells with mature tight junctions was seen (Figure 8B). Consistent with this morphology, by day 3, epithelial barrier function, as quantified by transepithelial electrical resistance (TEER) measurements, was higher in Transwell monolayers in differentiation medium compared to growth medium (Figure 8C). This experiment is an example of how organoids grown using the BOBA, SOBA, or SOBA filament approach can generate an in vitro model of an alternative source of intestinal organoids.

SOBA filament organoids can be used to generate organoid-derived Transwell monolayers, which offer the advantage of access to both the apical and basolateral sides, but are difficult to scale up because they require large numbers of cells for seeding. By enabling large-scale organoid expansion, the suspended BME hydrogel culture method may facilitate the development and implementation of complex intestinal organoid-derived models with greater tissue fidelity and experimental advantages.

Examples 4 ：Generation of lung organoids

This example discloses the generation of lung organoids using the BME suspension method described herein.

method:

Human lung ATII cell isolation and organoid source. Unidentified human lung tissue from deceased donors was obtained through the Western Donor Network. Lung dissociation for single cells and organoid establishment has been described previously (Konishi et al., 2022). Briefly, tissue was washed with HBSS buffer and expanded with digestion buffer (collagenase type I: 450 units/mL; dispase: 5 units/mL; DNase I: 10 units/mL). After removal of the pleura and small airways, the remaining tissue was minced with a single-edged blade, transferred to a standard tube containing warm digestion buffer, and incubated at 37°C for a total of 1 hour with stirring and vigorous mixing every 15 minutes. Pass the solution through a 100 µm cell filter and pellet at 450 g for 10 minutes at 4°C. Resuspend the cell pellet in 5 mL ACK buffer to lyse red blood cells and incubate at room temperature for 3 to 5 minutes. Terminate the reaction by adding DMEM/F12+10%FBS. Then filter the cell suspension through a 40 µm cell filter and pellet at 450 g for 5 minutes at 4°C. The cells were then labeled with CD45 magnetic microbeads (Miltenyi Biotech, catalog number 130-045-801) and loaded onto a MACS® column, which was placed in the magnetic field of a MACS separator (according to the manufacturer's protocol). The magnetically labeled CD45+ cells were retained in the column and discarded, and the unlabeled cells that passed through the column were collected. These cells were washed in Gentle MACS buffer, pelleted at 450g for 5 minutes at 4°C, and incubated with DMEM + 10% FBS containing 50 nM Lysotracker Green (Invitrogen, catalog number L7526) at 37°C for 30 minutes. The following antibodies were added to the cells on ice for 30 minutes: pre-conjugated HTII280+Alexa 647 (Terrance Biotech; Invitrogen Catalog No. A20186); EpcamPE-Cy7 (BioLegend, Catalog No. 324222); CD45-Alexa700 (BioLegend, Catalog No. 304024), CD31-Alexa594 (BioLegend, Catalog No. 303126). The live/dead staining probe Sytox Blue (Invitrogen) was also added. Cells were then pelleted, washed, resuspended in PBS + 2% FBS, and ATII cells were isolated by sorting against Sytox Blue-, CD45-, CD31-, EpCAM+, HTII-280+, lysotracker Green+. ATII cells were resuspended in MATRIGEL® (reduced growth factor, without phenol red; Corning, catalog number 356231), plated in 50 µl domes in 6-well plates (60 x 10 ⁴ cells/mL, or 3000 cells/50 µl MATRIGEL®), fixed at 37°C for 30 min, and then overlaid with 3 ml serum-free feeder-free (SFFF) medium (Konishi et al., 2022) containing 10 µM ROCK inhibitor Y27632 (Selleckchem, catalog number S1049) for 3 days. After the first 2 to 3 days of culture, SFFF medium without ROCK inhibitor was replaced every 2 to 3 days.

Organoid maintenance. Organoid cultures were passaged every 10 to 14 days. For passaging, cultures were first incubated with Accutase (Cell Stem Cell Technologies, catalog #07922) for 15 minutes to soften the MATRIGEL®. Domes and organoids were then collected in 15 ml tubes and incubated at 37°C for 15 minutes with agitation. After centrifugation at 450 g for 5 minutes at 4°C, cells were treated with TrypLE Express (Gibco, catalog #12604-021) for 10 minutes at 37°C. TrypLE Express was inactivated by diluting with PBS and the cells were pelleted at 450 x g for 5 minutes at 4°C. Cells were resuspended in MATRIGEL® on ice at 60 x ¹⁰⁴ cells/mL, plated in 50 µL domes in 6-well plates, and fixed at 37°C for 30 minutes. Domes were covered with SFFF medium containing 10 µM ROCK inhibitor for the first 3 days and then SFFF medium without ROCK inhibitor was added and changed every 2 to 3 days.

Suspended hydrogel BOBA cultures. Single organoids in MATRIGEL® were prepared on ice as described above. Pre-warmed SFFF medium containing 10 µM ROCK inhibitor was added to a 6-well plate (4 mL/well) and kept in a 37°C incubator. To generate suspended BME droplets or BOBA, the organoid-MATRIGEL® solution was dispensed in 10 µL volumes directly into the warm medium using an electronic continuous pipette (reference) with a wide-bore or cut pipette tip (opening approximately 2 mm). During the dispensing process, the tip was immediately dipped below the liquid surface and then lifted after each dispense to ensure droplet separation. After 3 days, add SFFF medium and change it every 2 to 3 days. For medium exchange, place a sterile 70 µm cell filter into the well, tilt the plate and gently aspirate 3 mL of medium through the filter.

result:

As shown in Figure 9, lung organoids were successfully generated using alveolar type II (ATII) stem cells isolated from lung tissue embedded in hydrogel suspended in culture medium.

Examples 5 ：Generation of Mammary Organoids

This example provides a method for generating mammary organoids using the BME suspension method described herein.

method:

Human mammary organoids were sourced. Mammary organoids were sourced as previously described by Sachs et al. Cell 172(1-2):373-389.e10 (2018), which is incorporated herein in its entirety. Mammary tissue was cut into 1-3 ^mm3 pieces, minced, and washed with 10 mL AdDF+++ (Advanced DMEM/F12 containing 1x Glutamax, 10 mM HEPES, and antibiotics). Tissues were then digested in 10 mL organoid medium containing 1 to 2 mg/ml collagenase (Sigma, C9407) at 37°C on an orbital shaker for 1 to 2 hours. Sequentially shear the digested tissue suspension using a plastic and flamed glass Pasteur pipette and after each shearing step, filter the suspension over a filter ( e.g. , 100 μm filter). Perform the subsequent shearing step on the tissue pieces retained after filtration with approximately 10 ml of AdDF+++. Add 2% FCS to the filtered suspension and then centrifuge at 400 rcf . Resuspend the pellet in 10 ml of AdDF+++ and centrifuge again at 400 rcf . If a red precipitate is visible, lyse the erythrocytes in 2 mL erythrocyte lysis buffer (Roche, 11814389001) at room temperature for 5 minutes, then add 10 ml AdDF+++ and centrifuge at 400 rcf . Resuspend the precipitate in 10 mg/ml cold BME ( e.g. , CULTREX® Reduced Growth Factor BME Type 2 (Trevigen, 3533-010-02)) and allow 40 µL of the BME-cell suspension to solidify on a pre-warmed 24-well suspension plate (Greiner, M9312) at 37°C for 20 minutes. When gelation is complete, add 400 µL of organoid medium to each well and transfer the plate to a humidified 37°C/5% CO ₂ incubator with 2% or ambient O _2. Change the medium every 4 days. Organoid culture medium contained R-Spondin 1 conditioned medium (10%) or R-Spondin 3 (250 ng/ml) with neuromodulin 1 (5 nM), FGF7 (5 ng/ml), FGF10 (20 ng/ ml), EGF (5 ng/ml), Noggin (100 ng/ml), A83-01 (500 nM), Y-27632 (5 µM), SB202190 (500 nM), B27 supplement (1x), N-acetylcysteine (1.25 mM), niacinamide (5 mM), GlutaMax 100x (1x), HEPES (10 nM), penicillin/streptomycin (100 U/ml/100 µg), primocin (50 µg/ml) and Advanced DMEM/F12 (1x).

Organoid maintenance. Organoids were passaged every 1 to 4 weeks. Cystic organoids were resuspended in 2 mL of cold AdDF+++ and mechanically sheared by a flamed glass Pasteur pipette. Delicate organoids were dissociated by resuspending in 2 mL of TrypLE Express (Invitrogen, 12605036), incubated at room temperature for 1 to 5 minutes, and mechanically sheared by a flamed glass Pasteur pipette. After adding 10 mL of AdDF+++ and centrifuging at 300 rcf or 400 rcf, organoid fragments were resuspended in cold BME and replated at the above ratios ( e.g. , 1:1 to 1:6) to form new organoids.

Suspended hydrogel BOBA cultures. Prepare single organoid cells or organoid fragments in BME on ice as described above. Add pre-warmed organoid medium to 6-well plates (5 mL/well), 100 cm dishes (15 to 30 mL/dish), or 50 mL conical tubes (30 mL/tube) and keep in a 37°C warm bead bath. To generate suspended BME droplets or BOBA, dispense the organoid cell-BME solution in 10 µL volumes directly into the warm medium using an electronic continuous pipette (Integra VIAFLO 300) with a wide-bore or cut pipette tip (opening approximately 2 mm). During dispensing, the tip is dipped immediately below the surface of the liquid and then lifted after each dispense to ensure droplet separation. For larger format cultures, transfer BOBA to flasks by serological pipette or decanting. Change medium every 2 to 4 days. In 6-well dish cultures, place a sterile 70 µm cell filter in the well, tilt the dish and gently aspirate 4 mL of medium through the filter. In flask cultures, tilt the flask at an angle, place BOBA in a corner, and replace approximately 2/3 of the volume of used medium with a serological pipette.

Suspended hydrogel SOBA and SOBA fragment cultures. Prepare single organoid cells or organoid fragments in BME on ice as described above. Add pre-warmed organoid medium to 6-well plates (5 mL/well), 100 cm dishes (15-30 mL/dish) and keep on a warm bead bath. To generate suspended SOBA filaments, gently draw the cell-BME solution into a syringe with a 15-gauge blunt-tip needle and extrude directly into the warm medium while moving the submerged needle in a linear, serpentine, or spiral motion in the XY plane. To generate SOBA fragments, gently triturate the SOBA filament culture twice using a 10 mL serological pipette or a wide-bore P1000 pipette tip. Additional medium was added to give a final BME to medium ratio of 1:10. Medium changes were performed as described above for BOBA cultures.

Immunofluorescence Sample Preparation and Confocal Microscopy. 24-well dome cultures were fixed with 2% paraformaldehyde (PFA) in PBS. Domes were detached from the plate using a spatula and transferred to a microcentrifuge tube using a cut P1000 pipette tip. For BOBA cultures, 500 µL of culture was transferred to a microcentrifuge tube using a cut P1000 pipette tip, the medium was removed and 2% PFA in PBS was added. Samples were incubated in fixative at room temperature for 15 to 30 minutes and then washed three times in PBS. Samples were stained in microcentrifuge tubes with primary antibodies diluted in blocking/permeabilization buffer (3% BSA, 0.1% Triton X-100, 0.02% sodium azide in PBS) for at least 4 hours at room temperature and then washed three times in PBS. Samples were then incubated with secondary antibodies, DAPI, and AlexaFluor 660 agaricus diluted in blocking/permeabilization buffer for at least 2 hours at room temperature. Images were collected on a Stellaris 8 confocal microscope (Leica) using a 40X objective and 3D reconstruction was performed using Imaris image analysis software (Oxford Instruments).

Transcriptome analysis. For RNA isolation, the RNeasy Micro Plus kit (Qiagen) was used. RLT+ lysis buffer was added to BME domes or pelleted BOBA and stored at -80°C. RNA isolation was performed using QiaCube Connect (Qiagen), and RNA was quantified using Nanodrop 8000 (ThermoFisher). Bulk mRNA-seq (NovaSeq PE150) and analysis were performed by Novogene. Reads were aligned using HISAT2 (Mortazavi et al. 2008), differential gene expression analysis was performed using DESeq2 (Anders et al. 2014), and statistical significance was calculated using a negative binomial distribution model with Benjamini-Hochberg FDR correction. Stem cell and proliferation markers (EpCAM and CD49f ⁺ ) and differentiation markers (cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (k5), cytokeratin 14 (K14), smooth muscle actin (SMA)) were analyzed.

Examples 6 ：Generation of pancreatic organoids

This example provides a method for generating pancreatic organoids using the BME suspension method described herein.

method:

Human pancreatic organoids source and maintenance. Pancreatic organoids were generated as previously described by Georgakopoulos et al. BMC Developmental Biology 20:4 (2020), which is incorporated herein in its entirety. Approximately 5 mg of pancreatic tissue was minced manually and dissociated with a gentleMACS dissociator (Miltenyi Biotec) for a total of 2 minutes. The minced tissue was washed twice in wash medium and digested in 40 mL of digestion solution and placed at 37°C for 1 to 2 hours. The wash solution consisted of Dulbecco's modified Eagle's medium (DMEM), high glucose, GlutaMAX, pyruvate (Life Technologies) supplemented with 1% fetal bovine serum (FBS) (Life Technologies) and 1% penicillin/streptomycin (10,000 U/mL) (Life Technologies), and the digestion solution consisted of collagenase type I (Sigma-Aldrich) and dispase II (Life Technologies) at a concentration of 0.125 mg/mL in DMEM containing 0.1 mg/mL DNase I (Sigma-Aldrich). The detached tubes were either manually picked with a pipette or the entire digestion mixture was filtered with a 100 μm pore nylon cell filter (Falcon). Duct fragments were washed in basal medium (Advanced DMEM/F12 (Life Technologies) supplemented with 1% penicillin/streptomycin, 1% Glutamax 100x (Life Technologies), and HEPES (Life Technologies) 10 mM) and spun at 200 g for 5 min. Cell pellets were mixed with reduced growth factor BME 2-RGF (Basement Membrane Extract Type 2 3533-010-02; AMSBIO, CULTREX®), seeded in 24-well plates, and overlaid with optimized human pancreatic organoid expansion medium. Optimized human pancreatic organoid expansion medium included basal medium supplemented with 1X N2 and 1X B27 (both from GIBCO), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 10% RSPO1 conditioned serum-free medium, 10 nM human [Leu ¹⁵ ]-gastrin I (Sigma-Aldrich), 50 ng/mL EGF (Peprotech), 25 ng/mL Noggin (Peprotech), 100 ng/mL FGF10 (Peprotech), 10 mM niacinamide (Sigma-Aldrich), 5 μM A83.01 (Tocris), 10 μM FSK (Tocris), and 3 μM PGE2 (Tocris). During the first 7 days, optimized human pancreatic organoid expansion medium was supplemented with 10 μM Rho kinase inhibitor (Y27632, Sigma-Aldrich). After 14 days, cells were passaged as previously described by Broutier et al. Nat Protoc. 11:1724–43 (2016), which is incorporated herein in its entirety.

Suspended hydrogel BOBA cultures. Prepare single organoid cells or organoid fragments in BME on ice as described above. Add pre-warmed medium to 6-well plates (5 mL/well), 100 cm dishes (15 to 30 mL/dish), or 50 mL conical tubes (30 mL/tube) and keep in a 37°C warm bead bath. To generate suspended BME droplets or BOBA, dispense the organoid cell-BME solution in 10 µL volumes directly into the warm medium using an electronic continuous pipette (Integra VIAFLO 300) with a wide-bore or cut pipette tip (opening approximately 2 mm). During dispensing, the tip is dipped immediately below the surface of the liquid and then lifted after each dispense to ensure droplet separation. For larger format cultures, transfer BOBA to flasks by serological pipette or decanting. Change medium every 2 to 4 days. In 6-well dish cultures, place a sterile 70 µm cell filter in the well, tilt the dish and gently aspirate 4 mL of medium through the filter. In flask cultures, tilt the flask at an angle, place BOBA in a corner, and replace approximately 2/3 of the volume of used medium with a serological pipette.

Suspended hydrogel SOBA and SOBA fragment cultures. Prepare single organoid cells or organoid fragments in BME on ice as described above. Add pre-warmed medium to 6-well plates (5 mL/well), 100 cm dishes (15-30 mL/dish) and keep on a warm bead bath. To generate suspended SOBA filaments, gently draw the cell-BME solution into a syringe with a 15-gauge blunt-tip needle and extrude directly into the warm medium while moving the submerged needle in a linear, serpentine, or spiral motion in the XY plane. To generate SOBA fragments, gently triturate the SOBA filament culture twice using a 10 mL serological pipette or a wide-bore P1000 pipette tip. Additional medium was added to give a final BME to medium ratio of 1:10. Medium changes were performed as described above for BOBA cultures.

Immunofluorescence Sample Preparation and Confocal Microscopy. 24-well dome cultures were fixed with 2% paraformaldehyde (PFA) in PBS. Domes were detached from the plate using a spatula and transferred to a microcentrifuge tube using a cut P1000 pipette tip. For BOBA cultures, 500 µL of culture was transferred to a microcentrifuge tube using a cut P1000 pipette tip, the medium was removed and 2% PFA in PBS was added. Samples were incubated in fixative at room temperature for 15 to 30 minutes and then washed three times in PBS. Samples were stained in microcentrifuge tubes with primary antibodies diluted in blocking/permeabilization buffer (3% BSA, 0.1% Triton X-100, 0.02% sodium azide in PBS) for at least 4 hours at room temperature and then washed three times in PBS. Samples were then incubated with secondary antibodies, DAPI, and AlexaFluor 660 agaricus diluted in blocking/permeabilization buffer for at least 2 hours at room temperature. Images were collected on a Stellaris 8 confocal microscope (Leica) using a 40X objective and 3D reconstruction was performed using Imaris image analysis software (Oxford Instruments).

Transcriptome analysis. For RNA isolation, the RNeasy Micro Plus kit (Qiagen) was used. RLT+ lysis buffer was added to BME domes or pelleted BOBA and stored at -80°C. RNA isolation was performed using QiaCube Connect (Qiagen), and RNA was quantified using Nanodrop 8000 (ThermoFisher). Bulk mRNA-seq (NovaSeq PE150) and analysis were performed by Novogene. Reads were aligned using HISAT2 (Mortazavi et al. 2008), differential gene expression analysis was performed using DESeq2 (Anders et al. 2014), and statistical significance was calculated using a negative binomial distribution model with Benjamini-Hochberg FDR correction. Stem cell and proliferation markers ( CD133 , LGR5 , PDX1 , SOX9 , ALDH1A1 , NEUROG3 , and NKX6.1 ) and differentiation markers ( keratin 19 (KRT19) , MUC1 , INS , GCG , and AMY ) were analyzed.

Examples 7 ：Generation of liver organoids

This example provides a method for generating liver organoids using the BME suspension method described herein.

method:

Human liver organoids source and maintenance. Liver organoids were generated as previously described by Huch et al. Cell 160(1-2):299-312 (2015), which is incorporated herein in its entirety. Liver biopsies ranging in size from 0.5 to 1 ^cm3 were obtained from donor and explant livers during liver transplantation. Hepatocytes were isolated from liver biopsies by mincing the liver biopsies, rinsing twice in DMEM (GIBCO) containing 1% FCS, and incubating with digestion solution (2.5 mg/ml collagenase D (Roche) and 0.1 mg/ml DNase I (Sigma) in EBSS (Hyclone, Thermoscientific)) at 37°C for 20 to 40 minutes. The digestion was stopped by adding cold DMEM containing 1% FCS. The digestion suspension was filtered through a 70 µm nylon cell filter and spun at 300 to 400 × g for 5 min. The resulting pellet was resuspended in DMEM containing 1% FCS and kept cold. If material remained on the filter, this was further digested in Accutase (GIBCO) at 37°C for 10 min before stopping and collecting the cells as described above. The different fractions were then pooled and washed with cold Advanced DMEM/F12 and spun at 300 to 400 × g for 5 min. The cell pellet is mixed with a hydrogel ( e.g. , MATRIGEL® (BD Biosciences) or reduced growth factor BME 2 (basement membrane extract, type 2, Pathclear)) and approximately 3,000 to 10,000 cells are plated per well in a plate ( e.g. , a 48-well plate). The medium is then added after the MATRIGEL® or BME solidifies. The culture medium was based on AdDMEM/F12 (Invitrogen) supplemented with 1% N2 (GIBCO) and 1% B27 (GIBCO), 1.25 mM N-acetylcysteine (Sigma), 10 nM gastrin (Sigma), and growth factors: 50 ng/ml EGF (Peprotech), 10% RSPO1 conditioned medium (homemade), 100 ng/ml FGF10 (Peprotech), 25 ng/ml HGF (Peprotech), 10 mM niacinamide (Sigma), 5 µM A83.01 (Tocris), and 10 µM FSK (Tocris). During the first 3 days after isolation, cultures were established by supplementing the medium with 25 ng/ml Noggin (Peprotech), 30% Wnt medium (as described by Barker et al. Cell Stem Cell 6:25-36 (2010)), and 10 µM (Y27632, Sigma Aldrich) or hES cell selective recovery solution (Stemgent). The medium was then changed to medium without Noggin, Wnt, Y27632, and hES cell selective recovery solution. After 10 to 14 days, organoids were removed from MATRIGEL® or BME, mechanically dissociated into small fragments, and transferred to fresh medium. Passage every 7 to 10 days for at least 6 months at a split ratio ( e.g. , 1:4 to 1:8). To prepare frozen stocks, lyse organoid cultures and mix with Recovery Cell Culture Freezing Medium (GIBCO) and freeze according to standard procedures. Thaw cultures using standard thawing procedures and culture as described above. Supplement media with Y-27632 (10 μM) within the first 3 days after thawing.

For hepatocyte differentiation, hepatic organoids were plated and cultured in the above hepatic medium supplemented with BMP7 (25 ng/ml) for 7 to 10 days. The cultures were split and plated in this BMP7-supplemented medium for at least about 2 to 4 days. Subsequently, the medium was changed to differentiation medium, which included AdDMEM/F12 medium supplemented with 1% N2 and 1% B27 and containing EGF (50 ng/ml), gastrin (10 nM, Sigma), HGF (25 ng/ml), FGF19 (100 ng/ml), A8301 (500 nM), DAPT (10 µM), BMP7 (25 ng/ml), and dexamethasone (30 µM). Change the differentiation medium every 2 to 3 days over a period of 11 to 13 days.

Suspended hydrogel BOBA cultures. Prepare single organoid cells or organoid fragments in BME on ice as described above. Add pre-warmed medium to 6-well plates (5 mL/well), 100 cm dishes (15 to 30 mL/dish), or 50 mL conical tubes (30 mL/tube) and keep in a 37°C warm bead bath. To generate suspended BME droplets or BOBA, dispense the organoid cell-BME solution in 10 µL volumes directly into the warm medium using an electronic continuous pipette (Integra VIAFLO 300) with a wide-bore or cut pipette tip (opening approximately 2 mm). During dispensing, the tip is dipped immediately below the surface of the liquid and then lifted after each dispense to ensure droplet separation. For larger format cultures, transfer BOBA to flasks by serological pipette or decanting. Replace medium with medium every 2 to 3 days. In 6-well dish cultures, place a sterile 70 µm cell filter in the well, tilt the dish and gently aspirate 4 mL of medium through the filter. In flask cultures, tilt the flask at an angle, place BOBA in a corner, and replace approximately 2/3 of the volume of used medium with a serological pipette.

Suspended hydrogel SOBA and SOBA fragment cultures. Prepare single organoid cells or organoid fragments in BME on ice as described above. Add pre-warmed medium to 6-well plates (5 mL/well), 100 cm dishes (15-30 mL/dish) and keep on a warm bead bath. To generate suspended SOBA filaments, gently draw the cell-BME solution into a syringe with a 15-gauge blunt-tip needle and extrude directly into the warm medium while moving the submerged needle in a linear, serpentine, or spiral motion in the XY plane. To generate SOBA fragments, gently triturate the SOBA filament culture twice using a 10 mL serological pipette or a wide-bore P1000 pipette tip. Additional medium was added to give a final BME to medium ratio of 1:10. Medium changes were performed as described above for BOBA cultures.

Immunofluorescence Sample Preparation and Confocal Microscopy. 24-well dome cultures were fixed with 2% paraformaldehyde (PFA) in PBS. Domes were detached from the plate using a spatula and transferred to a microcentrifuge tube using a cut P1000 pipette tip. For BOBA cultures, 500 µL of culture was transferred to a microcentrifuge tube using a cut P1000 pipette tip, the medium was removed and 2% PFA in PBS was added. Samples were incubated in fixative at room temperature for 15 to 30 minutes and then washed three times in PBS. Samples were stained in microcentrifuge tubes with primary antibodies diluted in blocking/permeabilization buffer (3% BSA, 0.1% Triton X-100, 0.02% sodium azide in PBS) for at least 4 hours at room temperature and then washed three times in PBS. Samples were then incubated with secondary antibodies, DAPI, and AlexaFluor 660 agaricus diluted in blocking/permeabilization buffer for at least 2 hours at room temperature. Images were collected on a Stellaris 8 confocal microscope (Leica) using a 40X objective and 3D reconstruction was performed using Imaris image analysis software (Oxford Instruments).

Transcriptome analysis. For RNA isolation, the RNeasy Micro Plus kit (Qiagen) was used. RLT+ lysis buffer was added to BME domes or pelleted BOBA and stored at -80°C. RNA isolation was performed using QiaCube Connect (Qiagen), and RNA was quantified using Nanodrop 8000 (ThermoFisher). Bulk mRNA-seq (NovaSeq PE150) and analysis were performed by Novogene. Reads were aligned using HISAT2 (Mortazavi et al. 2008), differential gene expression analysis was performed using DESeq2 (Anders et al. 2014), and statistical significance was calculated using a negative binomial distribution model with Benjamini-Hochberg FDR correction. Stem cell and proliferation markers ( LGR5 ) and differentiation markers ( ALB , CYP3A4 , and HNF4A for hepatocytes, and KRT19 , KRT7 , and SOX9 for ductal cells) were analyzed.

Examples 8 ：Organoids squeezed out of a syringe BME Assembly (SOBA) Cultivation method

This example provides methods for generating intestinal organoids using SOBA filaments and SOBA filament fragments and maintaining such intestinal organoids. The SOBA method facilitates and speeds up culture preparation because a large volume of cell-BME solution can be loaded into a single syringe to generate SOBA hydrogel filaments. Overall, the SOBA method enables large-scale organoid scalability, improves organoid culture uniformity, and saves labor, time, and resources.

Material:

The method uses the following materials: 10 cm or 15 cm culture dishes, 5 mL syringes, 15-gauge blunt-tip needles (sterilized by immersion in 70% EtOH), flasks (T25-T225 or 6-well plates/culture dishes are compatible), 50 mL conical tubes, centrifuge, incubator with 5% CO ₂ , TC inverted light microscope, and 37°C water bath.

Drugs:

CULTREX® Reduced Growth Factor Basement Membrane Extract Type 2 (BME, R&D Systems 3533-010-2; must be kept on ice) or MATRIGEL®; TrypLE Express; PBS (without Ca ²⁺ /Mg ²⁺ ); Organoid Growth Medium: Human Intestinal Organoid Growth Medium (OGM, Stem Cell Tech 06010) and Primocin® (1:1000, InvivoGen ant-pm-1); and Organoid Passaging Medium: Organoid Growth Medium and 10 µM Y-27632 (10 µM; 1:1000 of a 10 mM stock solution in PBS).

Low retention pipette tips can be used in all steps to prevent organoid loss. If low retention tips are not available and organoid loss is unacceptable, tubes and/or tips can be coated with 1% BSA/PBS prior to use.

method:

Cell preparation . Gastrointestinal organoids were generated as described herein and cultured for 7 to 14 days in organoid growth medium, which included human Intesticult Organoid Growth Medium (OGM, Stem Cell Tech 06010) and Primocin® (1:1000, InvivoGen ant-pm-1). Viable cells were counted (about 50% to 70% viability was usually observed) and cells were washed by resuspending in 10 mL DMEM or PBS and centrifuging at 500 x g for 3 minutes, as appropriate. This improved the viability percentage, but there was some overall cell loss. To create the cell-BME solution, resuspend the organoid cell pellet in BME on ice at a density of 6 x 10 ⁵ cells/mL BME (this is equivalent to 3 x 10 ⁴ cells per well of a 24-well plate; seeding density may need to be adjusted for individual rows). Gently resuspend the cell pellet in 1 mL of BME using a P1000 pipette without introducing air bubbles. Then add additional BME in 1 mL increments, triturating gently each time.

Suspended hydrogel SOBA and SOBA fragment cultures. Pre-warmed organoid subculture medium (pre-warmed in 37°C water or bead bath) consisting of organoid growth medium and 10 µM Y-27632 (10 µM; 1:1000 of a 10 mM stock in PBS) is added to the culture dishes (30 mL medium in a 10 cm dish or 60 mL medium in a 15 cm dish) and kept on a warm bead bath to maintain temperature. SOBA production can be performed at room temperature on the benchtop without the need for a warm bead bath if performed rapidly.

To generate suspended SOBA filaments, load cold cell-BME solution into a syringe with a 15-gauge (or wider) blunt-tip needle or large-volume pipette tip. Extrude the cell-BME solution directly into warm medium while moving the submerged needle or tip in a linear, serpentine, or spiral motion in the X-Y plane to generate SOBA filaments. Organoids have been successfully grown in filaments 1-5 mm in diameter, with wider filaments generated by slower dispensing speeds and slower X-Y motions. Wider and longer filaments improve gravity settling of SOBA during medium changes. SOBA filaments may not form well if the medium is not warm enough or the BME solution is too cold. If the medium is not warm enough, it may not be able to warm the BME enough to polymerize quickly enough during the dispensing process. If the BME-cell solution is too cold, the warm medium may not immediately warm the BME to a sufficient temperature for proper solidification. The BME-cell solution may be briefly warmed in the hands or may be removed from ice to room temperature for 30 seconds to a minute (depending on the volume of BME) prior to generating the SOBA.

To transfer the SOBA filament and medium to a flask or other culture vessel, use a wide-opening serological pipette to slowly aspirate and dispense the SOBA filament to prevent fragmentation of the SOBA filament. Then add the Organoid Subculture Medium to the culture vessel to achieve a final BME to medium ratio of 1:10.

Medium changes . To allow SOBA filaments to settle by gravity, (a) support the culture vessel ( e.g. , flask) vertically and tilt diagonally for 5 minutes (e.g., flask can be supported in a tube rack or ice bucket), or (b) gently decant the culture into a 50 mL conical tube, where transfers of SOBA filaments by serological pipette should be limited to minimize fragmentation. If SOBA fragments are very small, they may remain floating in the medium, in which case, transfer the culture to a conical tube to facilitate settling of the SOBA. Centrifuge the tube at 500 x g for 3 minutes, turn off the brake to promote sedimentation, and dilute the culture with PBS and centrifuge again. In some cases, the culture may be diluted with PBS and centrifuged again. Using a serological pipette, replace approximately 50% to 75% of the medium with fresh organoid subculture medium every 2 to 3 days. If the culture was transferred to a conical tube, gently decant the culture back into the original culture vessel.

Passaging SOBA cultures. Organoids are typically ready for passage 7 to 14 days after inoculation of a single organoid cell. Initially, allow SOBA fragments to settle by gravity. As discussed above, to allow SOBA filaments to settle by gravity, (a) support the culture vessel ( e.g. , flask) vertically and tilt it diagonally for 5 minutes, or (b) gently decant the culture into a 50 mL conical tube. Remove as much medium as possible using a serological pipette. If SOBA fragments are still floating in the medium, dilute the culture, transfer to a conical tube, and dilute with PBS. Centrifuge the tube at 500 x g for 3 minutes with the brake off to promote sedimentation.

Pre-warmed TrypLE Express is added to a conical tube at a ratio of at least 10 mL TrypLE Express to 1 mL BME in SOBA culture medium, and the solution is triturated with a serological pipette to break up SOBA threads. The solution is then incubated in a 37°C water bath for 30 minutes to 1 hour, with vigorous trituration with a serological pipette during the incubation period as appropriate. After incubation, the solution is triturated vigorously with a serological pipette and visually inspected for organoid dissociation ( e.g. , using an inverted microscope). TrypLE Express is then inactivated by adding PBS to the conical tube.

Using this technique, live cells can be counted, diluted, or aliquoted as desired. In certain embodiments, cells can be passaged, frozen, or used in experiments as desired. * * * * * * * * *

Although the subject matter and advantages of the present disclosure have been described in detail, it should be understood that various changes, substitutions and modifications may be made herein without departing from the spirit and scope of the present disclosure. In addition, the scope of the present case is not intended to be limited to the specific embodiments of the processes, machines, manufactures and material compositions, means, methods and steps described in the specification. A person with ordinary knowledge in the art will easily understand from the invention of the subject matter disclosed herein that a process, machine, manufacture, material composition, means, method or step that currently exists or will be developed in the future can be used according to the subject matter disclosed herein to perform substantially the same function or achieve substantially the same result as the corresponding embodiment described herein. Therefore, the appended claims are intended to include within the scope of such claims such processes, machines, manufacture, compositions of matter, means, methods or steps.

Various patents, patent applications, publications, product descriptions, protocols, and serial accession numbers are cited throughout this application, the contents of which are incorporated herein by reference in their entirety for all purposes.

Figures 1A-1H show the scaling of intestinal organoids in suspension in BME hydrogel. (A) Schematic, (B) photographs, and (C) bright field microscopy of human intestinal organoids in conventional dome cultures (top) and suspended BOBA (organoid bead assemblies embedded in BME) cultures (bottom). Scale bar for micrographs is 500 µm. (D) Diameter of organoids in dome and BOBA cultures in bright field images. (E) Percentage of Ki67-positive cells in confocal images. Data presented are mean ± SD, Student's t test, n = 3, 10 fields each. (F) 3D reconstructed confocal images of colon organoids in dome or BOBA culture. Ki67 is green, nuclei are blue, actin is white, and the scale bar is 20 µm. (G, H) Total viable cells, cells per ^cm2 surface area, or cells per µL BME for (G) colon and (H) ileum organoids. All data presented are mean ± SD, Student’s t test, *p ≤ 0.05, ****p ≤ 0.0001, n = 3 experiments. Figures 2A-2C show organoid differentiation in BOBA culture. (A) Bright field images of colon organoids in dome (top) and BOBA (bottom) cultures show comparable morphology of proliferating and differentiating organoids. Scale bar, 100 µm. (B) Bulk RNA-seq shows that colon organoids cultured in dome and BOBA similarly downregulate stem cell and progenitor cell markers and upregulate differentiation markers following transition from growth medium to differentiation medium. (C) Markers specific for differentiated epithelial cell types (MUC2 for goblet cells, FABP1 for intestinal epithelial cells, and CHGA for intestinal endocrine cells) are expressed in organoids cultured in both dome (top) and BOBA (bottom) formats. Nuclei are blue, actin is white, and the scale bar is 10 µm. Figures 3A-3C show that BME volume and culture vessel can affect organoid growth in suspended BME hydrogel culture. (A) Bright field images of intestinal organoids in surface-attached domes (50 µL BME in 0.5 mL medium) in a 24-well plate or in BOBA culture (0.5, 1, or 2 mL BME in 5 mL medium) in a 6-well plate. Scale bar is 200 µm. (B, C) Quantification of organoid diameter, total viable cells, viable cells per ^cm2 surface area, and viable cells per µL BME in (B) 6-well plates or (C) 25 ^cm2 flasks. Data presented are mean ± SD, one-way ANOVA multiple comparison test, n = 3 experiments; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Figures 4A-4E show organoid size homogeneity in BOBA culture. (A) Schematic depicting the imaging strategy used for homogeneity analysis. (B, C) Bright field images of colon organoids in the deepest plane of a 50 µl BME dome or 10 µl suspended BOBA hydrogel. Scale bars are (B) 200 µm and (C) 1 mm. (D) Quantification of organoid diameter in the horizontal ROI of the dome or BOBA hydrogel according to position across the X-axis. Data presented are mean ± SD, and n = 3 replicates in a representative experiment of 3 experiments. (E) Quantification of mean organoid diameter at the edge or core of the dome and BOBA culture. All data presented are mean ± SD, two-way ANOVA, and n = 3 replicates in a representative experiment of 3 experiments. Figures 5A-5B show gene expression in organoids in dome and BOBA culture. (A, B) Bulk RNA-seq analysis shows differences in gene expression of (A) stem cell and proliferation markers and (B) intestinal epithelial cell markers between colon organoids in dome culture or suspended BOBA culture after 7 days of culture in growth medium. Data presented are means ± SD of normalized counts determined by DESeq2, n = 3 replicates, and statistical analysis was performed by negative binomial distribution model with BH adjusted p value, *padj ≤ 0.05, **padj ≤ 0.01. Figures 6A-6D show that alternative suspended BME hydrogel culture formats have comparable organoid growth. (A) Photographs and (B) bright field images of colon organoids in 6-well plate cultures in BOBA, SOBA (syringe-extruded BME organoid assemblies), or SOBA fragment formats. The alternative formats enable faster culture preparation for scale-up. Scale bar is 1 mm. (C) Quantification of organoid diameter and (D) viable cells per well. Data presented are mean ± SD, one-way ANOVA multiple comparison test, n = 3 experiments. Figures 7A-7E show the use of suspended BME hydrogel organoid cultures for mid-production screening. (A) Schematic diagram of the experiment. SOBA chip organoid cultures in 225 ^cm2 flasks were ground to achieve a homogenous organoid suspension and then seeded into 96-well plates. (B) Bright field image of SOBA chip cultures grown in 225 ^cm2 flasks. Scale bar is 1 mm. (C) Bright field image of randomly selected wells across a 96-well plate showing comparable organoid density between wells. Scale bar is 1 mm. (D) Cell Titer Glo 3D (CTG) viability readouts show similar well-to-well variability between dome and suspended BME cultures in 96-well plates. Data presented are mean ± SD. Student’s t test. (E) Representative dose-response viability curves (CTG assay) of suspended BME organoids treated with diacerein, sorafenib, SN-38, or docetaxel for 3 days. Data presented are mean ± SD, n = 4. Figures 8A-8C show the generation of Transwell monolayers using suspended BME hydrogel organoids . (A) Schematic diagram of the experiment. SOBA chip organoid cultures in 225 ^cm2 flasks were digested into single-cell suspensions and then seeded into 96-well BME-coated Transwell chambers at confluence. (B) Bright field images of SOBA chip organoid-derived Transwell monolayers 3 days after seeding. Transwells were set up in either monolayer growth medium or monolayer differentiation medium. Scale bar is 100 µm. (C) Transepithelial electrical resistance (TEER) of Transwell monolayers cultured in either monolayer growth medium (filled circles ) or monolayer differentiation medium (open circles). Data presented are mean ± SD, n = 6 wells. FIG9 provides images of lung AT2 organoids embedded in hydrogel suspended in BOBA medium. FIG10 provides a schematic depicting exemplary conditions for generating hydrogel droplets, referred to herein as basement membrane organoid bead assemblies (BOBAs). Organoids or dissociated organoid cells suspended in cold liquid hydrogel are dispensed as droplets into a room temperature or warmer culture medium, which allows the hydrogel to solidify immediately upon dispensing into the culture medium. FIG. 11 provides a schematic diagram depicting exemplary conditions for producing hydrogel filaments, which are referred to herein as syringe-extruded organoid basement membrane assemblies (SOBAs). Organoids or dissociated organoid cells suspended in cold liquid hydrogel are extruded (by a syringe, pipette, or any other dispensing device) as filaments into a room temperature or warmer culture medium, which allows the hydrogel to solidify immediately upon dispensing into the culture medium.

Claims (117)

A method for producing tissue-derived epithelial organoids, comprising: (a)   contacting tissue-derived epithelial stem cells with hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture; (b)   suspending the hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture; and (c)   culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to produce tissue-derived epithelial organoids. The method of claim 1, wherein a plurality of tissue-derived epithelial stem cells are contacted with the hydrogel to produce the hydrogel-tissue-derived epithelial stem cell mixture. The method of claim 2, wherein the plurality of tissue-derived epithelial stem cells comprises about 1 × 10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1 × 10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. The method of claim 2 or 3, wherein the plurality of tissue-derived epithelial stem cells are contained in tissue fragments, organoid fragments, or a combination thereof. The method of any one of claims 1 to 3, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells are isolated from primary epithelial tissue. The method of any one of claims 1 to 5, wherein the hydrogel solidifies upon contact with the culture medium. The method of any one of claims 1 to 6, wherein suspending the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium comprises immersing a dispensing device containing the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium and dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the culture medium. A method as claimed in any one of claims 1 to 7, wherein the temperature of the culture medium is about 25°C to about 50°C. A method as claimed in any one of claims 1 to 8, wherein the temperature of the culture medium is about 30°C to about 50°C. The method of any one of claims 1 to 9, wherein the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is about 2°C to about 25°C. The method of any one of claims 1 to 10, wherein the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is about 2°C to about 20°C. The method of any one of claims 1 to 11, wherein the hydrogel is selected from the group consisting of: synthetic hydrogels, natural hydrogels, and combinations thereof. The method of claim 12, wherein the natural hydrogel comprises basement membrane extract (BME) or extracellular matrix (ECM) components. A method as in any one of claims 1 to 13, wherein the hydrogel has a storage modulus G' that is equal to or greater than the loss modulus G''. The method of any one of claims 1 to 14, wherein the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width and/or diameter greater than about 0.1 mm. The method of claim 15, wherein the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometry comprising a length, width, and/or diameter of about 0.1 mm to about 1,000 mm. The method of any one of claims 1 to 16, wherein the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the culture medium as droplets. The method of any one of claims 1 to 16, wherein the suspended hydrogel-tissue-derived epithelial stem cell mixture has a filamentous structure. The method of claim 18, wherein the filamentous structure has a linear, serpentine or spiral shape. The method of any one of claims 1 to 19, further comprising fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce fragmented structures comprising the tissue-derived epithelial organoids. A method for producing a suspended culture of tissue-derived epithelial organoids, comprising: (a)   introducing a mixture comprising hydrogel and tissue-derived epithelial stem cells into a culture medium to produce a suspended mixture; and (b)   culturing the suspended mixture in the culture medium to produce the tissue-derived epithelial organoids in a suspended state. The method of claim 21, wherein the mixture introduced into the culture medium comprises the hydrogel and a plurality of tissue-derived epithelial stem cells. The method of claim 22, wherein the plurality of tissue-derived epithelial stem cells comprises about 1 × 10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1 × 10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. The method of claim 22 or 23, wherein the plurality of tissue-derived epithelial stem cells are contained in tissue fragments, organoid fragments, or a combination thereof. The method of any one of claims 21 to 23, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells are isolated from native epithelial tissue. The method of any one of claims 21 to 25, wherein the hydrogel solidifies upon contact with the culture medium. A method as in any of claims 21 to 26, wherein introducing the mixture into the culture medium comprises immersing a dispensing device containing the mixture in the culture medium and dispensing the mixture into the culture medium. A method as claimed in any one of claims 21 to 27, wherein the temperature of the culture medium is about 25°C to about 50°C. A method as claimed in any one of claims 21 to 28, wherein the temperature of the culture medium is about 30°C to about 50°C. A method as claimed in any one of claims 21 to 29, wherein the temperature of the mixture is about 2°C to about 25°C. A method as claimed in any one of claims 21 to 30, wherein the temperature of the mixture is about 2°C to about 20°C. The method of any one of claims 21 to 31, wherein the hydrogel is selected from the group consisting of: synthetic hydrogels, natural hydrogels, and combinations thereof. The method of claim 32, wherein the natural hydrogel comprises basement membrane extract (BME) or extracellular matrix (ECM) components. A method as in any one of claims 21 to 33, wherein the hydrogel has a storage modulus G' that is equal to or greater than the loss modulus G''. The method of any of claims 21 to 34, wherein the suspended mixture has a geometric shape comprising a length, width, and/or diameter greater than about 0.1 mm. The method of claim 35, wherein the suspended mixture has a geometric shape comprising a length, width, and/or diameter of about 0.1 mm to about 1,000 mm. The method of any one of claims 21 to 36, wherein the mixture is introduced into the culture medium as droplets. A method as claimed in any one of claims 21 to 36, wherein the mixture is introduced into the culture medium in a filamentous structure. The method of claim 38, wherein the filamentous structures have a linear, serpentine or spiral shape. The method of any one of claims 21 to 39, further comprising fragmenting the suspended mixture to produce fragmented structures comprising epithelial organoids of said tissue origin. The method of any one of claims 1 to 40, wherein the culture medium is in a container selected from the group consisting of a culture dish, a multiwell tray, a conical tube, a container, a culture bag, a bioreactor or a flask. A method for producing a suspension culture of tissue-derived epithelial organoids, comprising: (a)   contacting tissue-derived epithelial stem cells with hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture; (b)   depositing the hydrogel-tissue-derived epithelial stem cell mixture on a substrate; (c)   solidifying the hydrogel-tissue-derived epithelial stem cell mixture to produce a solidified hydrogel-tissue-derived epithelial stem cell mixture; (d)  suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture; and (e)   culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to produce tissue-derived epithelial organoids. The method of claim 42, wherein a plurality of tissue-derived epithelial stem cells are contacted with the hydrogel to produce the hydrogel-tissue-derived epithelial stem cell mixture. The method of claim 43, wherein the plurality of tissue-derived epithelial stem cells comprises about 1 × 10 ⁴ tissue-derived epithelial stem cells/ml hydrogel to about 1 × 10 ⁷ tissue-derived epithelial stem cells/ml hydrogel. The method of claim 43 or 44, wherein the plurality of tissue-derived epithelial stem cells are contained in tissue fragments, organoid fragments, or a combination thereof. The method of any one of claims 42 to 44, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells are isolated from native epithelial tissue. The method of any one of claims 32 to 46, further comprising removing the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate before suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the culture medium. The method of any of claims 42 to 47, wherein the hydrogel is selected from the group consisting of: synthetic hydrogels, natural hydrogels, and combinations thereof. The method of claim 48, wherein the natural hydrogel comprises basement membrane extract (BME) or extracellular matrix (ECM) components. A method as in any of claims 42 to 49, wherein the hydrogel has a storage modulus G' that is equal to or greater than the loss modulus G''. The method of any of claims 42 to 50, wherein the solidified hydrogel-tissue-derived epithelial stem cell mixture has a geometry comprising a length, width, and/or diameter greater than about 0.1 mm. The method of claim 51, wherein the solidified hydrogel-tissue-derived epithelial stem cell mixture has a geometry comprising a length, width, and/or diameter of about 0.1 mm to about 1,000 mm. The method of any one of claims 42 to 52, wherein the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate as droplets. The method of any one of claims 42 to 52, wherein the hydrogel-tissue-derived epithelial stem cell mixture is deposited on the substrate to have a filamentous structure. The method of claim 54, wherein the filamentous structure has a linear, serpentine or spiral shape. The method of any one of claims 42 to 55, further comprising fragmenting the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to produce fragmented structures comprising the tissue-derived epithelial organoids. The method of any one of claims 1 to 56, wherein the tissue-derived epithelial organoids have a uniform morphology compared to reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. The method of claim 57, wherein the tissue-derived epithelial organoids are of uniform size. The method of claim 58, wherein the average diameter of the tissue-derived epithelial organoids is more uniform than that of the reference tissue-derived epithelial organoids. The method of any of claims 1 to 59, wherein stem cells and/or proliferation markers are expressed at higher levels in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. The method of claim 60, wherein the stem cell and/or proliferation marker is selected from the group consisting of: MKI67, EpCAM, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. The method of any of claims 1 to 61, wherein a differentiation marker is expressed at a lower level in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. The method of claim 62, wherein the differentiation marker is selected from the group consisting of: keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. A tissue-derived epithelial organoid produced by the method of any one of claims 1 to 63. A composition comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium. The composition of claim 65, wherein the hydrogel has a geometry comprising a length, width, and/or diameter greater than about 0.1 mm. The composition of claim 66, wherein the hydrogel has a geometric shape comprising a length, width, and/or diameter of about 0.1 mm to about 1,000 mm. A composition as in any one of claims 65 to 67, wherein the hydrogel is in the form of droplets. A composition as in any one of claims 65 to 67, wherein the hydrogel has a filamentous structure. The composition of claim 69, wherein the filamentous structure has a linear, serpentine or helical shape. The composition of any of claims 65 to 70, wherein stem cell and/or proliferation markers are expressed at higher levels in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. The composition of claim 71, wherein the stem cell and/or proliferation marker is selected from the group consisting of: MKI67, EpCAM, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. The composition of any of claims 65 to 72, wherein a differentiation marker is expressed at a lower level in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. The composition of claim 73, wherein the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. A method for screening an agent, comprising: (a)   contacting a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids as claimed in claim 64, or a composition as claimed in any one of claims 65 to 74, with an agent; and (b)   analyzing the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for changes indicative of the effectiveness, disposition and/or toxicity of the agent. The method of claim 75, wherein the agent is in contact with the tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids for about 15 minutes to about 3 years. The method of claim 75 or 76, wherein the agent is a therapeutic agent. The method of claim 77, wherein the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof. A method for performing genomic screening comprising: (a)   providing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids as claimed in claim 64 or a composition as claimed in any one of claims 65 to 74; (b)   generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid; and (c)   analyzing the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for changes associated with the mutation. The method of claim 79, wherein the mutation is generated using a gene regulation system. The method of claim 80, wherein the gene regulation system is a gene editing system. The method of claim 81, wherein the gene editing system is a CRISPR system. The method of any one of claims 75 to 82, wherein the change is a change in a property selected from the group consisting of cell viability, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, repression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof. A method for generating an epithelial cell model, comprising: (a)   providing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids as claimed in claim 64; (b)   digesting the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids into single cells; and (c)   culturing the single cells in a culture medium to generate a cell monolayer. The method of claim 84, wherein the single cells are cultured on a permeable cell culture insert. The method of claim 84 or 85, wherein the culture medium is a differentiation medium. The method of claim 84 or 85, wherein the culture medium is a cell growth or stem cell promoting medium. A method for screening an agent, comprising: (a)   contacting the cell monolayer produced by the method of any one of claims 84 to 87 with an agent; and (b)   analyzing the cell monolayer for changes indicative of the effectiveness, disposition and/or toxicity of the agent. The method of claim 88, wherein the agent is in contact with the cell monolayer for about 15 minutes to about 3 years. The method of claim 88 or 89, wherein the agent is a therapeutic agent. The method of claim 90, wherein the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof. A method for performing genomic screening, comprising: (a)   providing a cell monolayer produced by the method of any one of claims 84 to 87; (b)   generating a mutation in the genome of one or more cells in the cell monolayer; and (c)   analyzing the cell monolayer for changes associated with the mutation. The method of claim 92, wherein the mutation is generated using a gene regulation system. The method of claim 93, wherein the gene regulation system is a gene editing system. The method of claim 94, wherein the gene editing system is a CRISPR system. The method of any one of claims 84 to 95, wherein the change is a change in a property selected from the group consisting of cell viability, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, repression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof. The method of any one of claims 1 to 63 and 75 to 96 or the composition of any one of claims 65 to 74, wherein the tissue fragment is a fragment from a tissue selected from the group consisting of tear glands, tonsils, salivary glands, gastrointestinal tissue, thyroid, lung, breast, liver, bile duct, stomach, kidney, pancreas, endometrium, fallopian tube, cervix, prostate, bladder, taste buds, ovaries, placenta and combinations thereof. The method of any one of claims 1 to 63 and 75 to 96 or the composition of any one of claims 65 to 74, wherein the tissue-derived epithelial stem cells are obtained from fragments of organoids selected from the group consisting of tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, gastric organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblastic organoids, and combinations thereof. A system for culturing tissue-derived epithelial organoids comprises tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium. The system of claim 99, wherein the tissue-derived epithelial organoid is an intestinal organoid. The system of claim 99 or 100, wherein the hydrogel has a geometry comprising a length, width, and/or diameter greater than about 0.1 mm. The system of any of claims 99 to 101, wherein the hydrogel has a geometric shape comprising a length, width, and/or diameter of about 0.1 mm to about 1,000 mm. A system as in any of claims 99 to 102, wherein the hydrogel is in the form of droplets. A system as in any one of claims 99 to 102, wherein the hydrogel has a filamentous structure. The system of claim 104, wherein the filamentary structure has a linear, serpentine, or helical shape. The system of any of claims 99 to 105, wherein stem cell and/or proliferation markers are expressed at higher levels in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. The system of claim 106, wherein the stem cell and/or proliferation marker is selected from the group consisting of: MKI67, EpCAM, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. The system of any of claims 99 to 107, wherein a differentiation marker is expressed at a lower level in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. The system of claim 108, wherein the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. The system of any of claims 99 to 109, wherein the tissue-derived epithelial organoid is selected from the group consisting of tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, gastric organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblastic lining organoids, and combinations thereof. A system as in any of claims 99 to 110, comprising one or more robots and/or automated components for generating and/or culturing epithelial organoids derived from the tissue. The system of claim 111, wherein the one or more robots and/or automated components include a liquid handling robot. A system for performing the method of any of claims 1 to 63 and 75 to 98, comprising one or more robots and/or automated components. The system of claim 113, wherein the one or more robots and/or automated components include a liquid handling robot. A method as in any of claims 1 to 63 and 75 to 98, wherein one or more of the steps of the method are performed by one or more robots and/or automated components. The method of claim 115, wherein the one or more robots and/or automated components include a liquid handling robot. The tissue-derived epithelial organoid of claim 64, wherein the tissue-derived epithelial organoid is selected from the group consisting of tear gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, gastric organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblastic lining organoids, and combinations thereof.