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WO2024263961A2 - Procédés de culture en suspension sans matrice - Google Patents

Procédés de culture en suspension sans matrice Download PDF

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Publication number
WO2024263961A2
WO2024263961A2 PCT/US2024/035082 US2024035082W WO2024263961A2 WO 2024263961 A2 WO2024263961 A2 WO 2024263961A2 US 2024035082 W US2024035082 W US 2024035082W WO 2024263961 A2 WO2024263961 A2 WO 2024263961A2
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culture media
liquid
pscs
hgs
composition
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PCT/US2024/035082
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WO2024263961A3 (fr
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Magdalena KASENDRA
Katherine BOYLIN
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Children's Hospital Medical Center
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0679—Cells of the gastro-intestinal tract
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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    • C12N2513/00—3D culture

Definitions

  • aspects of the present disclosure relate generally to suspension cultures of pluripotent stem cells (PSCs), PSC aggregates, differentiated cells, spheroids, and organoids derived from the PSCs and PSC aggregates.
  • PSCs pluripotent stem cells
  • the presently disclosed methods for making said PSCs, PSC aggregates, differentiated cells, spheroids and organoids may be performed without the use of basement membrane matrices during PSC maintenance and expansion, as well as during differentiation of PSCs and PSC aggregates into differentiated cells and organoids, for example definitive endoderm (DE), hindgut spheroids (HGS), and intestinal organoids (IO).
  • DE definitive endoderm
  • HHS hindgut spheroids
  • IO intestinal organoids
  • compositions comprising such PSCs, differentiated cells, spheroids and organoids. Even further, disclosed herein are method of treatment using such compositions.
  • HIO human intestinal organoids
  • hPSC human PSC
  • direct differentiation protocols may suffer from high inter- hPSC line, inter-experimental and inter-well variability as they rely on the spontaneous formation and detachment of spheroids at -Day 7 of culture.
  • the transition from two-dimensional (e.g., monolayer) culture conditions to three-dimensional (e.g., MatrigeL embedded) culture conditions can represent a potentially significant bottleneck of HIO manufacture.
  • aspects of the present disclosure relate generally to suspension culture methods and compositions of pluripotent stem cells (PSC), three-dimensional PSC aggregates, differentiated cells, spheroids and organoids derived from PSCs. These methods may be performed without the use of xenogeneic basement membrane matrices during PSC maintenance and expansion, as well as during differentiation of PSCs into differentiated cells and organoids, for example definitive endoderm (DE), hindgut spheroids (HGS), and intestinal organoids (IO).
  • PSC pluripotent stem cells
  • the disclosed methods and compositions utilize a suspension culture for each of the following phases in the manufacture of an organoid from a PSC: maintenance and expansion of PSC, differentiation of PSC into DE, differentiation of DE into spheroids (e.g., hindgut spheroids), and differentiation and maturation of spheroids into an organoid (e.g., an intestinal organoid).
  • maintenance and expansion of PSC differentiation of PSC into DE
  • differentiation of DE into spheroids e.g., hindgut spheroids
  • differentiation and maturation of spheroids into an organoid e.g., an intestinal organoid
  • compositions for each of the aforementioned phases.
  • such compositions are based on three- dimensional expansion of PSCs, PSC aggregates, DE, spheroid, and/or organoids, via a suspension culture.
  • Embodiment 1 A method comprising: (a) inoculating a liquid culture media with PSCs; (b) culturing, in a bioreactor, the liquid culture media that is inoculated with the PSCs such that three-dimensional PSC aggregates form in the liquid culture media, wherein the culturing in the bioreactor comprises suspending the PSCs in the liquid culture media; (c) passaging the PSCs by: (i) dissociating at least a portion of the three-dimensional PSC aggregates into single cells; and (ii)inoculating a second liquid culture media with the dissociated three-dimensional PSC aggregates of (i) with PSCs.
  • Embodiment 2 The method of embodiment 1, wherein the liquid culture media and the second liquid culture media are free of materials of animal or human origin; optionally wherein the liquid culture media and the second liquid culture media are free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 3 The method of any one of the preceding embodiments, wherein the PSCs are passaged when the diameter of a majority of the three-dimensional PSC aggregates formed is not more than 500 pm.
  • Embodiment 4 The method of any one of the preceding embodiments, wherein the PSCs are passaged when the diameter of a majority of the three-dimensional PSC aggregates formed is not more than 400 pm.
  • Embodiment 5 The method of any one of the preceding embodiments, wherein the PSCs are passaged when the diameter of a majority of the three-dimensional PSC aggregates formed is not more than 300 pm.
  • Embodiment 6 The method of any one of the preceding embodiments, wherein at least 80% of the three-dimensional PSC aggregates are dissociated into single cells.
  • Embodiment 7 The method of any one of the preceding embodiments, optionally wherein at least 90% of PSC aggregates are dissociated into single cells.
  • Embodiment 8 The method of embodiment 1, where the method comprises passaging the PSCs two or more times by culturing the PSCs of inoculated second liquid culture media until additional three-dimensional PSC aggregates form.
  • Embodiment 9 The method of any one of the preceding embodiments, wherein the liquid culture media of (a) and/or the second liquid culture media of (c)(ii) is inoculated at a density of about 100,000-220,000 PSCs/ml.
  • Embodiment 10 The method of any one of the preceding embodiments, wherein the liquid culture media of (a) and/or the second liquid culture media of (c)(ii) is inoculated at a density of about 180,000-220,000 PSCs/ml.
  • Embodiment 11 The method of any one of the preceding embodiments, wherein the passaging occurs after a period of time following the inoculation in (a) that is between about 40-168 hours.
  • Embodiment 12 The method of any one of the preceding embodiments, wherein the passaging occurs after a period of time following the inoculation in (a) that is between about 40-84 hours.
  • Embodiment 13 The method of any one of the preceding embodiments, wherein the passaging occurs after a period of time following the inoculation in (a) that is between about 66-78 hours.
  • Embodiment 14 The method of any one of the preceding embodiments, wherein the method further comprises exchanging a portion of the culture media in the bioreactor of (a) after a period of time following the inoculation in (a) and/or (c)(ii) that is between about 36-60 hours.
  • Embodiment 15 The method of any one of the preceding embodiments, wherein the method further comprises exchanging a portion of the culture media in the bioreactor of (a) after a period of time following the inoculation in (a) and/or (c)(ii) that is between about 42-54 hours.
  • Embodiment 16 The method of any one of the preceding embodiments, wherein the method further comprises exchanging a portion of the liquid culture media in the bioreactor of (a) after a period of time following the inoculation in (a) and/or (c)(ii), wherein the portion of the liquid culture media exchanged is at least 50% of the liquid culture media in the bioreactor of (a).
  • Embodiment 17 The method of any one of the preceding embodiments, the method further comprising, prior to the inoculation in (a): culturing the PSCs on the surface of a substrate; and collecting the PSCs from the surface of the substrate for use in the inoculation of (a) when the PSCs are in a logarithmic growth phase and/or at 35-55% confluency, optionally wherein said collecting comprises dissociating the PSCs prior to the inoculation of (a).
  • Embodiment 18 The method of embodiment 17, wherein the PSCs are collected from the surface of the substrate for use in the inoculation of (a) when the PSCs are in a logarithmic growth phase and/or at 40-50% confluency.
  • Embodiment 19 The method of any one of the preceding embodiments, wherein the dissociation is chemical, enzymatic and/or mechanical dissociation.
  • Embodiment 20 The method of any one of the preceding embodiments, wherein the dissociation is enzymatic, optionally wherein the enzyme comprises a proteolytic and/or a collagenolytic enzyme, optionally wherein the enzyme is Accutase.
  • Embodiment 21 The method of any one of the preceding embodiments, wherein the bioreactor of (a) and/or (b) comprises a rotating chamber comprising the liquid culture media, wherein the rotation speed of the rotating chamber is selected such that the number of PSCs in the liquid culture media at (c) is at least 2 or 2.5 -fold the number of PSCs used to inoculate the liquid culture media, optionally wherein the number of PSCs in the liquid culture media at (c) is at least 2 or 2.5-fold the number of PSCs used to inoculate the liquid culture media for at least two passages of the PSCs.
  • Embodiment 22 The method of any one of the preceding embodiments, wherein the liquid culture media is a serum-free media; optionally, wherein the media comprises recombinant human basic fibroblast growth factor (rh bFGF) and/or recombinant human transforming growth factor P (rh TGF ).
  • rh bFGF human basic fibroblast growth factor
  • rh TGF human transforming growth factor P
  • Embodiment 23 The method of any one of the preceding embodiments, wherein, following one or more passages, the portion of PSCs expressing Oct4, SSEA1 and TRA 1-60 at levels at least as high as the average expression level of the PSCs used in the inoculation in (a) is at least 85%.
  • Embodiment 24 The method of embodiment 23, wherein, following the one or more passages, the portion of PSCs expressing Oct4, SSEA1 and TRA 1-60 at levels at least as high as the average expression level of the PSCs used in the inoculation in (a) is at least 95%.
  • Embodiment 25 The method of any one of the preceding embodiments, wherein the PSCs express SOX2 and KLF4.
  • Embodiment 26 A method for differentiating PSCs into definitive endoderm (DE) in a three dimensional suspension culture, the method comprising: (d) culturing, in a bioreactor, a liquid culture media inoculated with PSCs; wherein the culturing of the liquid culture media inoculated with PSCs of (d) comprises suspending the PSCs in the liquid culture media; and (e) culturing, in a bioreactor, the PSCs of (d) in liquid definitive endoderm differentiation culture media for a period of time sufficient to differentiate the PSCs into DE, wherein the culturing of the PSCs of (d) in the liquid definitive endoderm differentiation culture media comprises suspending the PSCs in the liquid definitive endoderm differentiation culture media.
  • Embodiment 27 The method of embodiment 26, wherein the liquid culture media or the liquid definitive endoderm differentiation culture is free of materials of animal or human origin; optionally wherein the culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 28 The method of embodiment 26 or 27, wherein the culturing in (d) is for a period of time between about 18-54 hours.
  • Embodiment 29 The method of any one of embodiments 26-28, wherein the culturing in (d) is for a period of time between about 24-48 hours.
  • Embodiment 30 The method of any one of embodiments 26-29, wherein the liquid culture media inoculated with PSCs cultured in (d) is the PSC inoculated culture media of (c)(ii) of any one of embodiments 1-24.
  • Embodiment 31 The method of any one of embodiments 26-30, wherein the period of time sufficient to differentiate the PSCs into DE is a period of time that is between about 48-96 hours.
  • Embodiment 32 The method of any one of embodiments 26-31, wherein the period of time sufficient to differentiate the PSCs into DE is a period of time that is between about 60-84 hours.
  • Embodiment 33 The method of any one of embodiments 26-32, wherein the period of time sufficient to differentiate the PSCs into DE is a period of time that is between about 66-78 hours.
  • Embodiment 34 The method of any one of embodiments 26-33, wherein culturing the PSCs in the liquid definitive endoderm differentiation culture media for a period of time sufficient to differentiate the PSCs into DE comprises culturing the PSCs in a culture media comprising a nodal signaling pathway activator and/or a Wnt signaling pathway activator for a first period, then culturing the PSCs in a culture media comprising the nodal signaling pathway activator and/or the Wnt signaling pathway activator and a serum or serum replacement for a second period, and then culturing the PSCs in a culture media comprising the nodal signaling pathway activator and/or the Wnt signaling pathway activator and the serum or serum replacement for a third period.
  • Embodiment 35 The method of embodiment 34, wherein the culture media in which the PSCs are cultured for the first period further comprises a BMP activator.
  • Embodiment 36 The method of embodiment 34 or 35, wherein the culture media in which the PSCs are cultured for the second period, and the culture media in which the PSCs are cultured for the third period, comprise: the nodal signaling pathway activator and/or the Wnt signaling pathway activator and a serum; optionally wherein the serum is FBS.
  • Embodiment 37 The method of embodiment 34 or 35, wherein the culture media in which the PSCs are cultured for the second period, and the culture media in which the PSCs are cultured for the third period, comprise: the nodal signaling pathway activator and/or the Wnt signaling pathway activator and a serum replacement; optionally wherein the serum replacement is knockout serum replacement (KSR).
  • KSR knockout serum replacement
  • Embodiment 38 The method of any one of embodiments 34-37, wherein each of the first, second and third period of time is between about 18-30 hours.
  • Embodiment 39 The method of any one of embodiments 34-38, wherein each of the first, second and third period of time is between about 20-28 hours.
  • Embodiment 40 The method of any one of embodiments 26-39, wherein the efficiency of DE induction is at least about 35%.
  • Embodiment 41 The method of any one of embodiments 26-40, wherein the efficiency of DE induction is at least about 45-55%.
  • Embodiment 42 The method of any one of embodiments 26-41, wherein the DE expresses Soxl7 and FoxA2.
  • Embodiment 43 A method for differentiating definitive endoderm (DE) into hindgut spheroids (HGS) in a three dimensional suspension culture, the method comprising: (f) culturing, in a bioreactor, DE in liquid hindgut differentiation culture media for a period of time sufficient to differentiate the DE into HGS, wherein the culturing of the DE comprises suspending the DE in the liquid hindgut differentiation culture media.
  • DE definitive endoderm
  • HGS hindgut spheroids
  • Embodiment 44 The method of embodiment 43, wherein the liquid hindgut differentiation culture media is free of materials of animal or human origin; optionally wherein the liquid hindgut differentiation culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 45 The method of embodiment 43, wherein the DE cultured in (f) is the DE of any one of embodiments 25-39.
  • Embodiment 46 The method of any one of embodiments 43-45, wherein the period of time sufficient to differentiate the DE into HGS is a period of time that is between about 60-120 hours.
  • Embodiment 47 The method of any one of embodiments 43-46, wherein the period of time sufficient to differentiate the DE into HGS is a period of time that is between about 84-108 hours.
  • Embodiment 48 The method of any one of embodiments 43-47, wherein the period of time sufficient to differentiate the DE into HGS is a period of time that is between about 90-102 hours.
  • Embodiment 49 The method of any one of embodiments 43-48, wherein the liquid hindgut differentiation culture media is changed after a period of time that is between about 20-28 hours.
  • Embodiment 50 The method of any one of embodiments 43-49, wherein the liquid hindgut differentiation culture media is changed after a period of time that is between about 22-26 hours.
  • Embodiment 51 The method of any one of embodiments 43-50, wherein the liquid hindgut differentiation culture media comprises a Wnt signaling pathway activator, an FGF signaling pathway activator, and optionally FBS.
  • Embodiment 52 The method of embodiment 51, wherein the Wnt signaling pathway activator comprises CHIR99021.
  • Embodiment 53 The method of embodiment 51 or 52, wherein the FGF signaling pathway activator comprises FGF4.
  • Embodiment 54 The method of any one of embodiments 51-53, wherein the FGF signaling pathway activator is FGF4 at a concentration between about 50-750 ng/ml.
  • Embodiment 55 The method of any one of any one of embodiments 51-54, wherein the Wnt pathway activator is CHIRON 99021 at a concentration between about 0.5 - 6 pM.
  • Embodiment 56 A method for differentiating hindgut spheroid (HGS) into intestinal organoids (IO) in a three dimensional suspension culture, the method comprising:
  • Embodiment 57 The method of embodiment 56, wherein the liquid IO maturation culture media is free of materials of animal or human origin; optionally wherein the culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 58 The method of embodiment 56 or 57, wherein the HGS cultured in (g) is the HGS of any one of embodiments 40-52.
  • Embodiment 59 The method of any one of embodiments 56-58, wherein the period of time sufficient to differentiate the HGS into IO is a period of time that is between about 12-30 days.
  • Embodiment 60 The method of any one of embodiments 56-59, wherein the period of time sufficient to differentiate the HGS into IO is a period of time that is between about 15-28 days.
  • Embodiment 61 The method of any one of embodiments 56-60, wherein the IO maturation culture media is changed after a period of time that is between about 24-54 hours.
  • Embodiment 62 The method of any one of embodiments 56-61, wherein the IO maturation culture media is changed after a period of time that is between about 46-50 hours.
  • Embodiment 63 The method of any one of embodiments 56-62, wherein the IO maturation culture media comprises one or more of EGF, R-spondin, Noggin, Gremlin 1, and/or Epiregulin (EREG).
  • EGF EGF
  • R-spondin Noggin
  • Gremlin 1 EGF
  • EREG Epiregulin
  • Embodiment 64 The method of embodiment 63, wherein the concentration of EGF, R-sponding, Noggin, Gremlin 1, and/or EREG is between about 25-150 ng/ml.
  • Embodiment 65 The method of embodiment 63 or 64, wherein the concentration of EGF R-sponding, Noggin, Gremlin 1, and/or EREG is between about 50-100 ng/ml.
  • Embodiment 66 The method of any one of embodiments 56-65, wherein the HGS are not dissociated prior to culturing in the IO maturation culture media, wherein epithelial cells of the IO formed have a polarity wherein the apical surface is oriented to the outside of the IO.
  • Embodiment 67 Embodiment 67.
  • a method for differentiating hindgut spheroids (HGS) into intestinal organoids (IO) having an apical-in polarity in a three dimensional suspension culture comprising the method of any one of embodiments 56-66; wherein the method further comprises dissociating at least a portion of the HGS into HGS single cells prior to incubation in the IO maturation culture media; wherein the culturing of the HGS comprises suspending the dissociated HGS single cells and any non-dissociated HGS in the liquid IO maturation culture media; wherein epithelial cells of the IO formed from the dissociated HGS single cells have a polarity wherein the apical surface is oriented to the inside of the IO.
  • Embodiment 68 The method of embodiment 67, wherein the liquid IO maturation culture media is free of materials of animal or human origin; optionally wherein the liquid IO maturation culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 69 The method of embodiment 67 or 68, wherein at least 80% of the HGS are dissociated into single cells.
  • Embodiment 70 The method of any one of embodiments 67-69, wherein at least 90% of HGS are dissociated into single cells.
  • Embodiment 71 The method of any one of embodiments 67-70, wherein a concentration of dissociated HGS single cells are in the IO maturation culture media, wherein the concentration is between about 0.05x105 - 80x105 dissociated HGS single cells/ml of IO maturation culture media.
  • Embodiment 72 The method of any one of embodiments 67-71, wherein a concentration of dissociated HGS single cells are in the IO maturation culture media, wherein the concentration is between about 10x105-80x105 dissociated HGS single cells/ml of IO maturation culture media.
  • Embodiment 73 The method of any one of embodiments 67-72, wherein a concentration of dissociated HGS single cells are in the IO maturation culture media, wherein the concentration is between about 20x105-60x105 dissociated HGS single cells/ml of IO maturation culture media.
  • Embodiment 74 The method of any one of embodiments 67-73, wherein the dissociation is chemical, enzymatic and/or mechanical dissociation.
  • Embodiment 75 The method of any one of embodiments 67-74, wherein the dissociation is enzymatic, optionally wherein the enzyme comprises a proteolytic and/or a collagenolytic enzyme, optionally wherein the enzyme is Accutase.
  • Embodiment 76 The method of any one of embodiments 67-75, wherein the method further comprises transplanting the IO into a subject.
  • Embodiment 78 The method of embodiment 77, wherein the IO is transplanted under the kidney capsule of a non-human animal for a period of time that is between about 12- 20 weeks.
  • Embodiment 79 The method of any one of embodiments 77-78, wherein the IO is transplanted under the kidney capsule of a non-human animal for a period of time that is between about 16-20 weeks.
  • Embodiment 80 The method of embodiment 76, wherein transplanting the IO into the subject comprises transplanting the IO into an intestinal lumen of the subject for treatment of an intestine of the subject.
  • Embodiment 81 The method of any one of embodiments 76-80, wherein the IO is matured in vitro for a period of time prior to transplantation, optionally wherein the period of time is between about 7-28 days.
  • Embodiment 82 The method of any one of embodiments 76-81, wherein the IO is matured in vitro for a period of time prior to transplantation that is between about 14-28 days.
  • Embodiment 83 The method of any one of embodiments 76-82, wherein the IO is matured in vitro for a period of time prior to transplantation that is between about 21-28 days.
  • Embodiment 84 The method of any one of embodiments 26-42, the method further comprising differentiating the DE into a spheroid, optionally wherein the differentiating comprises: (h) culturing, in a bioreactor, the DE in liquid differentiation culture media for a period of time sufficient to differentiate the DE into a spheroid, wherein the culturing of the DE comprises suspending the DE in the liquid differentiation culture media; optionally wherein the spheroid is a foregut or a hindgut spheroid.
  • Embodiment 85 The method of embodiment 84, wherein the liquid differentiation culture media is free of materials of animal or human origin; optionally wherein the liquid differentiation culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 87 The method of any one of embodiments 84-86, wherein the liquid organoid maturation culture media is free of materials of animal or human origin; optionally wherein the liquid organoid maturation culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 88 The method of any one of the preceding embodiments, wherein the PSC is an induced PSC (iPSC) or an embryonic stem cells (ESC).
  • iPSC induced PSC
  • ESC embryonic stem cells
  • Embodiment 89 The method of any one of the preceding embodiments, wherein the PSC is a human PSC, optionally a human iPSC (hiPSC).
  • the PSC is a human PSC, optionally a human iPSC (hiPSC).
  • Embodiment 90 A PSC or three-dimensional PSC aggregate made by the method of any one of embodiments 1-25 or 88-89.
  • Embodiment 91 DE made by the method of any one of embodiments 26-42 or 88-89.
  • Embodiment 92 An HGS made by the method of any one of embodiments 43- 552, or 88-89.
  • Embodiment 93 An IO made by the method of any one of embodiments 56-89.
  • Embodiment 94 An IO having an apical-in polarity, wherein epithelial cells of the IO have a polarity wherein the apical surface is oriented to the inside of the IO, optionally wherein the IO is a human IO (hlO).
  • Embodiment 95 The IO having an apical-in polarity made by the method of any one of embodiments 67-89.
  • Embodiment 96 A spheroid made by the method of any one of embodiments 84, 85, or -89.
  • Embodiment 97 An organoid made by the method of any one of embodiments 86-89.
  • Embodiment 98 A method of treatment comprising transplanting the IO of any one of embodiments 93-95, or cells derived therefrom, into an animal, optionally wherein the animal is suffering from a GI disease state; optionally wherein the animal is human.
  • Embodiment 99 A method of screening a compound for activity comprising contacting the IO of any one of embodiments 93-95, or cells derived therefrom, with the compound and measuring a response of the IO to the compound.
  • Embodiment 100 A method of screening a compound for activity comprising contacting the organoid of embodiment 97, or cells derived therefrom, with the compound and measuring a response of the organoid to the compound.
  • Embodiment 101 The method of any one of the preceding embodiments, wherein the method is free of any xenogenic material, optionally wherein the organoids are clinical grade and suitable for transplantation in a human.
  • Embodiment 102 The method of any one of the preceding embodiments, wherein the bioreactor of (a), (b), (d), (e), (f), (g), (h), and/or (i) comprises a rotating chamber comprising the liquid culture media, the second liquid culture media, the liquid definitive endoderm differentiation culture media, liquid hindgut differentiation culture media, liquid IO maturation culture media, liquid differentiation culture media, and/or liquid organoid maturation culture media; wherein the rotating chamber is a cylindric section which is rotated around its longitudinal axis, thereby suspending the PSCs and/or three-dimensional PSC aggregates in the liquid culture media, the second liquid culture media, the liquid definitive endoderm differentiation culture media, liquid hindgut differentiation culture media, liquid IO maturation culture media, liquid differentiation culture media, and/or liquid organoid maturation culture media; optionally wherein the chamber is oriented such that its longitudinal axis is parallel to the ground.
  • Embodiment 103 The method of any one of the preceding embodiments, wherein the bioreactor of (a), (b), (d), (e), (f), (g), (h), and/or (i) comprises a rotating chamber comprising the liquid culture media, the second liquid culture media, the liquid definitive endoderm differentiation culture media, liquid hindgut differentiation culture media, liquid TO maturation culture media, liquid differentiation culture media, and/or liquid organoid maturation culture media in a volume that is between about 5ml to about 50 L.
  • Embodiment 104 The method of any one of the preceding embodiments, wherein the bioreactor of (a), (b), (d), (e), (f), (g), (h), and/or (i) comprises a rotating chamber comprising the liquid culture media, the second liquid culture media, the liquid definitive endoderm differentiation culture media, liquid hindgut differentiation culture media, liquid IO maturation culture media, liquid differentiation culture media, and/or liquid organoid maturation culture media; wherein the rotation of the chamber is between about 3-7 rpm; and optionally wherein the rotational speed is a speed selected to keep the PSCs, the three-dimensional PSC aggregates, the spheroids, and/or the organoids in statical orbit.
  • Embodiment 105 The method of any one of the preceding embodiments, wherein the bioreactor of (a), (b), (d), (e), (f), (g), (h), and/or (i) comprises a rotating chamber comprising the liquid culture media, the second liquid culture media, the liquid definitive endoderm differentiation culture media, liquid hindgut differentiation culture media, liquid IO maturation culture media, liquid differentiation culture media, and/or liquid organoid maturation culture media; wherein the average shear stress on the PSCs, the three-dimensional PSC aggregates, the spheroids, and/or the organoids is less than about 5.0 dynes/cm 2 .
  • Embodiment 106 The method of any one of the preceding embodiments, wherein the liquid culture media, the second liquid culture media, the liquid definitive endoderm differentiation culture media, the liquid hindgut differentiation culture media, the liquid IO maturation culture media, the liquid differentiation culture media, and/or the liquid organoid maturation culture media comprises an anti-apoptotic agent.
  • Embodiment 107 The method of any one of the preceding embodiments, wherein the liquid culture media, the second liquid culture media, the liquid definitive endoderm differentiation culture media, the liquid hindgut differentiation culture media, the liquid IO maturation culture media, the liquid differentiation culture media, and/or the liquid organoid maturation culture media comprises an anti-adhesion agent.
  • Embodiment 108 The method of embodiment of any one of the preceding embodiments, wherein the liquid culture media comprises an anti-adhesion agent.
  • Embodiment 109 The method of embodiment 108, wherein the anti-adhesion agent is DSS, xantham gum, A-205804, I-CAM1, carboxymethyl cellulose, and/or Neural Organoid Basal Medium 2 (NOBM).
  • the anti-adhesion agent is DSS, xantham gum, A-205804, I-CAM1, carboxymethyl cellulose, and/or Neural Organoid Basal Medium 2 (NOBM).
  • NOBM Neural Organoid Basal Medium 2
  • Embodiment 110 The method of any one of embodiments 108-109, wherein the anti-adhesion agent is DSS at a concentration that is between about 1 pg/ml - 1000 pg/ml of the liquid culture media, the second liquid culture media, the liquid definitive endoderm differentiation culture media, the liquid hindgut differentiation culture media, the liquid IO maturation culture media, the liquid differentiation culture media, and/or the liquid organoid maturation culture media.
  • the anti-adhesion agent is DSS at a concentration that is between about 1 pg/ml - 1000 pg/ml of the liquid culture media, the second liquid culture media, the liquid definitive endoderm differentiation culture media, the liquid hindgut differentiation culture media, the liquid IO maturation culture media, the liquid differentiation culture media, and/or the liquid organoid maturation culture media.
  • Embodiment 111 A composition for a three-dimensional expansion and maintenance of pluripotent stem cell (PSC) cultures, the composition comprising: a liquid culture media comprising recombinant human basic fibroblast growth factor (rh bFGF), and/or a recombinant human transforming growth factor P (rh TGF ); and PSCs suspended in the culture media.
  • PSC pluripotent stem cell
  • Embodiment 112 The composition of embodiment 111, wherein the liquid culture media is a serum-free media, wherein the liquid culture media is free of materials of animal or human origin; optionally wherein the culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 113 The composition of embodiment 111 or 112, further comprising: an anti- apop to tic agent.
  • Embodiment 114 The composition of any one of embodiments 111-113, wherein the PSCs express Oct4, SSEA1, TRA 1-60, Sox 2, and/or TRA-1-81.
  • Embodiment 115 The composition of any one of embodiments 111-114, wherein the PSCs express Oct4, SSEA1, TRA 1-60, Sox 2, and TRA-1-81.
  • Embodiment 116 The composition of any one of embodiments 111-115, further comprising: an anti-adhesion agent.
  • Embodiment 117 The composition of embodiment 116, wherein the antiadhesion agent is one or both of DSS or xantham gum.
  • Embodiment 118 The composition of any one of embodiments 111-117, wherein the PSCs are suspended in the liquid culture media at a density that is about 50,000 - 1,000,000 PSCs/ml of culture media.
  • Embodiment 119 The composition of any one of embodiments 1 11-117, wherein the PSCs arc suspended in the liquid culture media at a density that is about 100,000 - 300,000 PSCs/ml of culture media
  • Embodiment 120 The composition of any one of embodiments 111-119, wherein the PSCs are suspended in the culture media at a density that is about 180,000 - 220,000 PSCs/ml of culture media.
  • Embodiment 121 A composition for differentiating PSCs into definitive endoderm (DE) in a three dimensional suspension culture, the composition comprising: a liquid DE differentiation culture media; and PSCs suspended in the liquid DE differentiation culture media.
  • DE definitive endoderm
  • Embodiment 122 The composition of embodiment 121, wherein the liquid DE differentiation culture media is a serum- free media, wherein the liquid DE differentiation culture media is free of materials of animal or human origin; optionally wherein the liquid DE differentiation culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 123 The composition of embodiment 121 or 122, wherein the PSCs have an average diameter of less than about 500pm.
  • Embodiment 124 The composition of any one of embodiments 121-123, wherein the PSCs have an average diameter of less than about 400pm.
  • Embodiment 125 The composition of any one of embodiments 121-124, wherein the PSCs have an average diameter of less than about 300pm.
  • Embodiment 126 The composition of any one of embodiments 121-125, wherein the liquid DE differentiation culture media comprises a nodal signaling pathway activator and/or a Wnt signaling pathway activator, at a concentration of about 10 to 200 ng/mL of liquid DE differentiation culture media.
  • Embodiment 127 The composition of embodiment 126, wherein the nodal signaling pathway activator or the Wnt signaling pathway activator is at a concentration of about 10 to 200 ng/mL of liquid DE differentiation culture media
  • Embodiment 128 The composition of any one of embodiments 126-127, wherein the nodal signaling pathway activator or the Wnt signaling pathway activator is at a concentration of about 50 to 150 ng/mL ng/mL of liquid DE differentiation culture media.
  • Embodiment 129 The composition of any one of embodiments 126-128, wherein the nodal signaling pathway activator or the Wnt signaling pathway activator at a concentration of about 100 to 200 ng/mL ng/mL of liquid DE differentiation culture media.
  • Embodiment 130 The composition of any one of embodiments 126-129, wherein the liquid DE differentiation culture media further comprises serum or a serum replacement at a concentration of about 0% to 20%.
  • Embodiment 131 The composition of any one of embodiments 126-130, wherein the liquid DE differentiation culture media further comprises serum or serum replacement at a concentration of about 2% to 5%.
  • Embodiment 132 The composition of any one of embodiments 111-131, further comprising: DE differentiated from the PSCs.
  • Embodiment 133 The composition of embodiment 132, wherein the DE differentiated from the PSCs expresses Soxl7 and/or FoxA2.
  • Embodiment 134 The composition of embodiment 132 or 133, wherein the DE differentiated from the PSCs expresses Soxl7 and FoxA2.
  • Embodiment 135 A composition for differentiating DE into hindgut spheroids (HGS) in a three dimensional suspension culture, the composition comprising: a liquid hindgut differentiation culture media comprising a Wnt signaling pathway activator, an FGF signaling pathway activator, and optionally FBS; and DE suspended in the liquid hindgut differentiation culture media.
  • HGS hindgut spheroids
  • Embodiment 136 The composition of embodiment 135, wherein the liquid hindgut differentiation culture media is free of materials of animal or human origin; optionally wherein the culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 137 The composition of embodiment 135 or 136, wherein the Wnt signaling pathway activator comprises CHIR99021, and wherein the FGF signaling pathway activator comprises FGF4.
  • Embodiment 138 The composition of any one of embodiments 135-137, wherein the FGF signaling pathway activator is at a concentration that is at least about 50 ng/ml of the liquid hindgut differentiation culture media.
  • Embodiment 139 The composition of any one of embodiments 135-138, wherein the FGF signaling pathway activator is at a concentration that is at least about 500 ng/ml of the liquid hindgut differentiation culture media.
  • Embodiment 140 The composition of any one of embodiments 135-139, wherein the Wnt pathway activator is at a concentration that is at least about 0.5 pM of the liquid hindgut differentiation culture media
  • Embodiment 141 A composition for differentiating HGS into intestinal organoids (IO) in a three dimensional suspension culture, the composition comprising: a liquid IO maturation culture media comprising EGF; and HGS suspended in the liquid IO maturation culture media.
  • IO intestinal organoids
  • Embodiment 142 The composition of embodiment 141, wherein the liquid IO maturation culture media is free of materials of animal or human origin; optionally wherein the liquid IO maturation culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 143 The composition of embodiment 141 or 142, wherein lumen of the HGS suspended in the liquid IO maturation culture media is exterior facing relative to the liquid IO maturation culture media.
  • Embodiment 144 The composition of any one of embodiments 141-143, wherein the concentration of EGF is at least about 25 ng/ml.
  • Embodiment 145 The composition of any one of embodiments 141-144, wherein the concentration of EGF is at least about 100 ng/ml.
  • Embodiment 146 The composition of any one of embodiments 141-145, wherein at least a portion of the HGS suspended in the liquid IO maturation culture media comprises dissociated HGS single cells.
  • Embodiment 147 The composition of embodiment 146, wherein at least 80% of the HGS are dissociated HGS single cells; optionally wherein at least 90% of HGS are dissociated single cells.
  • Embodiment 148 The composition of any one of embodiments 146 or 147, wherein a concentration of the dissociated HGS single cells in the liquid IO maturation culture media is in a range that about 0.1x105-80x105 dissociated HGS single cells/ml of liquid IO maturation culture media.
  • Embodiment 149 The composition of any one of embodiments 146-148, wherein a concentration of the dissociated HGS single cells in the liquid IO maturation culture media is in a range that is about 20x105-60x105 dissociated HGS single cells/ml of liquid IO maturation culture media.
  • Embodiment 150 The composition of any one of embodiments 141-149, further comprising: IO differentiated from the HGS.
  • Embodiment 151 The composition of embodiment 150, wherein epithelial cells of the IO formed from the dissociated HGS single cells have a polarity such that an apical surface is oriented to the inside of the IO.
  • Embodiment 152 The composition of any one of embodiments 141-151, wherein the HGS express CdX2.
  • Embodiment 153 The composition of any one of embodiments 141-152, wherein the HGS expresses FOX-F1 but does not express SOX2.
  • Embodiment 154 The composition of embodiments 141-153, wherein the liquid IO maturation culture media further comprises noggin.
  • Embodiment 155 A composition comprising: a liquid culture media; and three- dimensional PSC aggregates suspended in the liquid culture media.
  • Embodiment 156 The composition of embodiment 155, wherein the liquid culture media is free of materials of animal or human origin; optionally wherein the liquid culture media is free of any extracellular matrix and/or basement membrane matrix.
  • Embodiment 157 The composition of embodiment 155 or 156, wherein at least a portion of the three-dimensional PSC aggregates are dissociated as single cells.
  • Embodiment 158 The composition of any one of embodiments 155-157, wherein an average size of diameters of the three-dimensional PSC aggregates is less than 400 pm.
  • Embodiment 159 The composition of any one of embodiments 155-158, wherein an average size of diameters of the three-dimensional PSC aggregates is less than 350 pm.
  • Embodiment 160 The composition of any one of embodiments 155-159, wherein an average size of diameters of the three-dimensional PSC aggregates is less than 300 pm.
  • Embodiment 161 The composition of any one of embodiments 155-160, further comprising: an anti-adhesion agent.
  • Embodiment 162 The composition of embodiment 161, wherein the antiadhesion agent is DSS, xantham gum, A-205804, I-CAM1, carboxymethyl cellulose, and/or Neural Organoid Basal Medium 2 (NOBM).
  • the antiadhesion agent is DSS, xantham gum, A-205804, I-CAM1, carboxymethyl cellulose, and/or Neural Organoid Basal Medium 2 (NOBM).
  • Embodiment 163 The composition of embodiment 161 or 162, wherein the anti-adhesion agent is at a concentration that is between about 1 pg/ml - 1000 g/ml of the liquid culture media.
  • FIG. 1 A depicts an embodiment of an experimental protocol to explore the impact of various culture conditions on the maintenance and expansion of PSCs.
  • FIG. IB depicts an embodiment of an illustration of the basic principle of the operation of a suspension culture where the chamber is rotated around its longitudinal axis when the longitudinal axis is oriented parallel to the ground.
  • the depicted rotating vessel bioreactor (cell culture systems), rotates continuously to keep the cells, PSC aggregates, spheroids and/or organoids suspended by counterbalancing the gravitation forces, thereby ideally keeping them in statical orbit.
  • cells grown in the rotating vessel bioreactor experience very low shear forces.
  • FIG. 2 depicts the results of an embodiment of a study examining the impact of the inoculation density of PSC in the suspension culture media on the formation of PSC aggregates and cell death at various time points following culture inoculation.
  • FIG. 3 depicts the results of an embodiment of a study comparing dissociation reagents’ ability to form single cell cultures from PSC aggregates which form viable and homogenous PSC aggregates 24 and 96 hours post-inoculation of the single cells.
  • FIGs. 4A-4C depict the results of an embodiment of a study examining how the stage of growth of the PSC two-dimensional culture used to inoculate the suspension culture.
  • FIG. 4A depicts an embodiment of an iPSC growth curve and two timepoints for harvesting iPSCs for inoculation of the suspension culture: dO refers to the day at which the PSC line would usually be passaged (80-90% confluency) - note that this day may differ for different PSC lines (usually ranging from 4-6 days post-seeding); d-1 refers to one day before PSC culture reached “passage ready” confluency when monolayer is at 40-50% confluency.
  • dO refers to the day at which the PSC line would usually be passaged (80-90% confluency) - note that this day may differ for different PSC lines (usually ranging from 4-6 days post-seeding);
  • d-1 refers to one day before PSC culture reached “passage ready” confluency when monolayer is at
  • FIG. 4B depicts an embodiment of photographs of a starting two-dimensional PSC culture at d-1 and dO, the resulting PSC aggregates at day 4 (d4) of suspension culture inoculated with the d-1 or dO PSC.
  • FIG. 4C depicts an embodiment of a graph of the PSC aggregate size distribution at d4 of the suspension culture using either d-1 or dO PSC inoculates.
  • FIG. 5 depicts the results of an embodiment of a study comparing how the size of PSC aggregates in a suspension culture at the time of their passaging impacts the yield of PSC aggregates formed after reinoculation. Passaging of PSC aggregates at Day 3 (when the diameter of the majority of aggregates is ⁇ 400 pm) results in a successful propagation of the culture. Passaging of PSC aggregates at Day 4, when their diameter exceeds 400 pm results in a much lower yield of aggregates.
  • FIGs. 6A-6C depict the results of an embodiment of a study comparing how the bioreactor rotation speed impacts the yield of PSC aggregates formed.
  • FIG. 6A depicts an embodiment of PSC aggregates on day 3 (d3) of suspension culture at various bioreactor chamber rotation speeds.
  • FIG. 6B depicts an embodiment of a graph of the PSC aggregate size distribution at d3 of the suspension at various bioreactor chamber rotation speeds.
  • FIG. 6C depicts an embodiment of a chart comparing the output of PSC cells at the first (Pl), second (P2) and third (P3) passaging of suspension cultures at various bioreactor chamber rotation speeds.
  • FIG. 7 depicts the results of an embodiment based on a study comparing the use of anti- apop to tic agents.
  • the use of anti-apoptotic agents increase cell (e.g., PSCs) recovery with each passage.
  • the anti-apoptotic agent CEPT was found to provide higher cell recovery than the anti-apoptotic agent ROCKi.
  • FIG. 8 depicts the results of an embodiment of a study comparing how culture media, mTeSR 1 (research media) and mTeSR AOF (animal product free media), impacts PSC expansion and maintenance at the first (Pl), second (P2) and third (P3) passaging of a suspension culture.
  • FIGs. 9A and 9B depict the results of an embodiment of a study comparing how culture media, mTcSR 1 (research media; FIG. 9A) and mTcSR AOF (animal product free media; FIG. 9B), impacts stem cell markers: Oct4, SSEA4 and TRA 1-60.
  • FIG. 10 depicts the results of an embodiment of a study comparing how cell line (research-grade, PSC cell line 72.3; clinical-grade PSC cell line FF3 produced under GMP) impact the production of PSC aggregates at the first (Pl), second (P2) and third (P3) passaging of a suspension culture.
  • cell line search-grade, PSC cell line 72.3; clinical-grade PSC cell line FF3 produced under GMP
  • FIGs. 11A and 1 IB depict the results of an embodiment of a study comparing the impact of two-dimensional (2D) culturing (FIG. 11 A) to suspension culturing (3D) (FIG. 11B) on the expression of stem cell markers Oct4, SSEA4 and TRA 1-60 in research -grade PSC cell line 72.3.
  • FIGs. 12A and 12B depict the results of an embodiment of a study comparing the impact of two-dimensional (2D) culturing (FIG. 12A) to suspension culturing (3D) (FIG. 12B) on the expression of stem cell markers Oct4, SSEA4 and TRA 1-60 in clinical-grade PSC cell line FF3 produced under GMP.
  • FIGs. 13A-13B depict the result of an embodiment based on a study investigating the formation of three-dimensional PSC aggregates from PSCs, where the three-dimensional PSC aggregates are able to retain their pluripotency.
  • PSCs are able to form three-dimensional PSC aggregates progressing from Day 1 to Day 4.
  • number of such aggregates increases from 600 in Day 1 to -1400 in Day 4.
  • markers of pluripotency such as OCT4 and SSEA4 are evident in the three- dimensional PSC aggregates in the confocal imaging..
  • FIGs. 14A-14B depict the results of an evaluation of pluripotent 3D PSC aggregates across multiple lines and passages. As shown in FIG. 14A, there was a marked formation of three-dimensional PSC aggregates across passages for iPSC line 72.3 and ESC line Hl. FIG. 14B shows an increase in cell count as well as an increase in diameter of the PSC aggregates across the passages for both lines. Furthermore, FIG. 14B shows that an expression of pluripotency markers (e.g., OCT4 and SSEA-4) remained at least 90% across all the passages and lines tested for three-dimensional PSC aggregates, similar to the expression in conventional, two-dimensionally grown PSC monolayers.
  • FIG. 14A shows an expression of pluripotency markers (e.g., OCT4 and SSEA-4) remained at least 90% across all the passages and lines tested for three-dimensional PSC aggregates, similar to the expression in conventional, two-dimensionally grown PSC monolayers.
  • FIG. 15 depicts the results an evaluation comparing the pluripotency of PSC lines grown in three-dimensional suspension culture according to methods described herein to PSC lines grown in conventional 2D monolayers.
  • OCT4, SOX2, and KLF4 there is an increased pluripotency of PSC lines (Hl and 72.3) grown in three-dimensional suspension culture in comparison to those lines grown in 2D monolayers.
  • FIG. 16 depicts an embodiment of an experimental protocol for the matrix-free suspension culture production of HIOs from hiPSCs.
  • FIG. 17 depicts the results of an embodiment of a study examining the impact of acclimatization of PSCs to suspension culturing on the production of HIOs at various time points.
  • FIGs. 18A and 18B depict the results of an embodiment of a study comparing the efficiency of DE induction in two-dimensional (2D) culturing (FIG. 18 A) and suspension culturing (3D) (FIG. 18B) by examining expression of definitive endoderm markers Sox 17 and FoxA2.
  • FIG. 19 depicts the results of an embodiment demonstrating the effect of exposure to Activin A at different time points (e.g., at 24 hours, at 48 hours, or at 72 hours post-passaging) to DE induction efficiency in 3D suspension culture.
  • time points e.g., at 24 hours, at 48 hours, or at 72 hours post-passaging
  • 3D PSC aggregates exposed to Activin A at 48 hour post-passaging showed the highest DE differentiation efficiency, based on the expression of DE markers FoxA2 and Soxl7.
  • FIG. 20 depicts the results of an embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the DE stage.
  • the size of the 3D PSC aggregates at the time of the exposure to Activin determines DE induction efficiency.
  • FIG. 20 shows that smaller size of the 3D PSC aggregate (e.g., less than 500 pm (e.g., less than about 400 pm (e.g., less than about 350 pm (e.g., less than about 300 pm))))) ensures better DE induction.
  • 500 pm e.g., less than about 400 pm (e.g., less than about 350 pm (e.g., less than about 300 pm)
  • FIG. 21 depicts the results of an embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the HGS stage.
  • the effects were demonstrated by the expression of CDX2, which is a marker for intestinal tissue differentiation.
  • CDX2 is a marker for intestinal tissue differentiation.
  • FIG. 21 there is a strong expression of CDX2 at the hindgut stage of differentiation of 3D PSC aggregates with diameters smaller than 300 pm at the time of DE induction.
  • CDX2 there is a weaker and more sparse expression of CDX2 for 3D PSC aggregates having diameters greater than 300 m at the time of DE induction.
  • the results further confirm the importance that size of the 3D PSC aggregates has on differentiation efficiency.
  • FIG. 22 depicts the results of an embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the HIO stage. The effects were demonstrated by the expression of CDX2, a marker for intestinal tissue differentiation.
  • CDX2 a marker for intestinal tissue differentiation.
  • differentiation of 3D PSC aggregates having diameters no more than 300 pm resulted in apical- in HIO formation and substantially homogenous expression of CDX2 across generated HIOs.
  • differentiation of 3D PSC aggregates having diameters at least 300 pm resulted in mixed apical-out and apical-out structures and epithelial structures with weak or no CDX2 expression.
  • FIGs. 23-27 depict the results of an embodiment based on a study investigating the effect of dextran sulfate sodium (DSS), an anti-adhesion agent, mediating the size of 3D PSC aggregates.
  • DSS dextran sulfate sodium
  • FIG. 23 shows the effects of varied concentrations of DSS on the size of 3D PSC aggregates, with a concentration of 10 pg/ml having the highest effect of reducing the size of the 3D PSC aggregates.
  • FIG. 24 further shows that the effects of DSS on reducing aggregate size were noted across different PSC lines (72.3, FF3, Hl, and Hl GFP).
  • FIG. 25 further shows that a DSS concentration of 10 pg/ml had the highest reduction in the average diameter across the lines, while also increasing the yield of aggregates being formed.
  • FIGS. 26 and 27 further explore the effect of 10 pg/ml DSS on average size of PSC aggregates across different lines.
  • FIG. 26 shows that 10 pg/ml of DSS is sufficient to induce smaller diameter of PSC aggregates, causing an average of -200 pm decrease in mean diameter of PSC aggregates upon DSS treatment, in comparison to untreated controls.
  • FIG. 27 also shows that this effect is consistent across different iPSC and ESC lines, and leads to the shift in the frequency of PSC aggregate size distribution.
  • FIG. 28 depicts the results of an embodiment based on a study investigating the effect of DSS on the pluripotency of 3D PSC aggregates, as measured by pluripotency markers SOX2 and OCT4.
  • DSS had no negative effect on the pluripotency of the 3D PSC aggregates any of the concentrations tested.
  • the study also investigated the effect of DSS on the viability of 3D PSC aggregates, as measured by the release of a viability marker lactate dehydrogenase (LDH). The study found that DSS at concentrations at or below 1000 pg/ml has no negative effect on the viability of PSC aggregates.
  • LDH lactate dehydrogenase
  • FIG. 29 depicts the results of an embodiment based on a study investigating the effects of various treatment regimes for applying DSS on the average size, numbers, and pluripotency of 3D PSC aggregates.
  • the treatment regimes that were tested included control (i.e., no treatment regime), at inoculation, and throughout.
  • the results show that Treatment with 10 ug/ml of DSS at the time of inoculation is sufficient to maintain PSC aggregates ⁇ 400 um, while having no negative effect on the PSC aggregate numbers or expression of pluripotency genes.
  • prolonged treatment (Throughout) with DSS lead to a decreased OCT4 expression.
  • FIG. 30 depicts the results of an embodiment based on a study investigating the effects of DSS on the average size of 3D PSC aggregates across passages. As shown in FIG. 30, the effect of DSS treatment on PSC aggregate size (a decrease in diameter) was maintained across multiple passages.
  • FIG. 31 depicts the results of an embodiment based on a study investigating the effects of DSS on the propensity of 3D PSC aggregates to differentiate.
  • the propensity is measured based on differentiation efficiency, which is marked by an expression of markers FOXA2 and SOX17.
  • similar differentiation efficiency of 3D PSC aggregates toward differentiation to DE was found, as noted by the expression of Soxl7 and FoxA2 in the presence and absence of DSS, demonstrating that there is no negative effect of DSS treatment on cells propensity to differentiation.
  • FIG. 32 depicts the results of an embodiment of an experiment demonstrating the development of well-patterned HIOs in suspension culture having either an apical-out epithelial cell polarity or an apical-in epithelial polarity.
  • FIG. 33 depicts the results of an embodiment based on the development of well- patterned HIOs in 3D suspension culture.
  • the successful development of correctly patterned HIOs in the suspension culture is confirmed by immunofluorescent staining.
  • the markers CDX2, ZO-1, and Viml signify the differentiation into HIOs.
  • FIGs. 34A and 34B depict the results of an embodiment of an experiment demonstrating the in vivo maturation of HIOs developed in suspension culture following transplantation under the kidney capsule of a mouse.
  • FIG. 34A is an embodiment of a photograph of an HIO 9 weeks post-transplant under the kidney capsule.
  • FIG. 34B is an embodiment of H & E staining of an HIO 9 weeks post-transplant under the kidney capsule.
  • FIG. 35 depicts the results of an embodiment of an experiment demonstrating that the polarity of epithelial cells in HIOs developed in suspension culture can be modified. Dissociation of hindgut spheroids at day 7 (+ dissociation) and their reaggregation in suspension culture leads to the apical surface on the inside of the HIO (apical-in). If the DE is not dissociated at day 7 (- dissociation), the result is the apical surface on the outside of the HIO (apical-out).
  • aspects of the present disclosure relate generally to suspension culture methods of pluripotent stem cells (PSC), differentiated cells, spheroids and organoids derived from PSCs, and compositions of the same.
  • PSC pluripotent stem cells
  • these methods may be performed and/or may facilitate industrial efficiency and scalability
  • these methods may be performed without the use of basement membrane matrices (e.g., xenogenic basement membrane matrices) during PSC maintenance and expansion, as well as during differentiation of PSCs into differentiated cells and organoids, for example definitive endoderm (DE), hindgut spheroids (HGS), and intestinal organoids (IO).
  • the methods may be xeno-free, and may be performed as per Good Manufacturing Practices (GMP).
  • the disclosed methods utilize a suspension culture for each of the following phases in the manufacture of an organoid from an PSC: maintenance and expansion of PSC, differentiation of PSC into DE, differentiation of DE into spheroids (e.g., hindgut spheroids), and differentiation and maturation of spheroids into an organoid (e.g., an intestinal organoid). Also disclosed are uses of the methods and compositions for transplantation and treatment.
  • one or more of the following aspects of current culture protocols are improved: improve the transition from two- dimensional (e.g., monolayer) culture conditions to three-dimensional (e.g., Matrigel-embedded) culture conditions to remove or reduce a potentially significant bottleneck; eliminate the need for the use of extracellular (basement membrane) matrix (e.g.
  • Matrigel for organoid (e.g., IO) production, thereby reducing variability driven by batch-to-batch variations in extracellular matrix compositions and concentrations, and advance the transition toward xeno-free manufacturing of human organoids; decrease the labor by eliminating the manual plating in Matrigel domes found in some current organoid culturing methods; eliminate the manual processing around day 14 of some current culturing methods; enable large scale production of organoids; decrease variability in the resulting cell, spheroid, and/or organoid.
  • organoid e.g., IO
  • organoids One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is typically enclosed within the organoid interior. Many applications require access to the apical or luminal surface of the organoid, because this is the mucosal surface that normally interfaces with the external environment and thus absorbs nutrients, interacts with GI microbes and uptakes drugs or toxins.
  • the apical surface also secretes mucins, antimicrobial peptides and enzymes that regulate interactions between gut luminal contents and the epithelium.
  • Embodiments disclosed herein relate to a method of suspension culture that enables full control of human organoid epithelial polarity and creation of apical-out organoids.
  • PSCs are induced PSCs (iPSCs) or an embryonic stem cells (ESCs).
  • ESCs embryonic stem cells
  • the PSC is human (hPSC).
  • the PSC is a human iPSC (hiPSC).
  • the method comprises: (a) inoculating a liquid culture media with PSCs (optionally hPSC or hiPSC); (b) culturing in a bioreactor the liquid culture media that is inoculated with the PSCs such that three-dimensional PSC aggregates form in the liquid culture media, wherein the culturing in the bioreactor comprises suspending the PSCs in the liquid culture media; and (c) passaging the PSCs by: (i) dissociating at least a portion of the three-dimensional PSC aggregates into single cells; and inoculating a second liquid culture media with the dissociated three-dimensional PSC aggregates of i.) with PSCs.
  • the diameter of the majority of PSC aggregates (e.g., the three-dimensional PSC aggregates) formed is, or is not more than, 500, 450, 400, 350, 300, 250 pm, or a range defined by any two of the preceding values; optionally wherein the diameter of the majority of PSC aggregates is not more than 500 pm, not more than 400 pm, not more than 350 pm, or not more than 300 pm.
  • the suspension comprises rotation of the chamber in which the culture media is contained, causing rotation of the culture media around an axis that is parallel to the ground.
  • At least 80, 85, 90, 95, 98 or 99% of the PSC aggregates are dissociated into single cells at (c)(i); optionally wherein at least 90% of PSC aggregates are dissociated into single cells at (c)(i).
  • the PSCs may be inoculated to form a density that is, is at least, or is not more than, 50,000, 100,000, 180,000, 200,000, 220,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,500,000, or 2,000,000 PSCs/ml of culture media in the bioreactor, or a range defined by any two of the preceding values; in some embodiments, the density is 50,000-1,000,000, 50,000-500,000, 100,000-300,000, 180,000- 220,000 or 200,000 PSCs/ml of culture media; in some embodiments, the density is 180,000- 220,000 or 200,000 PSCs/ml of culture media.
  • the second liquid culture media with the dissociated three- dimensional PSC aggregates of (i) may be inoculated with PSCs form a density that is, is at least, or is not more than, 50,000, 100,000, 180,000, 200,000, 220,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,500,00, or 2,000,000 PSCs/ml of culture media in the bioreactor, or a range defined by any two of the preceding values; in some embodiments, the density is 50,000-1,000,000, 50,000-500,000, 100,000-300,000, 100,000- 200,000, 180,000-220,000 or 200,000 PSCs/ml of culture media; in some embodiments, the density is 180,000-220,000 or 200,000 PSCs/ml of culture media.
  • the method comprises passaging the PSCs two or more times by culturing the PSCs of inoculated culture media of (c)ii.) until three-dimensional PSC aggregates form, and repeating (c).
  • the culture is passaged 2, 3, 4, or more times.
  • the PSCs maintain expression of markers of stem cells, for example Oct4, SSEA4 and/or TRA 1-60 (e.g., Oct4, SSEA4 and TRA 1-60).
  • At least 85, 90, or 95% of the cells maintain their status as PSCs as evidence by expression of markers of stem cells, for example Oct4, SSEA4 and/or TRA 1-60 (e.g., Oct4, SSEA4 and TRA 1-60) following at least 2, 3, 4 or more passages.
  • markers of stem cells for example Oct4, SSEA4 and/or TRA 1-60 (e.g., Oct4, SSEA4 and TRA 1-60) following at least 2, 3, 4 or more passages.
  • the culture media in (a) (the initial inoculation of the suspension culture) and/or (c)(ii) (re-inoculation of the suspension culture during passaging), is inoculated at a density of about 100,000-220,000, about 180,000-220,000, or about 200,000 PSCs/ml of culture media.
  • the passaging occurs after a period of time following the inoculation in (a) and/or (c)(ii) that is, is at least, or is not more than about 40, 48, 54, 60, 66, 72, 78, 84, 90, 96, or 168 hours, or a range defined by any two of the preceding values; optionally wherein the period of time is about 40- 54, 40-84, 60-84, 66-78, or 72 hours.
  • the PSCs are passaged one or more times (e.g., 3 times), and following one or more passages (e.g., following the third passage) the portion of PSCs expressing Oct4, SSEA1 and/or TRA 1-60 (e.g., Oct4, SSEA4 and TRA 1-60) at levels at least as high as the average expression level of the PSCs used in the initial inoculation of the suspension culture in (a) is, or is at least, 85, 90, 93, 95, 97, 98 or 99%, or a range defined by any two of the preceding values.
  • the portion of PSCs expressing Oct4, SSEA1 and/or TRA 1-60 at levels at least as high as the average expression level of the PSCs used in the inoculation in (a) is at least 95%.
  • the PSCs are passaged one or more times. In some embodiments, the PSCs are passaged at least 3 times.
  • the portion of PSCs expressing Oct4, SSEA1 and/or TRA 1-60 at levels at least as high as the average expression level of the PSCs used in the initial inoculation of the suspension culture in (a) is, or is at least, 85, 90, 93, 95, 97, 98 or 99%, or a range defined by any two of the preceding values.
  • the portion of PSCs expressing Oct4, SSEA1 and/or TRA 1-60 at levels at least as high as the average expression level of the PSCs used in the inoculation in (a) is at least 95%.
  • the PSCs are passaged one or more times (e.g., at least 3 times), and the PSCs express SOX2 and/or KLF4 (e.g., SOX2 and KLF4).
  • the method further comprises exchanging a portion of the culture media in the bioreactor after a period of time following the inoculation in (a) and/or (c)(ii).
  • the period of time is, is at least, or is not more than, 36, 42, 48, 54 or 60 hours, or a range defined by any two of the preceding values; optionally wherein the period of time is 42-54, or 48 hours.
  • the portion of the culture media exchanged is, or is at least, 50, 60, 70, 80, 90, of 100% of the culture media in the bioreactor, or a range defined by any two of the preceding values; optionally wherein the portion is at least 80% or 90% of the culture media in the bioreactor.
  • the PSCs used to inoculate the suspension culture are first cultured on the surface of a substrate, e.g., on the surface of a culture flask (an adherent culture), also referred to herein as a two-dimensional or 2D culture. PSCs cultured on the surface of a substrate such as a culture flask typically form a monolayer of cells on the surface of the substrate.
  • the method further comprises prior to the inoculation of the suspension culture with PSCS in (a), culturing the PSCs on the surface of a substrate, and collecting the PSCs from the surface of the substrate for use in the inoculation of the suspension culture of (a) when the PSCs are in a logarithmic growth phase and/or at 35-55% or 40-50% confluency.
  • the collecting comprises dissociating the PSCs prior to the inoculation of the suspension culture in (a).
  • Methods for dissociation of cells in adherent cultures may include but are not limited to chemical, enzymatic and/or mechanical dissociation.
  • the dissociation is chemical, e.g., via use of EDTA.
  • the dissociation is enzymatic.
  • the enzyme used for dissociation comprises a proteolytic and/or a collagenolytic enzyme, for example Accutase.
  • Devices for the suspension culturing of cells may include bioreactors.
  • Use of bioreactors for suspension culturing may result in the generation of variable level of shear stress (e.g. shear stress can vary between 0.1-10 dynes/cm 2 in spinning flask depending on rotation speed, vessel size and shape, and culture medium volume).
  • PSCs e.g., human PSCs, are sensitive to high shear stress that may cause unexpected cell death and differentiation.
  • a bioreactor having reduced and/or low shear stress e.g., relative to traditional suspension culture bioreactors (spinning flask and vertical wheel bioreactors) or a bioreactor that is shear stress-free is used.
  • the bioreactor having reduced and/or low and/or no shear stress functions by rotating continuously and the uplifting movement from the rotation being counteracted by gravity to suspend cells within the media.
  • the bioreactor comprises a rotating chamber comprising the suspension culture media.
  • the chamber is a vessel having the shape of a cylindric section (e.g., shaped like a petri dish, optionally where the depth of the dish is greater than a typical petri dish).
  • the chamber is rotated around its longitudinal axis, which causes the liquid culture media contained in the chamber to rotate, thereby suspending the cells and aggregates in the liquid culture media.
  • the chamber is oriented such that its longitudinal axis is parallel to the ground, such that cells (e.g., individual cells, aggregates, spheroids, organoids, etc.) within the chamber rises on one side of the chamber due to the rotation of the chamber and liquid culture media, and fall on the opposite side due to the force of gravity.
  • FIG. IB depicts an embodiment of an illustration of the operation of a suspension culture where the chamber is rotated around its longitudinal axis (not illustrated, but which is normal to the plane of the illustration, passing through the center of the chamber/vessel) when the longitudinal axis is oriented parallel to the ground.
  • the bioreactor comprises a rotating chamber comprising the culture media, wherein the volume of culture media in the chamber is, is at least, or is not more than, 5, 10, 20, 30, 40, 50 ml, or a range defined by any of the preceding values.
  • the volume of culture media in the chamber is 5-10 ml; optionally 10 ml.
  • the bioreactor comprises a rotating chamber comprising the culture media, wherein the rotation of the chamber is, is at least, or is not more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or a range defined by any two of the preceding values. In some embodiments, the rotation is not more than 40 rpm. In some embodiments, the rotational speed is a speed selected to keep the cells and/or aggregates suspended in statical orbit. In some embodiments, the rotation is 3-7 rpm.
  • the rotation speed of the chamber is selected such that the number of PSCs in the culture at the time of passaging in (c) is least 2 or 2.5-fold the number of PSCs used to inoculate the culture media. In some embodiments, the rotation speed of the chamber is selected such that the number of PSCs in the culture at passaging in (c) is least 2 or 2.5-fold the number of PSCs used to inoculate the culture media for at least two or at least three passages of the PSCs.
  • the bioreactor is configured such that the average shear stress on the cells and aggregates in the culture media while the culture media is in motion (e.g., the chamber is rotating) is less than 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, or 0.005 dynes/cm 2 . In some embodiments, the average shear stress on the cells and aggregates in the culture media is less than 0.1 dynes/cm 2 .
  • Culture media may be used for the expansion and maintenance of PSCs.
  • the culture media is a serum-free media.
  • the culture media comprises recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor 0 (rh TGF0).
  • the culture media is free of materials of animal or human origin.
  • the culture media is free of any extracellular matrix and/or basement membrane matrix (e.g., Matrigel or similar products).
  • the lack of the extracellular matric and/or basement membrane matrix allows for three-dimensional suspension of cultures.
  • any of the aforementioned culture media may be liquid culture media.
  • compositions for expansion and maintenance of pluripotent stem cell (PSC) cultures comprising: a liquid culture media comprising recombinant human basic fibroblast growth factor (rh bFGF), and/or a recombinant human transforming growth factor 0 (rh TGF0); and PSCs suspended in the liquid culture media.
  • the liquid culture media is a serum- free media, wherein the liquid culture media is free of materials of animal or human origin; optionally wherein the culture media is free of any extracellular matrix and/or basement membrane matrix.
  • the composition further comprises an anti- apop to tic agent (e.g., ROCK inhibitor (ROCKi) and/or CEPT).
  • an anti- apop to tic agent e.g., ROCK inhibitor (ROCKi) and/or CEPT.
  • the PSCs express Oct4, SSEA1 and/or TRA 1-60. In at least one embodiment, the PSCs express Oct4, SSEA1 and TRA 1-60. In some embodiments, the PSCs express the PSCs further express Oct4, SSEA1, TRA 1-60, Sox2, and/or TRA-1-81.
  • the composition further comprises an anti-adhesion agent.
  • the anti-adhesion agent may be one or both of DSS or xantham gum.
  • the PSCs are suspended in the liquid culture media at a density that is about 50,000 - 1,000,000 PSCs/ml of culture media. In some embodiments, the PSCs are suspended in the liquid culture media at a density that is about 100,000 - 300,000 PSCs/ml of culture media. In some embodiments, the PSCs are suspended in the culture media at a density that is about 100,000 - 220,000 PSCs/ml of culture media. In some embodiments, the PSCs are suspended in the culture media at a density that is about 180,000 - 220,000 PSCs/ml of culture media.
  • PSCs are induced PSC (iPSC) or an embryonic stem cells (ESC).
  • iPSC induced PSC
  • ESC embryonic stem cells
  • the PSC is human (hPSC).
  • the PSC is a human iPSC (hiPSC).
  • the method comprises: (d) culturing in a biorcactor a liquid culture media inoculated with PSCs, wherein the culturing of the PSC inoculated culture media of (d) comprises suspending the PSCs in the liquid culture media; and (e) culturing in a bioreactor, the PSCs of (d) in liquid definitive endoderm differentiation culture media for a period of time sufficient to differentiate the PSCs into DE, wherein the culturing of the PSC inoculated culture media of (d) comprises suspending the PSCs in the liquid definitive endoderm differentiation culture media.
  • the culturing in (d) is for a period of time that is, is at least, is not more than, 18, 24, 30, 36, 42, 48, or 54 hours, or a range defined by any two of the preceding values; optionally wherein the period of time is 18-54 or 24-48 hours.
  • the liquid culture media inoculated with PSCs cultured in (d) is the PSC inoculated culture media of the passaged PSC culture of (c)(ii) described in the methods above, and elsewhere herein.
  • the differentiation is performed on the PCS suspension culture described above and elsewhere herein, where it is a continuation of any one of the methods described above and elsewhere herein, further comprising differentiating the PSCs of the PSC inoculated culture media of (c)(ii) into DE, optionally wherein the differentiating comprises: (d’) culturing the PSCs of the PSC inoculated culture media of (c)(ii) in a bioreactor, wherein the culturing comprises suspending the PSCs in the liquid culture media, and wherein the culturing is for a period of time that is, is at least, is not more than, 18, 24, 30, 36, 42, 48, or 54 hours, or a range defined by any two of the preceding values; optionally wherein the period of time is 18-54 or 24-48 hours; and (e) culturing in a bioreactor the PSCs of (d’) in definitive endoderm differentiation culture media for a period of time sufficient to differentiate the PSCs into
  • the liquid culture media and/or the liquid definitive endoderm media is cultured with PSCs at a density that is, is at least, or is not more than, 50,000, 100,000, 180,000, 200,000, 220,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,500,00, or 2,000,000 PSCs/ml of culture media, or a range defined by any two of the preceding values; optionally wherein the density is 50,000-1,000,000, 50,000- 500,000, 100,000-300,000, 180,000-220,000 or 200,000 PSCs/ml of culture media; [0230]
  • the period of time sufficient to differentiate the PSCs into DE is a period of time that is, is at least, is not more than, 48, 54, 60, 66, 72, 78, 84, 90 or 96 hours, or a range defined by any two of the preceding values; optionally wherein the period of time is 60-84, 66
  • culturing the PSCs in definitive endoderm differentiation culture media for a period of time sufficient to differentiate the PSCs into DE comprises: culturing the PSCs in culture media comprising a Wnt signaling pathway activator (e.g., CHIR99021) and/or a Nodal signaling pathway activator (e.g., Activin A), and optionally a BMP signaling pathway activator, for a first period; then culturing the PSCs in culture media comprising the Wnt signaling pathway activator and/or the Nodal signaling pathway activator, and optionally a serum (e.g., FBS) or serum replacement (e.g., Knockout Replacement Serum (KRS), HAS, etc.) for a second period, and then culturing the PSCs in culture media comprising the Wnt signaling pathway activator and/or the Nodal signaling pathway activator, and optionally the serum or the serum replacement for a third period.
  • each of the first, second and third periods of time is independently selected from a period of time that is, is at least, or is not more than, 18, 20, 22, 24, 26, 28 or 30 hours, or a range defined by any two of the preceding values; optionally wherein the first, second and third period of time is 20-28, 22-26, or 24 hours.
  • the efficiency of DE induction is, or is at least, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 95%, or a range defined by any two of the preceding values; optionally wherein the efficiency of DE induction is at least 45% or at least 50%; optionally wherein the efficiency of DE induction is 45-55%; optionally wherein the efficiency of DE induction is 80-95%.
  • the DE expresses Soxl7 and FoxA2.
  • the pluripotent stem cells are differentiated into definitive endoderm cells by contacting the pluripotent stem cells with Activin A, a BMP signaling pathway activator, or both.
  • the pluripotent stem cells are contacted with a concentration of Activin A that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng/mL, or any concentration of Activin A within a range defined by any two of the aforementioned concentrations, for example, 10 to 200 ng/mL, 10 to 100 ng/mL, 100 to 200 ng/mL, or 50 to 150 ng/mL.
  • the pluripotent stem cells arc contacted with Activin A at a concentration of 100 ng/mL, or about 100 ng/mL. In some embodiments, the pluripotent stem cells are contacted with a concentration of a BMP signaling pathway activator that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3,
  • the pluripotent stem cells are contacted with a BMP signaling pathway activator at a concentration of 15 ng/mL or about 15 ng/mL.
  • a bioreactor having reduced and/or low shear stress e.g., relative to traditional suspension culture bioreactors (spinning flask and vertical wheel bioreactors) or a bioreactor that is shear stress-free is used.
  • the bioreactor having reduced and/or low and/or no shear stress functions by rotating continuously and the uplifting movement from the rotation being counteracted by gravity to suspend cells within the media.
  • the bioreactor comprises a rotating chamber comprising the suspension culture media.
  • the chamber is a vessel having the shape of a cylindric section (e.g., shaped like a petri dish, optionally where the depth of the dish is greater than a typical petri dish).
  • the chamber is rotated around its longitudinal axis, which causes the liquid culture media contained in the chamber to rotate, thereby suspending the cells and aggregates in the liquid culture media.
  • the chamber is oriented such that its longitudinal axis is parallel to the ground, such that cells (e.g., individual cells, aggregates, spheroids, organoids, etc.) within the chamber rises on one side of the chamber due to the rotation of the chamber and liquid culture media, and fall on the opposite side due to the force of gravity.
  • the bioreactor comprises a rotating chamber comprising the culture media, wherein the volume of culture media in the chamber is, is at lease, or is not more than, 5, 10, 20, 30, 40, 50 ml, or a range defined by any of the preceding values. In some embodiments, the volume of culture media in the chamber is 5-10 ml; optionally 10 ml. In some embodiments, the bioreactor comprises a rotating chamber comprising the culture media, wherein the rotation of the chamber is, is at least, or is not more than, 1, 2, 3, 4,
  • the rotation is not more than 40 rpm. In some embodiments, the rotation is 3-7 rpm. In some embodiments, the rotational speed is a speed selected to keep the cell s and/or aggregates suspended in statical orbit.
  • the bioreactor is configured such that the average shear stress on the cells and aggregates in the culture media while the culture media is in motion (e.g., the chamber is rotating) is less than 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, or 0.005 dynes/cm2. In some embodiments, the average shear stress on the cells and aggregates in the culture media is less than 0.1 dynes/cm2.
  • the culture media is free of materials of animal or human origin. In some embodiments, the culture media is free of any extracellular matrix and/or basement membrane matrix (e.g. Matrigel or similar’ products).
  • extracellular matrix and/or basement membrane matrix e.g. Matrigel or similar’ products.
  • compositions for differentiating PSCs into definitive endoderm (DE) in a three dimensional suspension culture comprising: a liquid DE differentiation culture media; and PSCs suspended in the liquid DE differentiation culture media.
  • the liquid DE differentiation culture media is a serum-free media, wherein the liquid DE differentiation culture media is free of materials of animal or human origin; optionally wherein the liquid DE differentiation culture media is free of any extracellular matrix and/or basement membrane matrix.
  • the PSCs have an average diameter of less than about 400um. In some embodiments, the PSCs have an average diameter of less than about 300um.
  • the liquid DE differentiation culture media comprises Activin A at a concentration of about 10 to 200 ng/mL of liquid DE differentiation culture media. In some embodiment, the liquid DE differentiation culture media comprises Activin A at a concentration of about 50 to 150 ng/mL ng/mL of liquid DE differentiation culture media. In some embodiments, the liquid DE differentiation culture media comprises Activin A at a concentration of about 100 to 200 ng/mL ng/mL of liquid DE differentiation culture media. In another embodiment, the liquid DE differentiation culture media further comprises FBS at a concentration of about 0% to 20%. In some embodiments, the liquid DE differentiation culture media further comprises FBS at a concentration of about 0.2% to 10%. In some aspects, the liquid DE differentiation culture media further comprises FBS at a concentration of about 2% to 5%.
  • the composition further comprises DE differentiated from the PSCs.
  • the DE differentiated from the PSCs may express Soxl7 and/or FoxA2.
  • the DE differentiated from the PSCs expresses Sox 17 and FoxA2.
  • aspects of the disclosure include methods of differentiating DE into hindgut spheroids (HGS) in a three dimensional suspension culture.
  • the DE are derived from PSCs.
  • the PSCs are induced PSC (iPSC) or an embryonic stem cells (ESC).
  • the PSC is human (hPSC).
  • the PSC is a human iPSC (hiPSC).
  • the method comprises: (f) culturing in a bioreactor DE in liquid hindgut differentiation culture media for a period of time sufficient to differentiate the DE into HGS, wherein the culturing of the DE comprises suspending the DE in the liquid hindgut differentiation culture media.
  • the DE cultured in (f) is the DE made by any one of the methods disclosed above and elsewhere herein.
  • the differentiation is performed on the DE culture described above and elsewhere herein, where it is a continuation of any one of the methods described above and elsewhere herein, further comprising differentiating the DE into HGS, optionally wherein the differentiating comprises: (f) culturing in a bioreactor the DE in liquid hindgut differentiation culture media for a period of time sufficient to differentiate the DE into HGS, wherein the culturing of the DE comprises suspending the DE in the liquid hindgut differentiation culture media.
  • the period of time sufficient to differentiate the DE into HGS is a period of time that is, is at least, is not more than, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120 hours, or a range defined by any two of the preceding values; optionally wherein the period of time is 84-108, 90-102, or 96 hours.
  • the hindgut differentiation culture media is changed after a period of time that is, is at least, or is not more than, 18, 20, 22, 24, 26, 28 or 30 hours, or a range defined by any two of the preceding values; optionally wherein the first, second and third period of time is 20-28, 22-26, or 24 hours.
  • the hindgut differentiation culture media comprises a Wnt signaling pathway activator, an FGF signaling pathway activator, and optionally FBS.
  • the hindgut differentiation culture media comprises a Wnt signaling pathway activator, wherein the Wnt signaling pathway activator comprises CHIR99021, an FGF signaling pathway activator, wherein the FGF signaling pathway activator comprises FGF4, and optionally FBS.
  • the FGF signaling pathway activator is FGF4, optionally wherein the concentration is, is about, is at least, or is at least about 50 ng/ml, 100 ng/ml, 150 ng/ml, 200 ng/ml, 250 ng/ml, 300 ng/ml, 350 ng/ml, 400 ng/ml, 450 ng/ml, 500 ng/ml, 550 ng/ml, 600 ng/ml, 650 ng/ml, 700 ng/ml, or 750 ng/ml, or a range defined by any two of the preceding values, optionally 50-750 ng/ml, 50-100 ng/ml, or 50-500 ng/ml, or optionally at a concentration of 500 ng/ml.
  • the Wnt pathway activator is CHIRON 99021, optionally wherein the concentration is, is about, is at least, or is at least about 0.5 pM, 1 pM, 1.5 pM, 2 pM, 2.5 pM , 3 pM, 3.5 pM, 4 pM, 4.5 pM, 5 pM, 5.5 pM, or 6 pM, or a range defined by any two of the preceding values, optionally 0.5 - 6 pM, 0.5-3 pM, 3-6 pM, 2-4 pM, or optionally at a concentration of 3 pM.
  • a bioreactor having reduced and/or low shear stress e.g., relative to traditional suspension culture bioreactors (spinning flask and vertical wheel bioreactors) or a bioreactor that is shear stress-free is used.
  • the bioreactor having reduced and/or low and/or no shear stress functions by rotating continuously and the uplifting movement from the rotation being counteracted by gravity to suspend cells within the media.
  • the bioreactor comprises a rotating chamber comprising the suspension culture media.
  • the chamber is a vessel having the shape of a cylindric section (e.g., shaped like a petri dish, optionally where the depth of the dish is greater than a typical petri dish).
  • the chamber is rotated around its longitudinal axis, which causes the liquid culture media contained in the chamber to rotate, thereby suspending the cells and aggregates in the liquid culture media.
  • the chamber is oriented such that its longitudinal axis is parallel to the ground, such that cells (e.g., individual cells, aggregates, spheroids, organoids, etc.) within the chamber rises on one side of the chamber due to the rotation of the chamber and liquid culture media, and fall on the opposite side due to the force of gravity.
  • the bioreactor comprises a rotating chamber comprising the culture media, wherein the volume of culture media in the chamber is, is at lease, or is not more than, 5, 10, 20, 30, 40, 50 ml, or a range defined by any of the preceding values. In some embodiments, the volume of culture media in the chamber is 5-10 ml; optionally 10 ml. In some embodiments, the bioreactor comprises a rotating chamber comprising the culture media, wherein the rotation of the chamber is, is at least, or is not more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or a range defined by any two of the preceding values. In some embodiments, the rotation is not more than 40 rpm.
  • the rotation is 3-7 rpm.
  • the bioreactor is configured such that the average shear stress on the cells, aggregates, and/or spheroids in the culture media while the culture media is in motion (e.g., the chamber is rotating) is less than 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, or 0.005 dynes/cm 2 . In some embodiments, the average shear stress on the cells, aggregates, and/or spheroids in the culture media is less than 0.1 dynes/cm 2 .
  • the culture media is free of materials of animal or human origin. In some embodiments, the culture media is free of any extracellular matrix and/or basement membrane matrix (e.g. Matrigel or similar products).
  • extracellular matrix and/or basement membrane matrix e.g. Matrigel or similar products.
  • compositions for differentiating DE into hindgut spheroids (HGS) in a three dimensional suspension culture comprises: a liquid hindgut differentiation culture media comprising a Wnt signaling pathway activator, an FGF signaling pathway activator, and optionally FBS; and DE suspended in the liquid hindgut differentiation culture media.
  • the liquid hindgut differentiation culture media is free of materials of animal or human origin; optionally wherein the culture media is free of any extracellular matrix and/or basement membrane matrix.
  • the Wnt signaling pathway activator comprises CHIR99021, and wherein the FGF signaling pathway activator comprises FGF4.
  • the FGF signaling pathway activator is at a concentration that is at least about 50 ng/ml of the liquid hindgut differentiation culture media. In some embodiments, the FGF signaling pathway activator is at a concentration that is at least about 500 ng/ml of the liquid hindgut differentiation culture media. In some embodiments, the Wnt pathway activator is at a concentration that is at least about 0.5 M of the liquid hindgut differentiation culture media.
  • aspects of the disclosure include methods of differentiating HGS into intestinal organoids (IO) in a three dimensional suspension culture.
  • the HGS are derived from PSCs.
  • the PSCs are induced PSC (iPSC) or an embryonic stem cells (ESC).
  • the PSC is human (hPSC).
  • the PSC is a human iPSC (hiPSC).
  • the method comprises: (g) culturing in a bioreactor HGS in liquid IO maturation culture media for a period of time sufficient to differentiate the HGS into IO, wherein the culturing of the HGS comprises suspending the HGS in the liquid IO maturation culture media.
  • the HGS cultured in (g) is the HGS of any one of the methods described above and elsewhere herein for producing HGS.
  • the differentiation is performed on the HGS culture described above and elsewhere herein, where it is a continuation of any one of the methods described above and elsewhere herein, further comprising differentiating the HGS into IO, optionally wherein the differentiating comprises: (g) culturing in a bioreactor HGS in liquid IO maturation culture media for a period of time sufficient to differentiate the HGS into IO, wherein the culturing of the HGS comprises suspending the HGS in the liquid IO maturation culture media.
  • the period of time sufficient to differentiate the HGS into IO is a period of time that is, is at least, is not more than, about 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 days, or a range defined by any two of the preceding values; optionally wherein the period of time is 12-30, 12-20, 15-28, 15-20, or 20 days; optionally wherein the period of time is at least 12, 15, or 20 days.
  • the IO maturation culture media is changed after a period of time that is, is at least, or is not more than, about 24, 28, 32, 36, 38, 42, 44, 46, 48, 50, 52, or 54 hours, or a range defined by any two of the preceding values.
  • the first, second and third period of time is about 24-54, 42-54, 46-50, or 48 hours.
  • the IO maturation culture media comprises EGF, R- spondin, Noggin, Gremlin 1, and/or Epiregulin (EREG).
  • the concentration of EGF, R-spondin, Noggin, Gremlin 1, and/or Epiregulin (EREG) is, is about, is at least, or is at least about 25 ng/ml, 50 ng/ml, 75 ng/ml, 100 ng/ml, 125 ng/ml, 150 ng/ml, 175 ng/ml, or 200 ng/ml, or a range defined by any two of the preceding values, optionally 25-100 ng/ml, 50-150 ng/ml, 100 ng/ml, or optionally is at a concentration of 100 ng/ml.
  • the HGS are not dissociated prior to culturing in the IO maturation culture media, wherein epithelial cells of the IO formed have a polarity wherein the apical surface is oriented to the outside of the IO. In other words, the apical surface faces outward to the medium.
  • a bioreactor having reduced and/or low shear stress e.g., relative to traditional suspension culture biorcactors (spinning flask and vertical wheel bioreactors) or a bioreactor that is shear stress-free is used.
  • the bioreactor having reduced and/or low and/or no shear stress functions by rotating continuously and the uplifting movement from the rotation being counteracted by gravity to suspend cells within the media.
  • the bioreactor comprises a rotating chamber comprising the suspension culture media.
  • the chamber is a vessel having the shape of a cylindric section (e.g., shaped like a petri dish, optionally where the depth of the dish is greater than a typical petri dish).
  • the chamber is rotated around its longitudinal axis, which causes the liquid culture media contained in the chamber to rotate, thereby suspending the cells and aggregates in the liquid culture media.
  • the chamber is oriented such that its longitudinal axis is parallel to the ground, such that cells (e.g., individual cells, aggregates, spheroids, organoids, etc.) within the chamber rises on one side of the chamber due to the rotation of the chamber and liquid culture media, and fall on the opposite side due to the force of gravity.
  • the bioreactor comprises a rotating chamber comprising the culture media, wherein the volume of culture media in the chamber is, is at lease, or is not more than, 5, 10, 20, 30, 40, 50 ml, or a range defined by any of the preceding values.
  • the volume of culture media in the chamber is 5-10 ml; optionally 10 ml.
  • the bioreactor comprises a rotating chamber comprising the culture media, wherein the rotation of the chamber is, is at least, or is not more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or a range defined by any two of the preceding values. In some embodiments, the rotation is not more than 40 rpm. In some embodiments, the rotation is 3-7 rpm. In some embodiments, the rotational speed is a speed selected to keep the cells and/or aggregates suspended in statical orbit.
  • the bioreactor is configured such that the average shear stress on the cells, aggregates, spheroids and/or organoids in the culture media while the culture media is in motion (e.g., the chamber is rotating) is less than 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, or 0.005 dynes/cm2. In some embodiments, the average shear stress on the cells, aggregates, spheroids and/or organoids in the culture media is less than 0.1 dynes/cm2. [0251 ] In some embodiments, the culture media is free of materials of animal or human origin. In some embodiments, the culture media is free of any extracellular matrix and/or basement membrane matrix (e.g. Matrigel or similar products).
  • the culture media is free of materials of animal or human origin. In some embodiments, the culture media is free of any extracellular matrix and/or basement membrane matrix (e.g. Matrigel or similar products).
  • the epithelial cells of the IO have a polarity wherein the apical surface is oriented to the outside of the IO (aka, apical-out).
  • compositions for differentiating HGS into intestinal organoids (IO) in a three dimensional suspension culture comprise: a liquid IO maturation culture media comprising EGF; and HGS suspended in the liquid IO maturation culture media.
  • the liquid IO maturation culture media is free of materials of animal or human origin; optionally wherein the liquid IO maturation culture media is free of any extracellular matrix and/or basement membrane matrix.
  • the lumen of the HGS suspended in the liquid IO maturation culture media is exterior facing relative to the liquid IO maturation culture media.
  • the concentration of EGF is at least about 25 ng/ml. In some aspects, the concentration of EGF is at least about 100 ng/ml.
  • At least a portion of the HGS suspended in the liquid IO maturation culture media comprises dissociated HGS single cells.
  • at least 80% of the HGS are dissociated HGS single cells; optionally at least 90% of HGS are dissociated single cells.
  • a concentration of the dissociated HGS single cells in the liquid IO maturation culture media is in a range that about O.lxlO 5 - 80xl0 5 .
  • a concentration of the dissociated HGS single cells in the liquid IO maturation culture media is in a range that is about 20xl0 5 - 60xl0 5 dissociated HGS single cells/ml of liquid IO maturation culture media.
  • the composition further comprises: IO differentiated from the HGS.
  • epithelial cells of the IO formed from the dissociated HGS single cells have a polarity such that an apical surface is oriented to the inside of the IO.
  • the HGS express CdX2.
  • the HGS expresses FOX-F1 but does not express SOX2.
  • the liquid IO maturation culture media further comprises noggin.
  • aspects of the disclosure include methods of modifying the polarity of epithelial cells in IOs derived from HGSs in a three dimensional suspension culture.
  • the HGS are derived from PSCs.
  • the PSCs are induced PSC (iPSC) or an embryonic stem cells (ESC).
  • the PSC is human (hPSC).
  • the PSC is a human iPSC (hiPSC).
  • the method comprises: (g) culturing in a bioreactor HGS in liquid IO maturation culture media for a period of time sufficient to differentiate the HGS into IO, wherein the culturing of the HGS comprises suspending the HGS in the liquid IO maturation culture media, wherein the method further comprises dissociating at least a portion of the HGS into single cells prior to incubation in the IO maturation culture media, wherein the culturing of the HGS comprises suspending the dissociated HGS single cells and any non-dissociated HGS in the liquid IO maturation culture media, wherein epithelial cells of the IO formed from the dissociated HGS single cells have a polarity wherein the apical surface is oriented to the inside of the IO (aka, apical-in).
  • the dissociated HGS cultured in (g) is the HGS of any one of the methods described above and elsewhere herein for producing HGS.
  • the differentiation is performed on the HGS culture described above and elsewhere herein, where it is a continuation of any one of the methods described above and elsewhere herein, wherein the method further comprises dissociating at least a portion of the HGS into single cells prior to incubation in the IO maturation culture media, wherein the culturing of the HGS comprises suspending the dissociated HGS single cells and any nondissociated HGS in the liquid IO maturation culture media, wherein epithelial cells of the IO formed from the dissociated HGS single cells have a polarity wherein the apical surface is oriented to the inside of the IO.
  • a concentration of dissociated HGS single cells are in the IO maturation culture media, wherein the concentration is, is at least, is not more than, 0.05x 10 5 , 0.1 x 10 5 , 0.5x 10 5 , 1 x 10 5 , 2x 10 5 , 4x 10 5 , 6x 10 5 , 8x 10 5 , 16x 10 5 , 1 Ox 10 5 ,20x 10 5 , 40x 10 5 , or 80xl0 5 dissociated HGS single cclls/ml of IO maturation culture media, or a range defined by any two of the preceding values; optionally wherein the concentration is 0.1xl0 5 -80xl0 5 , IxlO 5 - 16xl0 5 , or 2X10 5 -6X10
  • the dissociation is chemical, enzymatic and/or mechanical dissociation.
  • the dissociation is chemical, e.g., EDTA.
  • the dissociation is enzymatic, optionally wherein the enzyme comprises a proteolytic and/or a collagenolytic enzyme, optionally wherein the enzyme is Accutase.
  • a bioreactor having reduced and/or low shear stress e.g., relative to traditional suspension culture bioreactors (spinning flask and vertical wheel bioreactors) or a bioreactor that is shear stress-free is used.
  • the bioreactor having reduced and/or low and/or no shear stress functions by rotating continuously and the uplifting movement from the rotation being counteracted by gravity to suspend cells within the media.
  • the bioreactor comprises a rotating chamber comprising the suspension culture media.
  • the chamber is a vessel having the shape of a cylindric section (e.g., shaped like a petri dish, optionally where the depth of the dish is greater than a typical petri dish).
  • the chamber is rotated around its longitudinal axis, which causes the liquid culture media contained in the chamber to rotate, thereby suspending the cells and aggregates in the liquid culture media.
  • the chamber is oriented such that its longitudinal axis is parallel to the ground, such that cells (e.g., individual cells, aggregates, spheroids, organoids, etc.) within the chamber rises on one side of the chamber due to the rotation of the chamber and liquid culture media, and fall on the opposite side due to the force of gravity.
  • the bioreactor comprises a rotating chamber comprising the culture media, wherein the volume of culture media in the chamber is, is at lease, or is not more than, 5, 10, 20, 30, 40, 50 ml, or a range defined by any of the preceding values. In some embodiments, the volume of culture media in the chamber is 5-10 ml; optionally 10 ml. In some embodiments, the bioreactor comprises a rotating chamber comprising the culture media, wherein the rotation of the chamber is, is at least, or is not more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or a range defined by any two of the preceding values. Tn some embodiments, the rotation is not more than 40 rpm.
  • the rotation is 3-7 rpm.
  • the rotational speed is a speed selected to keep the cells, aggregates, spheroids and/or organoids suspended in statical orbit.
  • the bioreactor is configured such that the average shear stress on the cells, aggregates, spheroids and/or organoids in the culture media while the culture media is in motion (e.g., the chamber is rotating) is less than 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, or 0.005 dynes/cm2.
  • the average shear stress on the cells, aggregates, spheroids and/or organoids in the culture media is less than 0.1 dynes/cm2.
  • the culture media is free of materials of animal or human origin. In some embodiments, the culture media is free of any extracellular matrix and/or basement membrane matrix (e.g. Matrigel or similar products).
  • extracellular matrix and/or basement membrane matrix e.g. Matrigel or similar products.
  • aspects of the disclosure relate to IO produced by the suspension culture methods described above and elsewhere herein.
  • the epithelial cells of the IO have a polarity wherein the apical surface is oriented to the inside of the IO (aka, apical-in).
  • aspects of the disclosure relate to IO, wherein the epithelial cells of the IO have a polarity wherein the apical surface is oriented to the inside of the IO (aka, apical-in).
  • aspects of the disclosure relate to methods of in vivo maturation of IOs by implantation of the IO under the kidney capsule of a subject (e.g., an animal).
  • the IO is derived from PSCs.
  • the PSCs are induced PSC (iPSC) or an embryonic stem cells (ESC).
  • the PSC is human (hPSC).
  • the PSC is a human iPSC (hiPSC).
  • the method comprises transplanting an IO under the kidney capsule of a non-human animal, optionally for a period of time that is, is at least, or is not more than, 6, 8, 10, 12, 14, 16, 18, or 20 weeks, or a range defined by any two of the preceding values, optionally, 6-20, 6-14, or 8-12 weeks.
  • the IO is the IO of any one of the methods described above and elsewhere herein for producing IO.
  • the IO is matured in vitro for a period of time prior to transplantation, optionally wherein the period of time is, is at least, or is not more than, 7, 10, 14, 16, 21, 25, or 28 days, or a range defined by any two of the preceding values, optionally 7- 28, 14-28, or 21-28 days.
  • aspects of the disclosure relate to methods of treatment of an intestine of a subject (e.g., due to intestinal lesions, damages, tissue loss, etc.) by implantation of the IO into the intestinal lumen of the subject (e.g., patient, human, animal, etc.).
  • the IO is derived from PSCs.
  • the PSCs are induced PSC (iPSC) or an embryonic stem cells (ESC).
  • the PSC is human (hPSC).
  • the PSC is a human iPSC (hiPSC).
  • the method comprises transplanting an IO into the intestinal lumen, optionally for a period of time that is, is at least, or is not more than, 6, 8, 10, 12, 14, 16, 18, or 20 weeks, or a range defined by any two of the preceding values, optionally, 6-20, 6-14, or 8-12 weeks.
  • the IO is the IO of any one of the methods described above and elsewhere herein for producing IO.
  • the IO is matured in vitro for a period of time prior to transplantation, optionally wherein the period of time is, is at least, or is not more than, 7, 10, 14, 16, 21, 25, or 28 days, or a range defined by any two of the preceding values, optionally 7-28, 14-28, or 21-28 days.
  • An aspect of the present disclosure is a method of differentiating the DE into a spheroid, optionally wherein the differentiating comprises: (f) culturing in a bioreactor the DE of any of the methods disclosed above and elsewhere herein in liquid differentiation culture media for a period of time sufficient to differentiate the DE into a spheroid, wherein the culturing of the DE comprises suspending the DE in the liquid differentiation culture media; optionally wherein the spheroid is a foregut or a hindgut spheroid.
  • Differentiation media for differentiating DE into spheroids are known in the art, and can be used in the methods disclosed herein.
  • An aspect of the present disclosure is a method of differentiating spheroids into organoids.
  • the method comprises culturing in a bioreactor the spheroids of any of the methods disclosed above and elsewhere herein in liquid organoid maturation culture media for a period of time sufficient to differentiate the spheroids into an organoid, wherein the culturing of the spheroid comprises suspending the spheroid in the liquid organoid maturation culture media; optionally wherein the organoid is selected from the group consisting of a liver, pancreatic, gastric, antral gastric, fundal gastric, intestinal, lung, or colonic organoid.
  • Differentiation media for differentiating spheroids e.g., foregut or hindgut
  • Differentiation media for differentiating spheroids are known in the art, and can be used in the methods disclosed herein.
  • a bioreactor having reduced and/or low shear stress e.g., relative to traditional suspension culture bioreactors (spinning flask and vertical wheel bioreactors) or a bioreactor that is shear stress-free is used.
  • the bioreactor having reduced and/or low and/or no shear stress functions by rotating continuously and the uplifting movement from the rotation being counteracted by gravity to suspend cells within the media.
  • the bioreactor comprises a rotating chamber comprising the suspension culture media.
  • the chamber is a vessel having the shape of a cylindric section (e.g., shaped like a petri dish, optionally where the depth of the dish is greater than a typical petri dish).
  • the chamber is rotated around its longitudinal axis, which causes the liquid culture media contained in the chamber to rotate, thereby suspending the cells and aggregates in the liquid culture media.
  • the chamber is oriented such that its longitudinal axis is parallel to the ground, such that cells (e.g., individual cells, aggregates, spheroids, organoids, etc.) within the chamber rises on one side of the chamber due to the rotation of the chamber and liquid culture media, and fall on the opposite side due to the force of gravity.
  • the bioreactor comprises a rotating chamber comprising the culture media, wherein the volume of culture media in the chamber is, is at lease, or is not more than, 5, 10, 20, 30, 40, 50 ml, or a range defined by any of the preceding values.
  • the volume of culture media in the chamber is about 5-50 ml. In some embodiments, the volume of culture media in the chamber is about 5-10 ml; optionally about 10 ml.
  • the bioreactor comprises a rotating chamber comprising the culture media, wherein the rotation of the chamber is, is at least, or is not more than, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or a range defined by any two of the preceding values. In some embodiments, the rotation is not more than about 40 rpm. In some embodiments, the rotation is 3-7 rpm. In some embodiments, the rotational speed is a speed selected to keep the cells, aggregates, spheroids and/or organoids suspended in statical orbit.
  • the bioreactor is configured such that the average shear stress on the cells, aggregates, spheroids and/or organoids in the culture media while the culture media is in motion (e.g., the chamber is rotating) is less than 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, or 0.005 dynes/cm2. In some embodiments, the average shear stress on the cells, aggregates, spheroids and/or organoids in the culture media is less than 0.1 dyncs/cm2.
  • the culture media is free of materials of animal or human origin. In some embodiments, the culture media is free of any extracellular matrix and/or basement membrane matrix (e.g. Matrigel or similar products).
  • extracellular matrix and/or basement membrane matrix e.g. Matrigel or similar products.
  • aspects of the disclosure relate to spheroids produced by the suspension culture methods described above and elsewhere herein. Aspects of the disclosure relate to organoids produced by the suspension culture methods described above and elsewhere herein.
  • the PSC is an induced PSC (iPSC) or an embryonic stem cells (ESC).
  • the PSC is a human PSC, optionally a human iPSC (hiPSC).
  • Pluripotent stem cells can be derived from any suitable source.
  • the source of pluripotent stem cells is a mammalian source, optionally human, non-human primate, rodent, porcine, and bovine.
  • the PSCs, definitive endoderm cells, spheroids, or organoids are genetically modified or edited according to methods known in the art.
  • gene editing using CRISPR nucleases such as Cas9 are explored in PCT Publications WO 2013/176772, WO 2014/093595, WO 2014/093622, WO 2014/093655, WO 2014/093712, WO 2014/093661, WO 2014/204728, WO 2014/204729, WO 2015/071474, WO 2016/115326, WO 2016/141224, WO 2017/023803, and WO 2017/070633, each of which is hereby expressly incorporated by reference in its entirety.
  • An aspect of the disclosure is PSCs or PSC aggregates made by any of the methods disclosed above and elsewhere herein.
  • An aspect of the disclosure is DE made by any of the methods disclosed above and elsewhere herein.
  • An aspect of the disclosure is HGS made by any of the methods disclosed above and elsewhere herein.
  • An aspect of the disclosure is IO made by any of the methods disclosed above and elsewhere herein.
  • An aspect of the disclosure is an 10 having an apical-in polarity, wherein epithelial cells of the TO have a polarity wherein the apical surface is oriented to the inside of the TO, optionally wherein the TO is a human TO (hlO).
  • the TO having an apical-in polarity is made by any of the methods disclosed above and elsewhere herein.
  • An aspect of the disclosure is a method of treatment comprising transplanting an IO made by any of the methods disclosed above and elsewhere herein, or cells derived therefrom, into an animal, optionally wherein the animal is suffering from a GI disease state.
  • the animal is human.
  • An aspect of the disclosure is the use of an IO made by any of the methods disclosed above and elsewhere herein, or cells derived therefrom, in the manufacture of a medicament for treatment of an animal comprising transplanting the IO, or cells derived therefrom, into the animal, optionally wherein the animal is suffering from a GI disease state.
  • the animal is human.
  • An aspect of the disclosure is a method of screening a compound for activity comprising contacting an IO made by any of the methods disclosed above and elsewhere herein, or cells derived therefrom, with the compound and measuring a response of the IO to the compound.
  • the IO is a model for an intestinal disease, and assessing the effects of the candidate compound or composition on the IO comprises assessing the effects of the candidate compound or composition on the disease.
  • the IO has been produced from cells derived from a subject.
  • the cells derived from the subject are induced pluripotent stem cells.
  • the subject has an intestinal disease.
  • the disclosure herein uses affirmative language to describe the numerous embodiments.
  • the disclosure also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.
  • the terms “individual”, “subject”, or “patient” as used herein have their plain and ordinary meaning as understood in light of the specification, and mean a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.
  • the term “mammal” is used in its usual biological sense.
  • primates including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.
  • an effective amount or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to that amount of a recited composition or compound that results in an observable effect.
  • Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that is effective to achieve the desired response for a particular subject and/or application.
  • the selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated.
  • a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.
  • inhibitor has its plain and ordinary meaning as understood in light of the specification, and may refer to the reduction or prevention of a biological activity.
  • the reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values.
  • delay has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of a biological event, to a time which is later than would otherwise be expected.
  • the delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values.
  • the terms inhibit and delay may not necessarily indicate a 100% inhibition or delay.
  • a partial inhibition or delay may be realized.
  • isolated has its plain and ordinary meaning as understood in light of the specification, and refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man.
  • Isolated substances and/or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and/or spanning the aforementioned values).
  • isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and/or spanning the aforementioned values).
  • a substance that is “isolated” may be “pure” (e.g., substantially free of other components).
  • isolated cell may refer to a cell not contained in a multi-cellular organism or tissue.
  • in vivo is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.
  • ex vivo is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside a living organism with little alteration of natural conditions.
  • in vitro is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.
  • nucleic acid or “nucleic acid molecule” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • oligonucleotides those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, or phosphoramidate.
  • nucleic acid molecule also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. “Oligonucleotide” can be used interchangeable with nucleic acid and can refer to either double stranded or single stranded DNA or RNA. A nucleic acid or nucleic acids can be contained in a nucleic acid vector or nucleic acid construct (e.g.
  • plasmid plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC)) that can be used for amplification and/or expression of the nucleic acid or nucleic acids in various biological systems.
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • HAC human artificial chromosome
  • the vector or construct will also contain elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.
  • elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.
  • a nucleic acid or nucleic acid molecule can comprise one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences can be joined in the same nucleic acid or nucleic acid molecule adjacently, or with extra nucleic acids in between, e.g.
  • downstream on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the 3 ’-end of a previous sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded.
  • upstream on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the 5 ’-end of a subsequent sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded.
  • nucleic acid has its plain and ordinary meaning as understood in light of the specification and refers to two or more sequences that occur in proximity either directly or with extra nucleic acids in between, e.g. linkers, repeats, or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths, but generally not with a sequence in between that encodes for a functioning or catalytic polypeptide, protein, or protein domain.
  • nucleic acids described herein comprise nucleobases.
  • Primary, canonical, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil.
  • Other nucleobases include but are not limited to purines, pyrimidines, modified nucleobases, 5- methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5 -bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.
  • peptide “polypeptide”, and “protein” as used herein have their plain and ordinary meaning as understood in light of the specification and refer to macromolecules comprised of amino acids linked by peptide bonds.
  • the numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available.
  • nucleic acid template By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g.
  • the term “downstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the C-terminus of a previous sequence.
  • upstream on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the N-terminus of a subsequent sequence.
  • purity of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual abundance of the substance, compound, or material relative to the expected abundance.
  • the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between.
  • Purity can be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof.
  • the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents.
  • Purity can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.
  • ELISA enzyme-linked immunosorbent assay
  • Yield of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual overall amount of the substance, compound, or material relative to the expected overall amount.
  • the yield of the substance, compound, or material is, is about, is at least, is at least about, is not more than, or is not more than about, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including all decimals in between.
  • Yield can be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.
  • intestinal organoid has its plain and ordinary meaning as understood in light of the specification and refers to three-dimensional cellular structures that present many properties of the small intestine of an organism.
  • intestinal organoids relate to those derived from human cells and exhibit the properties of a human small intestine.
  • intestinal organoids from other mammals are also encompassed.
  • Intestinal organoids as used herein are derived from pluripotent stem cells (e.g. embryonic stem cells or induced pluripotent stem cells) or an intermediate thereof (e.g.
  • intestinal organoids which are cellular structures derived from adult intestinal epithelium, and other so-called organoids produced from non-pluripotent adult intestinal stem cells, is that the intestinal organoids used herein contain both epithelium and mesenchyme. The mesenchyme performs an important supportive role for the epithelium, and greatly enhances the viability and robust function of the intestinal organoid.
  • the intestinal organoids used herein may exhibit a lumen with epithelial villus-like involutions closely resembling normal intestine, and peristaltic behavior.
  • the intestinal organoids used herein also contain specialized intestinal cell types, including enterocytes, Goblet cells, Paneth cells, and enteroendocrine cells.
  • References disclosing embodiments of intestinal organoids suitable for use herein include WO 2011/140441, WO 2016/061464, WO 2018/200481 , WO 2020/160371 , and WO 2021/030373, each of which are incorporated herein by reference in their entirety.
  • colonic organoid has its plain and ordinary meaning as understood in light of the specification and refers to three-dimensional cellular structures that present many properties of the colon of an organism.
  • colonic organoids relate to those derived from human cells and exhibit the properties of a human colon.
  • colonic organoids from other mammals are also encompassed.
  • Colonic organoids as used herein are derived from pluripotent stem cells (e.g. embryonic stem cells or induced pluripotent stem cells) or an intermediate thereof (e.g.
  • colonic organoids which are cellular structures derived from adult colon epithelium, and other so-called organoids produced from non- pluripotent adult colon stem cells, is that the colonic organoids used herein contain both epithelium and mesenchyme.
  • the mesenchyme performs an important supportive role for the epithelium, and greatly enhances the viability and robust function of the colonic organoid.
  • the colonic organoids used herein may exhibit a lumen with crypts but substantially free of villuslike structures.
  • the colonic organoids used herein also contain specialized colonic cell types, including a high number of Goblet cells (relative to intestinal organoids) and colonic enteroendocrine cells, but substantially free of Paneth cells.
  • References disclosing embodiments of colonic organoids suitable for use herein include WO 2018/106628, which is incorporated herein by reference in their entirety.
  • fragmentation fragmentation
  • fragmentation fragmentation
  • dissociation fragmentation
  • dissociated fragmentation of an organoid or other three- dimensional multicellular structure to produce a population of single cells and viable multicellular structures, fragments, or clumps, without excessively shearing or damaging the cells such that that all or the majority of dissociated organoid comprises intact and healthy cells.
  • fragmented does not generally refer to, e.g., non-living subcellular components or fragments of single cells, such as liberated intracellular contents or non-living vesicles, although these components may be present in embodiments of fragmented organoid compositions by way of natural apoptosis of cells or unintended damage during dissociation of organoids. Fragmentation or dissociation of the organoid may be done in a variety of methods generally known in the art. The process of fragmentation or dissociation may be such that some of the resultant cells are found as small multi-cellular clumps/fragments rather than as single cells.
  • the population of dissociated cells comprising multi-cellular clumps/fragments among single cells is contemplated for use herein.
  • the dissociated cell populations or compositions are present exclusively as multi-cellular clumps/fragments.
  • the dissociated cell populations or compositions are present exclusively as single cells without multi-cellular clumps/fragments.
  • the dissociated cell populations or compositions are predominantly (e.g. greater than 70%, 80%, or 90% of cells) multi-cellular clumps/fragments, with relatively few single cells.
  • the dissociated cell populations or compositions are present as a mixture of single cells and multicellular clumps/fragments.
  • enzyme dissociation has its plain and ordinary meaning as understood in light of the specification and refers to fragmentation or dissociation of an organoid or other three-dimensional multicellular structure using the catalytic activity of one or more enzymes.
  • a process generally well known in the art, enzymatic dissociation typically involves the use of proteolytic enzymes (e.g. trypsin), or enzymes specific for other molecules (e.g. hyaluronidase) involved in adherence to surface or intercellular bonds.
  • mechanical dissociation has its plain and ordinary meaning as understood in light of the specification and refers to fragmentation or dissociation of an organoid or other three-dimensional multicellular structure using a mechanical force.
  • a process generally well known in the art, mechanical dissociation may be accomplished, for example, through trituration through narrow bore channels, where the channels may be in the form of pipettes, needles, microfluidic channels, or the like.
  • the term “mucosa” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the most inner layer of the gastrointestinal tract.
  • the epithelium is the most inner layer of the mucosa, and is where epithelial cells and other specialized cells such as Goblet cells are found.
  • the epithelium also forms the villi structure of the intestine.
  • the epithelium is surrounded by connective tissue called the lamina limbal, and a thin layer of smooth muscle.
  • muscle as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the muscularis basement of the gastrointestinal tract. The muscularis regulates peristaltic behavior of the intestine and colon, and originates from the mesenchymal layer of the nascent gut tube during development.
  • regionality has its plain and ordinary meaning as understood in light of the specification and refers to the qualities and features that distinguish one cell type from another.
  • intestine and colon and other gastrointestinal organs
  • both organs originate from the same definitive endoderm but early specification results in the proper development and differentiation of the two organs and constituent cells commensurate with their function. Consequently, intestinal tissue exhibits a different regionality than colon tissue.
  • intestinal and colonic organoids used for engraftment in an intestinal injury model retain their respective qualities even after integration into the cell layers of a different organ (e.g. intestinal organoid into host colon tissue or colonic organoid into host intestinal tissue).
  • intestinal barrier has its plain and ordinary meaning as understood in light of the specification and refers to the cellular and mucosal barrier that separates the intraluminal contents of the gastrointestinal tract from the surrounding tissue and circulatory system, while still permitting nutrient exchange. This barrier is mediated by the intracellular junctions between the cells of the epithelium. During intestinal damage, this barrier can be disrupted, resulting in abnormal function of the intestine, passage of potentially pathogenic microorganisms or antigens into the body, and leaking of blood and molecules into the lumen.
  • “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and/or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity.
  • a “pharmaceutically acceptable” “diluent,” “excipient,” and/or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts.
  • a pharmaceutically acceptable diluent, excipient, and/or carrier is a diluent, excipient, and/or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs.
  • the term diluent, excipient, and/or “carrier” can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered.
  • Such pharmaceutical diluent, excipient, and/or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin.
  • Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions.
  • Suitable pharmaceutical diluents and/or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • a non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution.
  • the physiologically acceptable carrier may also comprise one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.
  • antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such
  • compositions can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents.
  • These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation typically suits the mode of administration.
  • Cryoprotectants are cell composition additives to improve efficiency and yield of low temperature cryopreservation by preventing formation of large ice crystals.
  • Cryoprotectants include but are not limited to DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methyl-formamide, dimethyl-formamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch.
  • Cryoprotectants can be used as part of a cry opreservation medium, which include other components such as nutrients (e.g.
  • cryoprotectant may be found at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within a range defined by any two of the aforementioned numbers.
  • Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, ure
  • excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, P-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof.
  • the amount of the excipient may be found in composition at a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w/w or any percentage by weight in a range defined by any two of the aforementioned numbers.
  • pharmaceutically acceptable salts has its plain and ordinary meaning as understood in light of the specification and includes relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like.
  • pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, and the like.
  • suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonatcs of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts.
  • the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; amino acids, including glycine, arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L- glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl aminoethane.
  • Proper formulation is dependent upon the route of administration chosen.
  • Techniques for formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques of administering a compound exist in the art including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, epicutaneous, aerosol, parenteral delivery, including intramuscular, subcutaneous, intra-arterial, intravenous, intraportal, intra- articular, intradermal, peritoneal, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.
  • a “carrier” has its plain and ordinary meaning as understood in light of the specification and refers to a compound, particle, solid, semi- solid, liquid, or diluent that facilitates the passage, delivery and/or incorporation of a compound to cells, tissues and/or bodily organs.
  • a “diluent” has its plain and ordinary meaning as understood in light of the specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable.
  • a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and/or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation.
  • a common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.
  • base membrane matrix or “extracellular matrix” as used herein has its plain and ordinary meaning in light of the specification and refers to any biological or synthetic compound, substance, or composition that enhances cell attachment and/or growth. Any extracellular matrix, as well as any mimetic or derivative thereof, known in the art can be used for the methods disclosed herein.
  • extracellular matrices include but are not limited to cell-based feeder layers, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-omithine, collagen, collagen IV, gelatin, fibronectin, vitronectin, laminin, laminin-511 elastin, tenascin, heparan sulfate, entactin, nidogen, osteopontin, perlecan, fibrin, basement membrane, Matrigel, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof.
  • a common basement membrane matrix that is used in laboratories arc those isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells.
  • EHS Engelbreth-Holm-Swarm
  • these basement membrane matrices are derived from non-human animals and therefore contain xenogeneic components that prevent its use towards humans. They are also not defined, which can lead to variability in manufacturing, as well as potentially harbor pathogens. Accordingly, in some embodiments, the methods for culturing cells may involve the use of synthetic and/or defined alternatives to these xenogeneic basement membrane matrices.
  • the use of non-xenogeneic basement membrane matrices or mimetics or derivatives thereof enables manufacturing of biological products better suited for human use.
  • passage and “passaging” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to the conventional approaches performed in biological cell culture methods to maintain a viable population of cells for prolonged periods of time.
  • cells are generally proliferative in cell culture, they undergo multiple cycles of mitosis until occupying the available space, which is typically a surface of a cell culture container (e.g., a plate, dish, or flask) submerged under culture medium.
  • the cells may grow out as a monolayer on a cell culture container surface or aggregate in a culture medium. If the growing cells occupy the entire available space of surface or form too large an aggregate, they cannot proliferate further and may exhibit senescent behavior.
  • the cells may be passaged by taking a fraction of the cells and seeding this fraction onto a fresh surface or into fresh culture medium following dissociation of the aggregates. This fraction of the cells will continue to proliferate and multiply until they occupy the available space of the new surface or again form aggregates, upon which this passaging can be repeated successively.
  • three-dimensional as used in “three-dimensional pluripotent stem cell (PSC) aggregate(s),” “three-dimensional suspension,” “suspension culture,” “three-dimensional culture,” three-dimensional culturing,” “three-dimensional expansion,” or “three-dimensional aggregates” refers to the ability of cells, PSCs, PSC aggregates, DE, spheroids, and/or organoids, to be able to grow, develop, reproduce, expand, and interact with their surrounding framework in three dimensions. Such growth, development, reproduction, expansion, and/or interaction may be facilitated by a suspension of said cells, PSCs, PSC aggregates, DE, spheroids, and/or organoids in the framework.
  • such growth, development, reproduction, expansion, and/or interaction may be facilitated by a suspension of beads or bead-like structures holding or otherwise providing a place for said cells, PSCs, PSC aggregates, DE, spheroids, and/or organoids in the framework.
  • two-dimensional PSC(s) two-dimensional culture
  • two-dimensional culturing two-dimensional monolayer
  • cells, PSCs, PSC aggregates, DE, spheroids, and/or organoids to be able to grow, develop, reproduce, expand, and interact with their surrounding framework in two dimensions (e.g., along a monolayer of a plate).
  • % w/w or “% wt/wt” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100.
  • % v/v or “% vol/vol” as used herein has its plain and ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.
  • totipotent stem cells also known as omnipotent stem cells
  • omnipotent stem cells has its plain and ordinary meaning as understood in light of the specification and are stem cells that can differentiate into embryonic and extra-embryonic cell types. Such cells can construct a complete, viable organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg arc also totipotent.
  • ESCs embryonic stem cells
  • ES cells embryonic stem cells
  • pluripotent stem cells has its plain and ordinary meaning as understood in light of the specification and encompasses any cells that can differentiate into nearly all cell types of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissues and nervous system). PSCs can be the descendants of inner cell mass cells of the preimplantation blastocyst or obtained through induction of a non-pluripotent cell, such as an adult somatic cell, by forcing the expression of certain genes.
  • Pluripotent stem cells can be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.
  • iPSCs induced pluripotent stem cells
  • hiPSC refers to human iPSCs.
  • iPSCs may be derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection may be achieved through viral transduction using viruses such as retroviruses or lentiviruses.
  • Transfected genes may include the master transcriptional regulators Oct-3/4 (POU5F1) and Sox2. although other genes may enhance the efficiency of induction. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar' to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection.
  • iPSCs include first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells.
  • a retroviral system is used to transform human fibroblasts into pluripotent stem cells using four pivotal genes: Oct3/4, Sox2. Klf4, and c-Myc.
  • a lentiviral system is used to transform somatic cells with 0CT4, SOX2, NANOG, and LIN28.
  • Genes whose expression arc induced in iPSCs include but arc not limited to Oct-3/4 POU5Fi , certain members of the Sox gene family (e.g., Soxl, Sox2, Sox3, and Soxl 5) certain members of the Klf family (e.g., Klfl, Klf2, Klf4, and Klf5).
  • certain members of the Myc family e.g., C-myc, L-myc, and N-myc
  • Nanog LIN28, Tert, Fbxl5, ERas, ECAT15-1, ECAT15-2, Tell, P-Catenin, ECAT1, Esgl, Dnmt3L, ECAT8, Gdf3, Fthll7, Sall4, Rexl, UTF1, Stella, Stat3, Grb2, Prdml4, Nr5al, Nr5a2, or E-cadherin, or any combination thereof.
  • precursor cell has its plain and ordinary meaning as understood in light of the specification and encompasses any cells that can be used in methods described herein, through which one or more precursor cells acquire the ability to renew itself or differentiate into one or more specialized cell types.
  • a precursor cell is pluripotent or has the capacity to becoming pluripotent.
  • the precursor cells are subjected to the treatment of external factors (e.g., growth factors) to acquire pluripotency.
  • a precursor cell can be a totipotent (or omnipotent) stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; an oligopotent stem cells and a unipotent stem cell.
  • a precursor cell can be from an embryo, an infant, a child, or an adult.
  • a precursor cell can be a somatic cell subject to treatment such that pluripotency is conferred via genetic manipulation or protein/peptide treatment.
  • Precursor cells include embryonic stem cells (ESC), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSC).
  • one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent.
  • pluripotent stem cells arc derived from embryonic stem cells, which are in turn derived from totipotent cells of the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro.
  • Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Methods for deriving embryonic stem cells from blastocytes are well known in the art. It would be understood by one of skill in the art that the methods and systems described herein are applicable to any stem cells.
  • Additional stem cells that can be used in embodiments in accordance with the present disclosure include but are not limited to those provided by or described in the database hosted by the National Stem Cell Bank (NSCB), Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF); WTSC cell Bank at the Wi Cell Research Institute; the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW- SCRMC); Novocell, Inc. (San Diego, Calif.); Cellartis AB (Goteborg, Sweden); ES Cell International Pte Ltd (Singapore); Technion at the Israel Institute of Technology (Haifa, Israel); and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania.
  • NSCB National Stem Cell Bank
  • UFSC cell Bank the University of Wisconsin Stem Cell and Regenerative Medicine Center
  • UW- SCRMC the University of Wisconsin Stem Cell and Regenerative Medicine Center
  • Novocell, Inc. San Diego, Calif.
  • Cellartis AB Goteborg, Sweden
  • Exemplary embryonic stem cells that can be used in embodiments in accordance with the present disclosure include but are not limited to SA01 (SA001); SA02 (SA002); ES01 (HES-1); ES02 (HES-2); ES03 (HES-3); ES04 (HES-4); ES05 (HES-5); ES06 (HES-6); BG01 (BGN-01); BG02 (BGN-02); BG03 (BGN-03); TE03 (13); TE04 (14); TE06 (16); UC01 (HSF1); UC06 (HSF6); WA01 (HI); WA07 (H7); WA09 (H9); WA13 (H13); WA14 (H14).
  • Exemplary human pluripotent cell lines include but are not limited to TkDA3-4, 1231A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34-1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF, or 317-12 cells.
  • cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type.
  • directed differentiation describes a process through which a less specialized cell becomes a particular specialized target cell type.
  • the particularity of the specialized target cell type can be determined by any applicable methods that can be used to define or alter the destiny of the initial cell. Exemplary methods include but are not limited to genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.
  • an adenovirus can be used to transport the requisite four genes, resulting in iPSCs substantially identical to embryonic stem cells. Since the adenovirus does not combine any of its own genes with the targeted host, the danger of creating tumors is eliminated.
  • non-viral based technologies are employed to generate iPSCs.
  • reprogramming can be accomplished via plasmid without any virus transfection system at all, although at very low efficiencies.
  • direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification.
  • generation of mouse iPSCs is possible using a similar methodology: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency.
  • the expression of pluripotency induction genes can also be increased by treating somatic cells with FGF2 under low oxygen conditions.
  • DE definitive endoderm
  • the term “definitive endoderm” or “DE” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the developmental cell type that gives rise to the gut tube and resultant gastrointestinal organs, including the esophagus, stomach, small intestine, colon, liver, and pancreas.
  • the anterior DE forms the foregut and its associated organs, including the liver and pancreas
  • the posterior DE forms the midgut and hindgut, which forms the small and large intestines and parts of the geni to urinary system.
  • Markers of DE include SOX 17 and FOXA2.
  • the Wnt and FGF signaling pathways establish regionalization between anterior and posterior patterning of the DE.
  • Pluripotent stem cells can be differentiated into definitive endoderm by known methods in the art.
  • definitive endoderm cells can be differentiated from pluripotent cells by contacting the definitive endoderm with the Nodal, Activin (e.g., Activin A or Activin B), and/or BMP subgroups of the TGF superfamily of growth factors.
  • the pluripotent stem cells are contacted with Nodal, Activin A, Activin B, a BMP signaling pathway activator, or any combination thereof, to differentiate the pluripotent stem cells to definitive endoderm.
  • the pluripotent stem cells are contacted with Activin A to differentiate the pluripotent stem cells to definitive endoderm.
  • the one or more growth factors are selected from the group consisting of Nodal, Activin A, Activin B, a BMP signaling pathway activator, or combinations of any of these growth factors.
  • the stem cells are contacted with Activin A and a BMP signaling pathway activator.
  • PSCs are treated with 100 ng/ml of Activin A for 3 days as previously described.
  • feeder cell has its plain and ordinary meaning as understood in light of the specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying on the cell surface.
  • Feeder cells are generally adherent cells and may be growth arrested.
  • feeder cells are growth-arrested by irradiation (e.g. gamma rays), mitomycin-C treatment, electric pulses, or mild chemical fixation (e.g. with formaldehyde or glutaraldehyde).
  • irradiation e.g. gamma rays
  • mitomycin-C treatment e.g. gamma rays
  • electric pulses e.g. with formaldehyde or glutaraldehyde
  • mild chemical fixation e.g. with formaldehyde or glutaraldehyde
  • Feeder cells may serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the culture medium, or synthesizing extracellular matrix proteins.
  • the feeder cells are allogeneic or xenogeneic to the supported target stem cell, which may have implications in downstream applications.
  • the feeder cells are mouse cells.
  • the feeder cells are human cells.
  • the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76/7 cells, human fibroblasts, human foreskin fibroblasts, human dermal fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells.
  • conditioned medium prepared from feeder cells is used in lieu of feeder cell co-culture or in combination with feeder cell co-culture.
  • feeder cells are not used during the proliferation of the target stem cells.
  • three-dimensional as used in “three-dimensional pluripotent stem cell (PSC) aggregate(s),” “three-dimensional suspension,” “three-dimensional culture,” two- dimensional culturing,” “three-dimensional expansion,” or “three-dimensional aggregates” refers to the ability of cells, PSCs, PSC aggregates, DE, spheroids, and/or organoids to be able to grow, develop, reproduce, expand, and interact with their surrounding framework in three dimensions. Such growth, development, reproduction, expansion, and/or interaction may be facilitated by a suspension of said cells, PSCs, PSC aggregates, DE, spheroids, and/or organoids in the framework.
  • PSC pluripotent stem cell
  • two-dimensional PSC(s) two-dimensional culture
  • two-dimensional culturing two-dimensional monolayer
  • cells, PSCs, PSC aggregates, DE, spheroids, and/or organoids to be able to grow, develop, reproduce, expand, and interact with their surrounding framework in two dimensions (e.g., along a monolayer of a plate).
  • iPSCs are used to produce definitive endoderm or other downstream cell types such as foregut endoderm, ventral foregut endoderm, and hepatic lineages.
  • human iPSCs hiPSCs are used to produce definitive endoderm or other downstream cell types such as foregut endoderm, ventral foregut endoderm, and hepatic lineages.
  • PSCs such as ESCs and iPSCs
  • the directed differentiation is done in a stepwise manner to obtain each of the differentiated cell types where molecules (e.g. growth factors, ligands, agonists, antagonists) are added sequentially as differentiation progresses.
  • the directed differentiation is done in a nonstepwise manner where molecules (e.g. growth factors, ligands, agonists, antagonists) are added at the same time.
  • directed differentiation is achieved by selectively activating certain signaling pathways in the PSCs or any downstream cells.
  • the embryonic stem cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a time that is, is about, is at least, is at least about, is not more than, or is not more than about, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours or any time within a range defined by any two of the aforementioned times, for example 6 hours to 300 hours, 24 hours to 120 hours, 48 hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours.
  • more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.
  • the embryonic stem cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10 ng/mL, 20 ng/mL, 50 ng/mL, 75 ng/mL, 100 ng/mL, 120 ng/mL, 150 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL, 1200 ng/mL, 1500 ng/mL, 2000 ng/mL, 5000 ng/mL, 7000 ng/mL, 10000 ng/mL, or 15000 ng/mL, or any concentration that is within a range defined by any two of the aforementioned concentrations, for example, 10 ng/mL to 15000 ng/mL, 100 ng/mL to 5000 ng/mL, 500 ng/m
  • concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is varied during the course of the treatment. In some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors arc added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can differ in concentrations.
  • the ESCs or iPSCs are cultured in growth media that supports the growth of stem cells.
  • the ESCs or iPSCs are cultured in stem cell growth media.
  • the stem cell growth media is RPMI 1640, DMEM, DMEM/F12, or Advanced DMEM/F12.
  • the stem cell growth media comprises fetal bovine serum (FBS).
  • the stem cell growth media comprises FBS at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any percentage within a range defined by any two of the aforementioned concentrations, for example 0% to 20%, 0.2% to 10%, 2% to 5%, 0% to 5%, or 2% to 20%.
  • the stem cell growth media does not contain xenogeneic components.
  • the growth media comprises one or more small molecule compounds, activators, inhibitors, or growth factors.
  • pluripotent stem cells are prepared from somatic cells.
  • pluripotent stem cells are prepared from biological tissue obtained from a biopsy.
  • the pluripotent stem cells are cryopreserved.
  • the somatic cells are cryopreserved.
  • pluripotent stem cells are prepared from PBMCs.
  • human PSCs are prepared from human PBMCs.
  • pluripotent stem cells are prepared from cryopreserved PBMCs.
  • PBMCs are grown on a feeder cell substrate.
  • PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate.
  • PBMCs are grown on an irradiated MEF feeder cell substrate.
  • definitive endoderm can further undergo anterior endoderm pattering, foregut specification and morphogenesis, dependent on FGF, Wnt, BMP, or retinoic acid, or any combination thereof.
  • human PSCs are efficiently directed to differentiate in vitro into liver epithelium and mesenchyme. It will be understood that molecules such as growth factors can be added to any stage of the development to promote a particular type of hepatic tissue formation.
  • siRNA and/or shRNA targeting cellular constituents associated with the FGF, Wnt, BMP, or retinoic acid signaling pathways are used to inhibit or activate these pathways.
  • the intestinal and colonic organoids disclosed herein are produced by a differentiation process from pluripotent stem cells (such as embryonic stem cells or induced pluripotent stem cells) or an intermediate thereof (such as definitive endoderm), and comprise epithelial cell types and mesenchymal cell types, along with intestinal or colonic specialized cell types.
  • pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells
  • an intermediate thereof such as definitive endoderm
  • Exemplary methods for making intestinal and colonic organoids can be found in U.S. Patents 9,719,068 and 10,781,425, U.S.
  • intestinal and colonic organoids are differentiated through the culture of definitive endoderm cells.
  • definitive endoderm cells can be differentiated from pluripotent cells by contacting the definitive endoderm with the Nodal, Activin, and/or BMP subgroups of the TGFP superfamily of growth factors.
  • the pluripotent stem cells are contacted with Nodal, Activin A, Activin B, a BMP signaling pathway activator, or any combination thereof, to differentiate the pluripotent stem cells to definitive endoderm.
  • the pluripotent stem cells are contacted with Activin A to differentiate the pluripotent stem cells to definitive endoderm.
  • Definitive endoderm can further be subjected to FGF/Wnt-induced posterior endoderm patterning to direct hindgut specification.
  • definitive endoderm is first contacted with a Wnt signaling pathway activator and an FGF signaling pathway activator to posteriorize the definitive endoderm to hindgut endoderm.
  • a Wnt signaling pathway activator and an FGF signaling pathway activator to posteriorize the definitive endoderm to hindgut endoderm.
  • hindgut endoderm grows as monolayer but also spontaneously buds off as clumps of cells called hindgut spheroids in suspension.
  • the Wnt signaling pathway activator comprises Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l , or Wntl6, or any combination thereof.
  • the Wnt signaling pathway activator is Wnt3a.
  • the Wnt signaling pathway activator comprises a glycogen synthase kinase- 3 (GSK3) inhibitor, which acts as a Wnt signaling pathway activator.
  • GSK3 inhibitor is CHIR99021.
  • the FGF signaling pathway activator comprises FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 (FGF19, FGF15/FGF19), FGF16, FGF17, FGF18, FGF20, FGF21, FGF22, FGF23, or any combination thereof.
  • the FGF signaling pathway activator is FGF4.
  • the hindgut endoderm and hindgut spheroids produced comprise CDX2+ polarized epithelium surrounded by CDX2+ mesenchyme, and lack Alb and Pdxl, which denote foregut endoderm.
  • the BMP signaling pathway regulates formation of distinct regional types of intestine. Inhibition of BMP signaling after the hindgut stage promotes a proximal intestinal fate (duodenum/jejunum). Activation of BMP signaling after the hindgut stage promotes a more distal intestinal cell fate (cecum/colon).
  • the hindgut endoderm is contacted with a BMP signaling pathway activator to differentiate the hindgut endoderm into an intestinal organoid.
  • the hindgut endoderm is contacted with a BMP signaling pathway inhibitor to differentiate the hindgut endoderm into a colonic organoid.
  • the BMP signaling pathway activator comprises BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, or IDE2, or any combination thereof.
  • the BMP signaling pathway activator comprises BMP2.
  • the BMP signaling pathway inhibitor comprises Noggin, RepSox, LY364947, LDN193189, or SB431542, or any combination thereof.
  • the BMP signaling pathway inhibitor comprises Noggin.
  • the methods disclosed herein include dissociation of cellular aggregates (e.g., PSC aggregates) and/or spheroids (e.g., hindgut spheroids), and/or organoids (e.g., intestinal and/or colonic organoids).
  • the dissociated cell population are prepared by chemical, enzymatic dissociation and/or mechanical dissociation of the aggregate, spheroid and/or organoid.
  • the dissociation is chemical, e.g., EDTA.
  • enzymatic dissociation comprises dissociating the aggregate, spheroid and/or organoid with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof.
  • the enzymatic dissociation comprises incubating the aggregate, spheroid and/or organoid with a proteolytic and/or a collagenolytic enzyme.
  • the enzymatic dissociation utilizes Accutase.
  • mechanical dissociation comprises passing the aggregate, spheroid and/or organoid through successively narrower bore channels.
  • the concentration of the dissociated cell population in the cell suspension is, is about, is at least, is at least about, is not more than, or is not more than about, 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , IO 10 , or 10 11 cells/mL, or any concentration of cells within a range defined by any two of the aforementioned concentrations, for example, 10 5 -10 n , 10 5 -10 8 , 10 9 - 10 11 or 1O 6 -1O 10 cells/mL.
  • the concentration of cells in the dissociated cell population that are mesenchymal cell types is, is about, is at least, is at least about, is not more than, or is not more than about, 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , IO 10 , or 10 11 cells/mL, or any concentration of cells within a range defined by any two of the aforementioned concentrations, for example, 10 5 -10 n , 10 5 -10 8 , 10 9 -10 n or 1O 6 -1O 10 cells/mL.
  • the concentration of cells in the dissociated cell population that are epithelial cell types is, is about, is at least, is at least about, is not more than, or is not more than about, 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , IO 10 , or 10 11 cells/mL, or any concentration of cells within a range defined by any two of the aforementioned concentrations, for example, 10 5 -10 n , 10 5 -10 8 , 10 9 -10 n or 1O 6 -1O 10 cells/mL.
  • the dissociated cell population is made up of multi-cellular fragments at a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the total cells in the dissociated cell population, or any percentage within a range defined by any two of the aforementioned percentages, for example, 30-100%, 50-100%, 75-100%, 90-100%, 30-75%, or 50-95%.
  • the dissociated cell population is in the form of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% multi-cellular fragments. In some embodiments, the dissociated cell population is in the form of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% multicellular fragments.
  • liver organoids have been explored previously in, for example, Ouchi et al. “Modeling Steatohepatitis in Humans with Pluripotent Stem Cell-Derived Organoids” Cell Metabolism (2019) 30(2):374-384; Shinozawa et al.
  • pluripotent stem cells, definitive endoderm, foregut endoderm, ventral foregut endoderm, or downstream liver cell types are contacted with a TGF- P pathway inhibitor.
  • the TGF- pathway inhibitor comprises one or more of A83-O1, RepSox, LY365947, and SB431542.
  • the cells are not treated with a TGF-P pathway inhibitor.
  • the TGF-P pathway inhibitor provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.
  • pluripotent stem cells, definitive endoderm, foregut endoderm, ventral foregut endoderm, or downstream liver cell types are contacted with an FGF pathway activator.
  • the FGF pathway activator comprises an FGF protein.
  • the FGF protein comprises a recombinant FGF protein.
  • the FGF pathway activator comprises one or more of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 (FGF19, FGF15/FGF19), FGF16, FGF17, FGF18, FGF20, FGF21, FGF22, or FGF23.
  • the cells are not treated with an FGF pathway activator.
  • the FGF pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.
  • pluripotent stem cells, definitive endoderm, foregut endoderm, ventral foregut endoderm, or downstream liver cell types are contacted with a Wnt pathway activator.
  • the Wnt pathway activator comprises a Wnt protein.
  • the Wnt protein comprises a recombinant Wnt protein.
  • the Wnt pathway activator comprises Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl 1, Wntl6, BML 284, IQ-1, WAY 262611, or any combination thereof.
  • the Wnt pathway activator comprises a GSK3 signaling pathway inhibitor.
  • the Wnt pathway activator comprises CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, or TWS119, or any combination thereof.
  • the Wnt pathway activator is CHIR99021.
  • the cells are not treated with a Wnt pathway activator.
  • the Wnt pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.
  • pluripotent stem cells, definitive endoderm, foregut endoderm, ventral foregut endoderm, or downstream liver cell types are contacted with a VEGF pathway activator.
  • the VEGF pathway activator comprises one or more of VEGF or GS4012.
  • the cells are not treated with a VEGF pathway activator.
  • the VEGF pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.
  • pluripotent stem cells, definitive endoderm, foregut endoderm, ventral foregut endoderm, or downstream liver cell types are contacted with an EGF pathway activator.
  • the EGF pathway activator comprises EGF.
  • the cells are not treated with an EGF pathway activator.
  • the EGF pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.
  • pluripotent stem cells, definitive endoderm, foregut endoderm, ventral foregut endoderm, or downstream liver cell types are contacted with ascorbic acid.
  • the cells are not treated with ascorbic acid.
  • Ascorbic acid as provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.
  • pluripotent stem cells, definitive endoderm, foregut endoderm, ventral foregut endoderm, or downstream liver cell types are contacted with a BMP pathway activator or BMP pathway inhibitor.
  • the BMP pathway activator comprises a BMP protein.
  • the BMP protein is a recombinant BMP protein.
  • the BMP pathway activator comprises BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, or IDE2, or any combination thereof.
  • the BMP pathway inhibitor comprises Noggin, RepSox, LY364947, LDN-193189, SB431542, or any combination thereof.
  • the cells are not treated with a BMP pathway activator or BMP pathway inhibitor.
  • the BMP pathway activator or BMP pathway inhibitor provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.
  • pluripotent stem cells, definitive endoderm, foregut endoderm, ventral foregut endoderm, or downstream liver cell types are contacted with a retinoic acid pathway activator.
  • the retinoic acid pathway activator comprises retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, or AM580, or any combination thereof.
  • the cells are not treated with a retinoic acid pathway activator.
  • the retinoic acid pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.
  • pluripotent stem cells are converted into liver cell types via a “one step” process.
  • one or more molecules that can differentiate pluripotent stem cells into DE culture e.g., Activin A
  • additional molecules that can promote directed differentiation of DE culture e.g., FGF4, CHIR99021, RA
  • pluripotent stem cells e.g., ESCs or iPSCs
  • the pluripotent stem cells are expanded in suspension culture as described above and elsewhere herein.
  • the pluripotent stem cells are expanded in cell culture comprising a ROCK inhibitor (e.g. Y-27632).
  • the iPSCs are differentiated into definitive endoderm cells.
  • the pluripotent stem cells are differentiated into definitive endoderm cells by contacting the pluripotent stem cells with Activin A, a BMP activator, or both.
  • the pluripotent stem cells are contacted with a concentration of Activin A that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng/mL, or any concentration of Activin A within a range defined by any two of the aforementioned concentrations, for example, 10 to 200 ng/mL, 10 to 100 ng/mL, 100 to 200 ng/mL, or 50 to 150 ng/mL.
  • the pluripotent stem cells are contacted with Activin A at a concentration of 100 ng/mL or about 100 ng/mL.
  • the pluripotent stem cells are contacted with a concentration of a BMP signaling pathway activator that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng/mL, or any concentration of a BMP signaling pathway activator within a range defined by any two of the aforementioned concentrations, for example, 1 to 200 ng/mL, 1 to 100 ng/mL, 25 to 200 ng/mL, 1 to 80 ng/mL, or 25 to 100 ng/mL.
  • the pluripotent stem cells are contacted with a BMP signaling pathway activator at a concentration of 100 ng/mL or
  • the TGF-b pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542.
  • the TGF-b pathway inhibitor is A83-01.
  • the TGF- b pathway inhibitor is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 100-1000 nM, 100-500 nM, 500-1000 nM, or 300-700 nM.
  • the TGF-b pathway inhibitor is provided at a concentration of, or of about, 500 nM.
  • the FGF pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF 19, FGF20, FGF21, FGF22, and FGF23.
  • FGF1 FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF 19, FGF20, FGF21, FGF22, and FGF23.
  • FGF pathway activator is FGF2.
  • the FGF pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-10 ng/mL, 1-5 ng/mL, 5-10 ng/mL, or 3-7 ng/mL. In some embodiments, the FGF pathway activator is provided at a concentration of, or of about, 5 ng/mL.
  • the Wnt pathway activator is selected from the group consisting of Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl 1, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and TWS119.
  • the Wnt pathway activator is CHIR99021. In some embodiments, the Wnt pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 pM, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-8 pM, 1-3 pM, 3-8 pM, or 2-4 pM. In some embodiments, the Wnt pathway activator is provided at a concentration of, or of about, 3 pM.
  • the VEGF pathway activator is selected from the group consisting of VEGF or GS4012. In some embodiments, the VEGF pathway activator is VEGF. In some embodiments, the VEGF pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-20 ng/mL, 1-10 ng/mL, 10-20 ng/mL, or 5-15 ng/mL. In some embodiments, the VEGF pathway activator is provided at a concentration of, or of about, 10 ng/mL.
  • the foregut endoderm cells of step c) are cultured in a media that further comprises EGF.
  • the EGF is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10-30 ng/mL, 10-20 ng/mL, 20-30 ng/mL, or 15-25 ng/mL.
  • the EGF is provided at a concentration of, or of about, 20 ng/mL.
  • the foregut endoderm cells of step c) are cultured in a media that does not comprise EGF.
  • the foregut endoderm cells of step c) are cultured in a media that further comprises ascorbic acid.
  • the ascorbic acid is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg/mL or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10-100 pg/mL, 10-50 pg/mL, 50-100 pg/mL, or 30-70 pg/mL.
  • the ascorbic acid is provided at a concentration of, or of about, 50 pg/mL.
  • the foregut endoderm cells of step c) are cultured in a media that does not comprise ascorbic acid.
  • the foregut endoderm cells of step c) are cultured in a media that further comprises a ROCK inhibitor (ROCKi).
  • the ROCK inhibitor is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-20 pM, 1-10 pM, 10-20 pM, or 5-15 pM.
  • the ROCK inhibitor is provided at a concentration of, or of about, 10 pg/mL.
  • the ROCK inhibitor is Y-27632
  • the foregut endoderm cells of step c) are cultured in a media that does not comprise the ROCK inhibitor.
  • Apoptotic Agents and Anti-Adhesion Agents in Culture Media are cultured in a media that does not comprise the ROCK inhibitor.
  • the culture media used in any one or more of the aforementioned processes may include an anti- apoptotic agent.
  • anti- apoptotic agent may include but are not limited to CEPT or ROCKi.
  • the use of the anti-apoptotic agent, such as CEPT may result in higher cell (e.g., PSC) recovery during passaging.
  • CEPT may be present in the culture medium at a concentration that is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-20 pM, 1-10 pM, 10-20 pM, or 5-15 pM.
  • the CEPT is provided at a concentration of, or of about, 10 pg/mL.
  • the culture media used in any one or more of the aforementioned processes may include an antiadhesion agent.
  • the anti-adhesion agent may include one or more of dextran sulphate sodium (DSS), xantham gum, A-205804, I-CAM1, carboxymethyl cellulose, and/or Neural Organoid Basal Medium 2 (NOBM).
  • the anti-adhesion agent may comprise DSS.
  • the anti-adhesion agent e.g., DSS
  • DSS may be present in the culture media at a concentration that is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 750, or 1000 pg/ml or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-1000 pg/ml, 1-500 pg/ml, 5-100 pg/ml, or 5-50 pg/ml.
  • anti-adhesion agent e.g., DSS
  • DSS is provided at a concentration of, or of about, 10 pg/mL of the culture media (e.g., the liquid culture media, the second liquid culture media, the liquid definitive endoderm differentiation culture media, the liquid hindgut differentiation culture media, the liquid IO maturation culture media, the liquid differentiation culture media, and/or the liquid organoid maturation culture media).
  • DSS is found to moderate (e.g., reduce) the average size of 3D PSC aggregates.
  • FIG. 1A depicts an embodiment of an experimental protocol to explore the impact of various culture conditions on the maintenance and expansion of PSCs.
  • cell culture media e.g., TeSR AOF
  • a low-shcar/shcar-frcc biorcactor (Clinostar, CellVivo) was utilized in the following experiments.
  • FIG. IB illustrates the operation of a suspension culture where the chamber is rotated around its longitudinal axis when the longitudinal axis is oriented parallel to the ground.
  • the depicted rotating vessel bioreactor (cell culture systems), rotates continuously to keep the cells, PSC aggregates, spheroids and/or organoids suspended by counterbalancing the gravitation forces, thereby ideally keeping them in statical orbit.
  • the bioreactor allows for suspension of cells in the liquid culture media.
  • the composition comprising the suspended cell culture undergo serial passaging (column 106).
  • an analysis of the composition may be performed during or after one or more passages (column 108). The analysis may include but is not limited to determination of aggregate size distribution, flow cytometry, immunohistochemistry, PCR, and/or tri-lineage differentiation.
  • FIG. 2 depicts the results of an embodiment of a study examining the impact of the inoculation density of PSC in the suspension culture media on the formation of PSC aggregates and cell death at various time points following culture inoculation.
  • the suspension culture media (10 ml) was inoculated with 2, 5 or 10 million PSCs.
  • the resulting PSC aggregates were examined after 1, 2, 3 or 4 days. While all of the seeding densities resulted in the formation of PSC aggregates, the 5M and 10M conditions resulted in significantly more cell death (not aggregating single cells), as well as more significantly larger aggregates (see markers 202 and 204 in FIG. 2) that could result in formation of the hypoxic core (see markers 206 and 208 in FIG. 2).
  • the 2M cell density 200K cells/ml culture media
  • FIG. 4A depicts an iPSC growth curve and the two timepoints the PSCs were harvested.
  • FIG. 4B depicts an embodiment of photographs of a stalling two-dimensional PSC adherent culture at d-1 and dO, and the resulting PSC aggregates at day 4 (d4) of suspension culture.
  • FIG. 4C depicts an embodiment of a graph of the PSC aggregate size distribution at d4 of the suspension culture using either d-1 or dO PSC inoculates. The results show that d-1 PSC inoculants resulted in significantly more PSC aggregates on d4 as compared to dO PSC inoculates.
  • FIGs. 6A-6C Experiments were conducted comparing how the bioreactor rotation speed impacts the yield of PSC aggregates formed (see FIGs. 6A-6C).
  • the Clinostar bioreactor utilizes a chamber holding 10 ml of culture media which is a cylindric section and which is rotated around its longitudinal axis The speed of rotation of the chamber can be varied.
  • FIG. 6A depicts an embodiment of PSC aggregates on day 3 (d3) of suspension culture at various bioreactor chamber rotation speeds.
  • FIG. 6B depicts an embodiment of a graph of the PSC aggregate size distribution at d3 of the suspension culture at various bioreactor chamber rotation speeds, and FIG.
  • 6C depicts an embodiment of a chart comparing the total cellular yield and fold cell expansion of PSC suspension cultures at the first (Pl), second (P2) and third (P3) passaging at various bioreactor chamber rotation speeds. While almost all bioreactor chamber rotation speeds (except for 80 rpm) resulted in successful formation of PSC aggregates, 5 and 40 rpm showed maintenance of consistent cellular expansion across 3 passages.
  • FIG. 8 depicts the results of PSC suspension culturing using mTeSR 1 (research media) or mTeSR AOF (animal product free media) at the first (Pl), second (P2) and third (P3) passaging of a suspension culture. Both media tested supported successful cellular expansion and maintenance of PSC aggregates, with similar levels of PSC expansion and aggregate formation efficiency across multiple passages.
  • FIGs. 9A and 9B depict the results of the impact of culture media, mTeSR 1 (research media; FIG.
  • FIG. 9A 9A and mTeSR AOF (animal product free media; FIG. 9B) on expression of stem cell markers: Oct4, SSEA4 and TRA 1-60. Both media tested, mTeSR 1 and mTeSR AOF supported successful maintenance of sternness (>90% cells expressing stem cell markers Oct4, SSEA4 and TRA 1-60) by PSC aggregates grown in suspension culture.
  • FIG. 10 depicts the results of a study comparing how cell line (research-grade, PSC cell line 72.3; clinical-grade PSC cell line FF3 produced under GMP) impact the production of PSC aggregates at the first (Pl), second (P2) and third (P3) passaging of a suspension culture.
  • Successful cellular expansion and maintenance of expression of stem cell markers (Oct4, SSEA4 and TRA 1-60) was observed in both cell lines grown in mTeSR AOF culture media across 3 passages.
  • FIGs. 11A and 11B depict the results of a study comparing the impact of two- dimensional (2D) culturing (FIG. 11 A) to suspension culturing (3D) (FIG.
  • FIGs. 12A and 12B depict the results of a study comparing the impact of two- dimensional (2D) culturing (FIG. 12A) to suspension culturing (3D) (FIG. 12B) on the expression of stem cell markers Oct4, SSEA4 and TRA 1-60 in clinical-grade PSC cell line FF3 produced under GMP.
  • FIGs. 13A-13B depict the result of an embodiment based on a study investigating the formation of three-dimensional PSC aggregates from PSCs, in which the three- dimensional PSC aggregates were found to retain their pluripotency.
  • PSCs are able to form three-dimensional PSC aggregates progressing from Day 1 to Day 4.
  • number of such aggregates increased from 600 in Day 1 to -1400 in Day 4.
  • markers of pluripotency such as OCT4 and SSEA4 were evident in the three-dimensional PSC aggregates as shown in the confocal imaging.
  • FIG. 14A-14B depict the results of an evaluation of pluripotent 3D PSC aggregates across multiple lines and passages.
  • FIG. 14A there was a marked formation of three-dimensional PSC aggregates across passages for iPSC line 72.3 and ESC line Hl.
  • FIG. 14B shows an increase in cell count as well as an increase in diameter of the PSC aggregates across the passages for both lines.
  • FIG. 14B shows that an expression of pluripotency markers (e.g., OCT4 and SSEA-4) remained at least 90% across all the passages and lines tested for three-dimensional PSC aggregates, similar to the expression in conventional, two-dimensionally grown PSC monolayers.
  • FIG. 14A shows an expression of pluripotency markers (e.g., OCT4 and SSEA-4) remained at least 90% across all the passages and lines tested for three-dimensional PSC aggregates, similar to the expression in conventional, two-dimensionally grown PSC monolayers.
  • FIG. 15 depicts the results an evaluation comparing the pluripotency of PSC lines grown in three-dimensional suspension culture according to methods described herein, compared to PSC lines grown in conventional 2D monolayers.
  • OCT4, SOX2, and KEF4 there is an increased pluripotency of PSC lines (Hl and 72.3) grown in three-dimensional suspension culture in comparison to those lines grown in 2D monolayers.
  • FIG. 16 depicts an embodiment of an experimental protocol for the matrix-free suspension culture production of HIOs from hiPSCs.
  • the protocol may involve formation of a 3D iPSC culture (e.g., using a bioreactor), including the formation of 3D PSC aggregates.
  • the acclimation to the 3D PSC culture may involve single cell dissociation.
  • the protocol may further involve a differentiation of the 3D iPSC to 3D definitive endoderm (DE) (e.g., via use of Activin A in the culture media for DE induction).
  • DE definitive endoderm
  • the protocol may further involve a differentiation of the 3D DE culture to hindgut spheroids (e.g., via use of CHIR99021 and/or FGF4 in the culture media).
  • the protocol may further involve the development of HIO (e.g., via single cell dissociation and the use of EGF).
  • FIG. 17 depicts the results of a study examining the impact of acclimatization of PSCs to suspension culturing on the production of HIOs at various time points. Comparison between cells which were differentiated toward HIOs either: a) right after their inoculation from 2D monolayers into 3D suspension culture (Not acclimatized); or b) after being subjected to 1 passage in 3D suspension culture (Acclimatized). Inoculation methods and culture conditions are provided in more detail below. The results show that lack of cell acclimatization to 3D suspension culture (by passaging and re-introduction into the 3D bioreactor culture) resulted in significant cellular death and failure in HIOs development. One passage in suspension culture was demonstrated to be sufficient to acclimatize cells to 3D suspension culture. Therefore, PSC acclimatization to suspension culture is important for successful formation of human intestinal organoids.
  • FIGs. 18A and 18B depict the results of a study comparing the efficiency of DE induction in two-dimensional (2D) aggregate monolayer culturing (FIG. 18 A) and suspension culturing (3D) (FIG. 18B) by examining expression of definitive endoderm markers Sox 17 and FoxA2.
  • DE was induced using the methods described in more detail below.
  • Successful DE induction in 3D suspension culture was confirmed by immunofluorescent staining and flow cytometry analysis.
  • the results show that comparable efficiency of DE induction (-50% +/+ Soxl7/FoxA2 cells) between 2D and 3D culture.
  • Experiments also showed Activin A to contribute to DE differentiation efficiency when applied at specific time points.
  • FIG. 18 A two-dimensional aggregate monolayer culturing
  • 3D FIG. 18B
  • FIG. 20 shows the results of an embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the DE stage.
  • the size of the 3D PSC aggregates at the time of the exposure to Activin determines DE induction efficiency.
  • FIG. 20 shows that smaller size of the 3D PSC aggregates (e.g., less than about 400 (e.g., less than about 300 m) in average diameter of the aggregates) ensures better DE induction.
  • FIG. 21 depicts the results of an embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the HGS stage.
  • FIG. 22 depicts the results of an embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the HIO stage. The effects were demonstrated by the expression of CDX2, a marker for intestinal tissue differentiation. As shown in FIG. 21,
  • differentiation of 3D PSC aggregates having diameters no more than about 300 pm resulted in apical -in HIO formation and substantially homogenous expression of CDX2 across generated HIOs.
  • differentiation of 3D PSC aggregates having diameters at least 300 pm resulted in mixed apical- out and apical-out structures and epithelial structures with weak or no CDX2 expression.
  • 3D PSC aggregates having a smaller size e.g., diameters of less than about 400 pm (e.g., less than about 300 pm) is favorable for intestinal tissue differentiation.
  • FIGs. 23-27 depicts the results of an embodiment based on a study investigating the effect of dextran sulfate sodium (DSS), an anti-adhesion agent, mediating the size of 3D PSC aggregates.
  • DSS dextran sulfate sodium
  • FIG. 23 shows the effects of varied concentrations of DSS on the size of 3D PSC aggregates, with a concentration of 10 pg/ml having the highest effect of reducing the size of the 3D PSC aggregates.
  • FIG. 24 further shows that the effects of DSS on reducing aggregate size were noted across different PSC lines (72.3, FF3, Hl, and Hl GFP).
  • FIG. 25 further shows that a DSS concentration of 10 g/ml had the highest reduction in the average diameter across the lines, while also increasing the yield of aggregates being formed.
  • FIGS. 26 and 27 further explore the effect of 10 pg/ml DSS on average size of PSC aggregates across different lines.
  • FIG. 26 shows that, 10 pg/ml of DSS is sufficient to induce smaller diameter of PSC aggregates, with an average of -200 pm decrease in mean diameter of PSCs upon DSS treatment in comparison to untreated controls.
  • FIG. 27 shows that this effect is consistent across different iPSC and ESC lines and leads to the shift in the frequency of PSC aggregate size distribution.
  • FIG. 28 depicts the results of an embodiment based on a study investigating the effect of DSS on the pluripotency of 3D PSC aggregates, as measured by pluripotency markers SOX2 and OCT4.
  • DSS had no negative effect on the pluripotency of the 3D PSC aggregates any of the concentrations tested.
  • the study also investigated the effect of DSS on the viability of 3D PSC aggregates, as measured by the release of a viability marker lactate dehydrogenase (LDH). The study found that DSS at concentrations at or below 1000 pg/ml has no negative effect on the viability of PSC aggregates.
  • LDH lactate dehydrogenase
  • FIG. 29 depicts the results of an embodiment based on a study investigating the effects of various treatment regimes for applying DSS on the average size, numbers, and pluripotency of 3D PSC aggregates.
  • the treatment regimes that were tested included control (i.e., no treatment regime), at inoculation, and throughout.
  • the results show that Treatment with 10 ug/ml of DSS at the time of inoculation is sufficient to maintain PSC aggregates ⁇ 400 um, while having no negative effect on the PSC aggregate numbers or expression of pluripotency genes.
  • prolonged treatment (Throughout) with DSS lead to a decreased OCT4 expression.
  • FIG. 30 depicts the results of an embodiment based on a study investigating the effects of DSS on the average size of 3D PSC aggregates across passages. As shown in FIG. 30, the effect of DSS treatment on PSC aggregate size (a decrease in diameter) was maintained across multiple passages.
  • FIG. 31 depicts the results of an embodiment based on a study investigating the effects of DSS on the propensity of 3D PSC aggregates to differentiate.
  • the propensity is measured based on differentiation efficiency, which is marked by an expression of markers FOXA2 and SOX17.
  • similar differentiation efficiency of 3D PSC aggregates toward differentiation to DE was found, as noted by the expression of Soxl7 and FoxA2 in the presence and absence of DSS, demonstrating that there is no negative effect of DSS treatment on cells propensity to differentiation.
  • FIG. 32 depicts the results of an experiment demonstrating the development of well-patterned HIOs in suspension culture having either an apical-out epithelial cell polarity or an apical-in epithelial polarity (see below for additional details). Induction of HIOs from DE was performed using the methods described in more detail below. Successful development of correctly patterned HIOs in suspension culture confirmed by immunofluorescent staining for CDx2, E-cadherin.
  • FIG. 33 depicts the results of an embodiment based on the development of well-patterned HIOs in 3D suspension culture. The successful development of correctly patterned HIOs in the suspension culture is confirmed by immunofluorescent staining.
  • FIGs. 34A and 34B depict the results of an experiment demonstrating the in vivo maturation of HIOs developed in suspension culture following transplantation under the kidney capsule of a mouse.
  • FIG. 34A is an embodiment of a photograph of an HIO 9 weeks post-transplant under the kidney capsule.
  • FIG. 34B is an embodiment of H & E staining of an HTO 9 weeks post-transplant under the kidney capsule.
  • HIOs developed using the described suspension culture methods showed efficient engraftment and appropriate maturation upon transplantation under the kidney capsule.
  • FIG. 35 depicts the results of an experiment demonstrating that the polarity of epithelial cells in HIOs developed in suspension culture can be modified.
  • the methods used for development of the HIOs are described in more detail below.
  • Dissociation of hindgut spheroids at day 7 (+ dissociation) and their reaggregation in suspension culture leads to the apical surface on the inside of the HIO (apical-in). If the DE is not dissociated at day 7 (- dissociation), the result is the apical surface on the outside of the HIO (apical-out).
  • bioreactor inoculation for iPSC maintenance may begin after one or more passages (e.g., at day 3 of a 5 day passage, at day 4 of the 5 day passage, at day 5 of the 5 day passage).
  • cells may be maintained in a 2D (e.g., monolayer) culture until they are deemed appropriate for inoculation.
  • the inoculation may begin with aspiration of media and the addition of a dissociation reagent (e.g., TrypLE, GCDR and/or Accutase) to each well of a multi-well plate containing the samples to be inoculated.
  • a dissociation reagent e.g., TrypLE, GCDR and/or Accutase
  • the contents of the multi-well plate may be incubated until enough cells have sloughed off of the plate.
  • the inoculation may further include collecting cells and adding to a conical tube. Media may be added to the tube and may be gently titrated to break up colonies further into a single cell suspension.
  • the culture may be spun down, supernatants may discarded, and culture media may be readded. The cells may be gently resuspended in the media to create single cell suspension.
  • a anti-adhesion agent such as lOuM (1 : 1000 dilution) of CEPT or ROCKi, may be added to the suspension to prevent cell death. Cells may then be counted to determine the amount needed to obtain an appropriate number or range of cells.
  • that appropriate number may be about 0.5xl0 A 6 million cells, lx!0 A 6 million cells, 1.5xl0 A 6 million cells, 2xlO A 6 million cells, 2.5xlO A 6 million cells, 3xl0 A 6 million cells, 3.5xl0 A 6 million cells, 4xlO A 6 million cells, 4.5xlO A 6 million cells, or 5xl0 A 6 million cells.
  • the appropriate range of cells may be a range formed by any two of the aforementioned number of cells.
  • composition comprising the culture media, the cells, and any of the aforementioned ingredients (anti-adhesion agents, dissociation agents, etc.) may then be added to a bioreactor (e.g., Clinostar) to fill the inner chamber.
  • a bioreactor e.g., Clinostar
  • an amount of anti-adhesion agent such as lOuM CEPT or ROCKi, may be added to the system, taking into account the amount of ROCKi added with the cells.
  • the bioreactor may be rotated (e.g., at about 5RPM) to allow the cells to aggregate (e.g., for about 24hr).
  • changes in the composition within the bioreactor may be visualized to ensure that the sample is free from any abnormalities (e.g., large clumps, contamination, etc.).
  • the bioreactor may be placed upright to allow cell aggregates to settle to the bottom of the bioreactor.
  • culture media may be slowly added to the bioreactor, while ensuring that bubbles are removed from the system, and excess media outside the bioreactor system is aspirated off.
  • the bioreactor may be rotated horizontally to disperse cells throughout inner chamber. The bioreactor speed may be adjusted to reduce or eliminate the possibility of aggregates hitting the walls of the inner chamber.
  • a dissociation agent e.g., TrypLE, GCDR and/or Accutase
  • the culture media may be pre-warmed (e.g., to about 37 C).
  • the contents of the bioreactor may be visualized to evaluate quality and size of aggregates.
  • the aggregates may be split before they reach a given size (e.g., split before the aggregates reach about 500pm, 450pm, 400pm, 350pm, or 300pm).
  • the aggregates may continue to be cultured based on the aforementioned steps until they arc deemed ready for passaging.
  • the passaging may begin by allowing the aggregates to settle (e.g., by placing the bioreactor vertically), and then exposing the inside of the bioreactor chamber (e.g., by turning the bioreactor onto its side and lifting the lid).
  • the cell aggregates may be gently transferred to one or more wells of a multi-well plate for size quantification.
  • the cell aggregates may be imaged for BF image analysis (described herein).
  • the aggregates may be transferred to a conical tube to remove and discard excess media.
  • a dissociation agent e.g., 1ml Accutase
  • the aggregates may be incubated in a warm bead bath for some time, while ensuring that the aggregates are mixed.
  • the aggregates may be periodically visualized to evaluate progress of dissociation.
  • an amount of media e.g., about 5 mL
  • the culture may be spun down, and supernatants may be removed.
  • the cells may be resuspended in culture media and an amount of anti-adhesion agent (e.g., IOUM CEPT or ROCKi) may be added.
  • the sample may be gently titrated to achieve single cell suspension. Cells and fold expansion may be counted and recorded.
  • the cells may be inoculated in a new bioreactor using the methods described herein.
  • iPSC Culture & Maintenance e.g., Using ClinoStar System
  • the process may begin by preparing the bioreactor and adding cells to the bioreactor as previously discussed.
  • a culture schedule may be following as shown in Table 1 below. If aggregates are larger than a specified size (e.g., 500pm 450pm 400pm, 350pm, or 300pm), passage may be performed prior to reaching d4.
  • Table 1 TeSR AOF 2D Media Change Schedule
  • the process may begin with inoculating the bioreactor with an appropriate number of cells to allow for growth over one or more passages.
  • the sample may be split on d3 and reinoculated back into the bioreactor to allow for 3D adjustment.
  • the process may further include creating media based on Table 2 as shown below.
  • DE Induction may begin about 48 hr post second inoculation.
  • DO media may be added following media changing protocols.
  • the starting material may be imaged.
  • DI media may be added following media changing protocols.
  • the material may be imaged to ensure minor changes.
  • the D2 media may be added following media changing protocols.
  • the material may be imaged to ensure minor changes.
  • the subset may be fixed in PFA for some time (e.g., about 30min-lhr). In some embodiments, the subset may change its fixing from PFA to PBS +/+ and be left to cool. The material may be imaged to ensure expression of Soxl7+/FoxA2+ aggregates.
  • a subset of cells may be obtained to perform DE FACS.
  • the subset may be tested to ensure 80% Soxl7+/FoxA2+ of Oct4- population.
  • the process may proceed with Mid-Hindgut Generation.
  • the process for mid-hindgut generation may involve making d3-d6 media according to Table 3.
  • the media may be changed daily and imaged.
  • the process for HiO maturation may begin with midhindgut dissociation.
  • aggregates at the bottom of the bioreactor may be collected (e.g., using a cut tip).
  • the aggregates may be moved to a conical tube.
  • the remaining media may be aspirated once the aggregates have settled again.
  • a pre-warmed dissociation agent e.g., TrypLE, GCDR and/or Accutase
  • the tube holding the sample may be placed in a warm bead bath.
  • the aggregates may be periodically resuspended until visually broken down.
  • media containing serum may be added to the mixture and gently pipetted. The sample may then be spun. Liquid may be aspirated. The aggregates may be resuspended aggregates in HiO Media as shown below in Table 4. The process may further involve counting and recording the amount generated.
  • an appropriate amount of cells may be added into a new bioreactor in HiO Media with an anti-adhesion agent (e.g., 1:1000 of CEPT or ROCKi).
  • the bioreactor may be rotated (e.g., at about 5RPM) overnight for aggregate formation.
  • the HiO Maturation process may involve changing media on a periodic bases (e.g., every other day or when yellow) with the media shown in Table 4.
  • aggregates may be moved into separate bioreactors if the sample aggregates become too dense.
  • Indicators of too dense cultures may include but are not limited to: media becoming acidic quickly, cells falling off due to shear stress, visually more than a large number of HiOs.
  • the sample may be monitored daily to ensure continued growth until about d34.
  • Table 4 HIO Maturation Media
  • the process for aggregate size BF analysis may begin by allowing aggregates to settle (e.g., by placing the bioreactor vertically). After aggregates settle, the process may involve exposing the inside of the bioreactor (e.g., by turning the bioreactor onto its side laying flat and remove the front panel). The process may further include gently transfer cell aggregates (e.g., using a pipet tip) to one or more wells of a multi-well plate for size quantification. The samples may be imaged, and the images may be saved (e.g., on Leica Software) and opened (e.g., on FIJI). [0420] In some embodiments, the image formats may be converted, and color imbalances may be removed or adjusted via thresholding. In some embodiments, masks may be applied or adjusted.
  • the process may further involve opening a software (e.g., MICROSOFT EXCEL) for size calculation, and calculating the diameter based on area measurement.
  • a software e.g., MICROSOFT EXCEL
  • Buckets may be created for each aggregate size range in increments (e.g., of about lOOum).
  • Appropriate visualizations e.g., bar graphs may be created.

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Abstract

Sont divulgués des procédés d'utilisation et de formation de cultures en suspension de cellules souches pluripotentes (PSC) et de cellules différenciées, de sphéroïdes et d'organoïdes dérivés de PSC (par exemple, à efficacité industrielle et/ou extensibilité), et des compositions les comprenant. Ces procédés peuvent être mis en œuvre en culture en suspension, sans utiliser de matrices de membrane basale, pendant la maintenance et l'expansion de PSC, ainsi que pendant la différenciation de PSC en cellules différenciées et en organoïdes, par exemple un endoderme définitif (DE), des sphéroïdes d'intestin postérieur (HGS) et des organoïdes intestinaux (IO). Selon certains aspects, les procédés peuvent être exempts de xéno-contaminants et peuvent être effectués selon de bonnes pratiques de fabrication (GMP). Sont également divulgués des procédés de commande de la polarité de cellules épithéliales dans des IO, la couche apicale étant orientée vers l'extérieur de l'organoïde, ou en variante vers l'intérieur de l'organoïde. Sont également divulguées des utilisations des procédés et des compositions pour la transplantation et le traitement.
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