Matrix-free suspension culture method

Abstract

Disclosed herein are methods for utilizing and forming suspension cultures of pluripotent stem cells (PSCs), and differentiated cells, spheroids, and organoids derived from PSCs (e.g., with industrial efficiency and / or scalability), as well as compositions comprising them. These methods can be carried out in suspension culture without the use of a basement membrane matrix during the maintenance and expansion of PSCs, and during the differentiation of PSCs into differentiated cells and organoids, such as endoderm (DE), hindgut spheroids (HGS), and intestinal organoids (IO). In some embodiments, the methods may be xenofree and may be carried out in accordance with Good Manufacturing Practices (GMP). Also disclosed herein are methods for controlling the polarity of epithelial cells in IOs, such that the apical layer is oriented outside the organoid, or alternatively inside the organoid. The use of methods and compositions for transplantation and therapeutic purposes is also disclosed.

Description

Technical Field

[0001] (Cross-reference) This application claims priority to U.S. Provisional Patent Application No. 63 / 510,087, filed on June 23, 2023, and U.S. Provisional Patent Application No. 63 / 582,207, filed on September 12, 2023, both entitled "METHODS OF MATRIX-FREE SUSPENSION CULTURE", the entireties of which are incorporated herein by reference.

[0002] (Field of the Invention) Aspects of the present disclosure generally relate to the suspension culture of pluripotent stem cells (PSCs), PSC aggregates, differentiated cells derived from PSCs and PSC aggregates, spheroids, and organoids. The methods of the present disclosure for generating such PSCs, PSC aggregates, differentiated cells, spheroids, and organoids can be performed without using a basement membrane matrix during the maintenance and expansion of PSCs and during the differentiation of PSCs and PSC aggregates into differentiated cells and organoids, such as definitive endoderm (DE), hindgut spheroids (HGS), and intestinal organoids (IO). Also disclosed herein is a method of controlling the polarity of epithelial cells in an IO, wherein the apical layer is oriented outside or alternatively inside the organoid. Further, compositions comprising such PSCs, differentiated cells, spheroids, and organoids are disclosed herein. Further, therapeutic methods using such compositions are disclosed herein.

Background Art

[0003] The direct differentiation of human intestinal organoids (HIOs) from human pluripotent stem cells (hPSCs) enables the generation of human intestinal tissue that recapitulates the structure of its in vivo counterpart and the presence and function of all known intestinal epithelial cell types (absorption, mucus, and hormone production). However, because the direct differentiation protocol relies on the spontaneous formation and detachment of spheroids on day -7 of culture, it can be plagued by high variability between hPSC lines, experiments, and wells. In addition, the transition from two-dimensional (e.g., monolayer) to three-dimensional (e.g., Matrigel-embedded) culture conditions can represent a potentially significant hurdle in HIO production. In addition to the low yield of HIO production and the need for the use of Matrigel (a highly variable animal-derived extracellular matrix), the high variability represents a potentially significant hurdle to the use of HIOs in basic research / translational research and potential therapeutic applications. Improved methods for culturing PSCs and differentiated cells, spheroids, and organoids, as well as the resulting compositions, are needed.

Summary of the Invention

[0004] Aspects of the present disclosure generally relate to suspension culture methods and compositions for pluripotent stem cells (PSCs), three-dimensional PSC aggregates, differentiated cells derived from PSCs, spheroids, and organoids. These methods can be carried out without using a heterologous basement membrane matrix during the maintenance and expansion of PSCs and during the differentiation of PSCs into differentiated cells and organoids, such as definitive endoderm (DE), hindgut spheroids (HGS), and intestinal organoids (IO). In some embodiments, the disclosed methods and compositions utilize suspension culture for each of the following stages in the production of organoids from PSCs: maintenance and expansion of PSCs, differentiation of PSCs into DE, differentiation of DE into spheroids (e.g., hindgut spheroids), and differentiation and maturation of spheroids into organoids (e.g., intestinal organoids).

[0005] Intermediates and resulting compositions for each of the aforementioned steps are also disclosed herein. In various embodiments, such compositions are based on the three-dimensional expansion of PSCs, PSC aggregates, DEs, spheroids, and / or organoids via suspension culture.

[0006] A method for controlling the polarity of epithelial cells in an organoid (IO), wherein the apical layer is oriented outward from the organoid, or alternatively, inward from the organoid, is disclosed herein.

[0007] Exceptional embodiments of this disclosure are provided in embodiments numbered as follows: Embodiment 1: A method comprising (a) inoculating PSCs into a liquid culture medium, (b) culturing the PSC-inoculated liquid culture medium in a bioreactor such that three-dimensional PSC aggregates are formed in the liquid culture medium, wherein the culturing in the bioreactor includes suspending the PSCs in the liquid culture medium, and (c) subculturing PSCs by (i) dissociating at least a portion of the three-dimensional PSC aggregates into single cells, and (ii) inoculating the dissociated three-dimensional PSC aggregates from (i) together with PSCs into a second liquid culture medium.

[0008] Embodiment 2: The method according to Embodiment 1, wherein the liquid culture medium and the second liquid culture medium do not contain any animal or human-derived material, and optionally, the liquid culture medium and the second liquid culture medium do not contain any extracellular matrix and / or basement membrane matrix.

[0009] Embodiment 3: The method according to Embodiment 1 or 2, wherein the PSCs are subcultured when the diameter of most of the formed three-dimensional PSC aggregates is 500 μm or less.

[0010] Embodiment 4: The method according to any one of Embodiments 1 to 3, wherein the PSCs are passaged when the diameter of most of the formed three-dimensional PSC aggregates is 400 μm or less.

[0011] Embodiment 5: The method according to any one of Embodiments 1 to 4, wherein the PSCs are passaged when the diameter of most of the formed three-dimensional PSC aggregates is 300 μm or less.

[0012] Embodiment 6: The method according to any one of Embodiments 1 to 5, wherein at least 80% of the three-dimensional PSC aggregate dissociates into single cells.

[0013] Embodiment 7: The method according to any one of Embodiments 1 to 6, wherein, optionally, at least 90% of the PSC aggregates dissociate into single cells.

[0014] Embodiment 8: The method according to Embodiment 1, wherein the method comprises subculturing the PSCs two or more times by culturing the PSCs in a second inoculated liquid culture medium until additional three-dimensional PSC aggregates are formed.

[0015] Embodiment 9: The method according to any one of Embodiments 1 to 8, wherein the liquid culture medium of (a) and / or the second liquid culture medium of (c)(ii) are inoculated at a density of approximately 100,000 to 220,000 PSCs / mL.

[0016] Embodiment 10: The method according to any one of Embodiments 1 to 9, wherein the liquid culture medium of (a) and / or the second liquid culture medium of (c)(ii) are inoculated at a density of approximately 180,000 to 220,000 PSCs / mL.

[0017] Embodiment 11: The method according to any one of Embodiments 1 to 10, wherein passage occurs after a period of approximately 40 to 168 hours after inoculation in (a).

[0018] Embodiment 12: The method according to any one of Embodiments 1 to 11, wherein passage occurs after a period of approximately 40 to 84 hours after inoculation in (a).

[0019] Embodiment 13: The method according to any one of Embodiments 1 to 12, wherein passage occurs after a period of approximately 66 to 78 hours after inoculation in (a).

[0020] Embodiment 14: The method according to any one of Embodiments 1 to 13, further comprising replacing a portion of the culture medium in the bioreactor of (a) after a period of about 36 to 60 hours following inoculation in (a) and / or (c)(ii).

[0021] Embodiment 15: The method according to any one of Embodiments 1 to 14, further comprising replacing a portion of the culture medium in the bioreactor of (a) after a period of about 42 to 54 hours following inoculation in (a) and / or (c)(ii).

[0022] Embodiment 16: The method according to any one of Embodiments 1 to 15, further comprising replacing a portion of the liquid culture medium in the bioreactor of (a) after a certain period following inoculation in (a) and / or (c)(ii), wherein the replaced portion of the liquid culture medium is at least 50% of the liquid culture medium in the bioreactor of (a).

[0023] Embodiment 17: The method according to any one of Embodiments 1 to 16, wherein the method comprises culturing PSCs on the surface of a substrate prior to inoculation in (a), and collecting the PSCs from the surface of the substrate for use in inoculation in (a) when the PSCs are in the logarithmic growth phase and / or have a confluence of 35-55%, and optionally further comprising such collection including dissociating the PSCs prior to inoculation in (a).

[0024] Embodiment 18: The method of Embodiment 17, wherein the PSCs are collected from the surface of the substrate for use in inoculation of (a) when the PSCs are in the logarithmic growth phase and / or have a confluence of 40-50%.

[0025] Embodiment 19: The method according to any one of Embodiments 1 to 18, wherein the dissociation is chemical, enzymatic, and / or mechanical dissociation.

[0026] Embodiment 20: The method according to any one of Embodiments 1 to 19, wherein the dissociation is enzymatic, and optionally the enzyme comprises a protease and / or a collagenase, and optionally the enzyme is Accutase.

[0027] Embodiment 21: The method according to any one of Embodiments 1 to 20, wherein the bioreactor of (a) and / or (b) includes a rotating chamber containing a liquid culture medium, the rotation speed of the rotating chamber is selected such that the number of PSCs in the liquid culture medium in (c) is at least twice or 2.5 times the number of PSCs used to inoculate the liquid culture medium, and optionally, the number of PSCs in the liquid culture medium in (c) is at least twice or 2.5 times the number of PSCs used to inoculate the liquid culture medium for at least two passages of the PSCs.

[0028] Embodiment 22: The method according to any one of Embodiments 1 to 21, wherein the liquid culture medium is a serum-free medium, and optionally the medium contains recombinant human basic fibroblast growth factor (rh bFGF) and / or recombinant human transforming growth factor β (rh TGFβ).

[0029] Embodiment 23: The method according to any one of Embodiments 1 to 22, wherein, after one or more passages, at least 85% of the PSCs express Oct4, SSEA1, and TRA 1-60 at levels at least the same as the mean expression levels of the PSCs used in the inoculation in (a).

[0030] Embodiment 24: The method according to Embodiment 23, wherein, after one or more passages, at least 95% of the PSCs express Oct4, SSEA1, and TRA 1-60 at levels at least the same as the mean expression levels of the PSCs used in the inoculation in (a).

[0031] Embodiment 25: The method according to any one of Embodiments 1 to 24, wherein the PSC expresses SOX2 and KLF4.

[0032] Embodiment 26: A method for differentiating PSCs into endoderm (DE) in a three-dimensional suspension culture, the method comprising: (d) culturing a liquid culture medium inoculated with PSCs in a bioreactor, wherein the culturing of the liquid culture medium inoculated with PSCs in (d) includes suspending the PSCs in the liquid culture medium; and (e) culturing the PSCs in (d) in a liquid endoderm differentiation culture medium in a bioreactor for a period sufficient to differentiate the PSCs into DE, wherein the culturing of the PSCs in (d) in the liquid endoderm differentiation culture medium includes suspending the PSCs in the liquid endoderm differentiation culture medium.

[0033] Embodiment 27: The method according to Embodiment 26, wherein the liquid culture medium or liquid endoderm differentiation culture does not contain any animal or human-derived material, and optionally the culture medium does not contain any extracellular matrix and / or basement membrane matrix.

[0034] Embodiment 28: The method according to Embodiment 26 or 27, wherein the culture in (d) is for a period of approximately 18 to 54 hours.

[0035] Embodiment 29: The method according to any one of Embodiments 26 to 28, wherein the culture in (d) is for a period of approximately 24 to 48 hours.

[0036] Embodiment 30: The method according to any one of Embodiments 26 to 29, wherein the liquid culture medium inoculated with PSCs cultured in (d) is the PSC inoculation culture medium of any one of Embodiments 1 to 24 (c)(ii).

[0037] Embodiment 31: The method according to any one of Embodiments 26 to 30, wherein the period is approximately 48 to 96 hours, which is sufficient to differentiate PSCs into DEs.

[0038] Embodiment 32: The method according to any one of Embodiments 26 to 31, wherein the period is approximately 60 to 84 hours, which is sufficient to differentiate PSCs into DEs.

[0039] Embodiment 33: The method according to any one of Embodiments 26 to 32, wherein the period sufficient to differentiate PSCs into DEs is approximately 66 to 78 hours.

[0040] Embodiment 34: The method according to any one of Embodiments 26 to 33, wherein culturing the PSCs in liquid endoderm differentiation culture medium for a period sufficient to differentiate the PSCs into DEs comprises: culturing the PSCs in a culture medium containing nodal signaling pathway activator and / or Wnt signaling pathway activator for a first period; culturing the PSCs in a culture medium containing nodal signaling pathway activator and / or Wnt signaling pathway activator and serum or serum substitute for a second period; and culturing the PSCs in a culture medium containing nodal signaling pathway activator and / or Wnt signaling pathway activator and serum or serum substitute for a third period.

[0041] Embodiment 35: The method according to Embodiment 34, wherein the culture medium in which the PSCs are cultured for a first period further comprises a BMP activator.

[0042] Embodiment 36: The method according to Embodiment 34 or 35, wherein the culture medium in which the PSCs are cultured for a second period and the culture medium in which the PSCs are cultured for a third period each contain nodal signaling pathway activator and / or Wnt signaling pathway activator and serum, and optionally the serum is FBS.

[0043] Embodiment 37: The method according to Embodiment 34 or 35, wherein the culture medium in which the PSCs are cultured for a second period and the culture medium in which the PSCs are cultured for a third period each comprises a nodal signaling pathway activator and / or a Wnt signaling pathway activator, and a serum substitute, wherein optionally the serum substitute is a knockout serum replacement (KSR).

[0044] Embodiment 38: The method according to any one of Embodiments 34 to 37, wherein each of the first, second, and third periods is approximately 18 to 30 hours.

[0045] Embodiment 39: The method according to any one of Embodiments 34 to 38, wherein each of the first, second, and third periods is approximately 20 to 28 hours.

[0046] Embodiment 40: The method according to any one of Embodiments 26 to 39, wherein the efficiency of DE induction is at least about 35%.

[0047] Embodiment 41: The method according to any one of Embodiments 26 to 40, wherein the efficiency of DE induction is at least about 45 to 55%.

[0048] Embodiment 42: The method according to any one of Embodiments 26 to 41, wherein the DE expresses Sox17 and FoxA2.

[0049] Embodiment 43: A method for differentiating endoderm (DE) into hindgut spheroid (HGS) in a three-dimensional suspension culture, the method comprising (f) culturing DE in liquid hindgut differentiation medium in a bioreactor for a period sufficient to differentiate the DE into HGS, wherein the culture of DE comprises suspending DE in liquid hindgut differentiation medium.

[0050] Embodiment 44: The method according to Embodiment 43, wherein the liquid hindgut differentiation culture medium does not contain any animal or human-derived material, and optionally, the liquid hindgut differentiation culture medium does not contain any extracellular matrix and / or basement membrane matrix.

[0051] Embodiment 45: The method according to Embodiment 43, wherein the DE cultured in (f) is the DE described in any one of Embodiments 25 to 39.

[0052] Embodiment 46: The method according to any one of Embodiments 43 to 45, wherein the period is approximately 60 to 120 hours, which is sufficient to differentiate DE into HGS.

[0053] Embodiment 47: The method according to any one of Embodiments 43 to 46, wherein the period is approximately 84 to 108 hours, which is sufficient to differentiate DE into HGS.

[0054] Embodiment 48: The method according to any one of Embodiments 43 to 47, wherein the period of time sufficient to differentiate DE into HGS is approximately 90 to 102 hours.

[0055] Embodiment 49: The method according to any one of Embodiments 43 to 48, wherein the liquid hindgut differentiation culture medium is replaced after a period of approximately 20 to 28 hours.

[0056] Embodiment 50: The method according to any one of Embodiments 43 to 49, wherein the liquid hindgut differentiation culture medium is replaced after a period of approximately 22 to 26 hours.

[0057] Embodiment 51: The method according to any one of Embodiments 43 to 50, wherein the liquid hindgut differentiation culture medium comprises Wnt signaling pathway activator, FGF signaling pathway activator, and optionally FBS.

[0058] Embodiment 52: The method according to Embodiment 51, wherein the Wnt signaling pathway activator includes CHIR99021.

[0059] Embodiment 53: The method according to Embodiment 51 or 52, wherein the FGF signaling pathway activator includes FGF4.

[0060] Embodiment 54: The method according to any one of Embodiments 51 to 53, wherein the FGF signaling pathway activator is FGF4 at a concentration of approximately 50 to 750 ng / mL.

[0061] Embodiment 55: The method according to any one of Embodiments 51 to 54, wherein the Wnt pathway activator is CHIRON 99021 at a concentration of approximately 0.5 to 6 μM.

[0062] Embodiment 56: A method for differentiating hindgut spheroids (HGS) into intestinal organoids (IO) in a three-dimensional suspension culture, wherein the method is (g) A method comprising culturing HGS in a liquid IO maturation culture medium in a bioreactor for a period sufficient to differentiate HGS into IO, wherein the culturing of HGS comprises suspending HGS in the liquid IO maturation culture medium.

[0063] Embodiment 57: The method according to Embodiment 56, wherein the liquid IO mature culture medium does not contain any animal or human-derived material, and optionally the culture medium does not contain any extracellular matrix and / or basement membrane matrix.

[0064] Embodiment 58: The method according to Embodiment 56 or 57, wherein the HGS cultured in (g) is the HGS described in any one of Embodiments 40 to 52.

[0065] Embodiment 59: The method according to any one of Embodiments 56 to 58, wherein the period sufficient to differentiate HGS into IO is approximately 12 to 30 days.

[0066] Embodiment 60: The method according to any one of Embodiments 56 to 59, wherein the period sufficient to differentiate HGS into IO is approximately 15 to 28 days.

[0067] Embodiment 61: The method according to any one of Embodiments 56 to 60, wherein the IO mature culture medium is replaced after a period of approximately 24 to 54 hours.

[0068] Embodiment 62: The method according to any one of Embodiments 56 to 61, wherein the IO mature culture medium is replaced after a period of approximately 46 to 50 hours.

[0069] Embodiment 63: The method according to any one of Embodiments 56 to 62, wherein the IO maturation culture medium comprises one or more of EGF, R-spongin, noggin, gremlin 1, and / or epiregulin (EREG).

[0070] Embodiment 64: The method according to Embodiment 63, wherein the concentrations of EGF, R-sponging, noggin, gremlin 1, and / or EREG are approximately 25 to 150 ng / mL.

[0071] Embodiment 65: The method according to Embodiment 63 or 64, wherein the concentrations of EGF R-sponging, noggin, gremlin 1, and / or EREG are approximately 50 to 100 ng / mL.

[0072] Embodiment 66: The method according to any one of Embodiments 56 to 65, wherein the HGS does not dissociate before being cultured in the IO maturation culture medium, and the epithelial cells of the formed IO have polarity in which the apical surface is oriented outward from the IO.

[0073] Embodiment 67. A method for differentiating hindgut spheroids (HGS) into intestinal organoids (IOs) having apical-in polarity in a three-dimensional suspension culture, wherein the method comprises the method according to any one of Embodiments 56 to 66, further comprising dissociating at least a portion of the HGS into HGS single cells before incubation in IO maturation culture medium, wherein the culture of HGS comprises suspending the dissociated HGS single cells and any undissociated HGS in liquid IO maturation culture medium, wherein the epithelial cells of the IOs formed from the dissociated HGS single cells have polarity in which the apical surface is oriented inward of the IOs.

[0074] Embodiment 68. The method according to Embodiment 67, wherein the liquid IO mature culture medium does not contain any animal or human-derived material, and optionally, the liquid IO mature culture medium does not contain any extracellular matrix and / or basement membrane matrix.

[0075] Embodiment 69. The method according to Embodiment 67 or 68, wherein at least 80% of the HGS dissociates into single cells.

[0076] Embodiment 70. The method according to any one of Embodiments 67-69, wherein at least 90% of the HGS dissociates into single cells.

[0077] Embodiment 71. The method according to any one of Embodiments 67 to 70, wherein a certain concentration of dissociated HGS single cells is present in IO mature culture medium, and the concentration is approximately 0.05 × 10⁵ to 80 × 10⁵ dissociated HGS single cells / mL relative to the IO mature culture medium.

[0078] Embodiment 72: The method according to any one of Embodiments 67 to 71, wherein a certain concentration of dissociated HGS single cells is present in IO mature culture medium, and the concentration is approximately 10 × 10⁵ to 80 × 10⁵ dissociated HGS single cells / mL relative to the IO mature culture medium.

[0079] Embodiment 73: The method according to any one of Embodiments 67 to 72, wherein a certain concentration of dissociated HGS single cells is present in IO mature culture medium, and the concentration is approximately 20 × 10⁵ to 60 × 10⁵ dissociated HGS single cells / mL relative to the IO mature culture medium.

[0080] Embodiment 74: The method according to any one of Embodiments 67 to 73, wherein the dissociation is chemical, enzymatic, and / or mechanical dissociation.

[0081] Embodiment 75: The method according to any one of Embodiments 67 to 74, wherein the dissociation is enzymatic, and optionally the enzyme comprises a protease and / or a collagenase, and optionally the enzyme is Accutase.

[0082] Embodiment 76: A method according to any one of embodiments 67 to 75, further comprising porting the method to target I / O.

[0083] Embodiment 77: The method of Embodiment 76, wherein the target of the IO is transplanted submucosa of a non-human animal for a period of approximately 6 to 20 weeks, which is optional.

[0084] Embodiment 78: The method according to Embodiment 77, wherein the IO is implanted under the kidney capsule of a non-human animal for a period of approximately 12 to 20 weeks.

[0085] Embodiment 79: The method according to Embodiment 77 or 78, wherein the IO is implanted under the kidney capsule of a non-human animal for a period of approximately 16 to 20 weeks.

[0086] Embodiment 80: The method according to Embodiment 76, wherein the implantation of IO to the target intestinal lumen is to treat the target intestine.

[0087] Embodiment 81: The method according to any one of Embodiments 76 to 80, wherein the IO matures in vitro for a certain period prior to transplantation, and optionally the period is about 7 to 28 days.

[0088] Embodiment 82: The method according to any one of Embodiments 76 to 81, wherein the IO matures in vitro for a period of approximately 14 to 28 days prior to transplantation.

[0089] Embodiment 83: The method according to any one of Embodiments 76 to 82, wherein the IO matures in vitro for a period of approximately 21 to 28 days prior to transplantation.

[0090] Embodiment 84: The method according to any one of Embodiments 26 to 42, wherein the method further comprises differentiating DE into spheroids, and optionally, differentiation comprises (h) culturing DE in a liquid differentiation medium in a bioreactor for a period sufficient to differentiate DE into spheroids, wherein the culturing of DE comprises suspending DE in a liquid differentiation medium, and optionally, culturing the spheroids being foregut or hindgut spheroids.

[0091] Embodiment 85: The method according to Embodiment 84, wherein the liquid differentiation medium does not contain any animal or human-derived material, and optionally, the liquid differentiation medium does not contain any extracellular matrix and / or basement membrane matrix.

[0092] Embodiment 86: The method according to Embodiment 84 or 85, wherein the method is (i) culturing the spheroids in a liquid organoid maturation culture medium in a bioreactor for a period of time sufficient to differentiate the spheroids into organoids, the culture of the spheroids further comprising suspending the spheroids in the liquid organoid maturation culture medium, and optionally the organoids are selected from the group consisting of liver, pancreas, stomach, gastric antrum, gastric fundus, intestine, lung, or colon organoids.

[0093] Embodiment 87: The method according to any one of Embodiments 84 to 86, wherein the liquid organoid maturation culture medium does not contain any animal or human-derived material, and optionally, the liquid organoid maturation culture medium does not contain any extracellular matrix and / or basement membrane matrix.

[0094] Embodiment 88: The method according to any one of Embodiments 1 to 87, wherein the PSC is an induced PSC (iPSC) or an embryonic stem cell (ESC).

[0095] Embodiment 89: The method according to any one of Embodiments 1 to 88, wherein the PSC is a human PSC, or optionally a human iPSC (human iPSC, hiPSC).

[0096] Embodiment 90: A PSC or three-dimensional PSC aggregate prepared by any one of Embodiments 1 to 25 or 88 to 89.

[0097] Embodiment 91: DE prepared by the method described in any one of Embodiments 26-42 and 88-89.

[0098] Embodiment 92: HGS prepared by any one of Embodiments 43-552 and 88-89.

[0099] Embodiment 93: An IO prepared by the method described in any one of Embodiments 56 to 89.

[0100] Embodiment 94: An IO having an inward polarity at its apical end, wherein the epithelial cells of the IO have a polarity in which their apical surface is oriented inward towards the IO, and optionally, the IO is a human IO (hIO).

[0101] Embodiment 95: An IO having an inward-facing polarity at its apex, manufactured by the method described in any one of Embodiments 67 to 89.

[0102] Embodiment 96: A spheroid prepared by the method of any one of Embodiments 84, 85, or ~89.

[0103] Embodiment 97: An organoid prepared by the method described in any one of Embodiments 86 to 89.

[0104] Embodiment 98: A treatment method comprising transplanting IO or cells derived therefrom as described in any one of Embodiments 93 to 95 into an animal, wherein the animal is optionally suffering from a GI disease condition, and optionally the animal is human.

[0105] Embodiment 99: A method for screening a compound for its activity, comprising contacting an IO or a cell derived therefrom as described in any one of Embodiments 93 to 95 with the compound, and measuring the response of the IO to the compound.

[0106] Embodiment 100: A method for screening a compound for its activity, comprising contacting an organoid or a cell derived therefrom as described in Embodiment 97 with the compound, and measuring the organoid's response to the compound.

[0107] Embodiment 101: The method according to any one of Embodiments 1 to 100, wherein the method does not include any heterogeneous material, and optionally the organoid is clinical grade and suitable for transplantation in humans.

[0108] Embodiment 102: The method according to any one of Embodiments 1 to 101, wherein the bioreactor of (a), (b), (d), (e), (f), (g), (h), and / or (i) comprises a rotating chamber containing liquid culture medium, second liquid culture medium, liquid endoderm differentiation medium, liquid hindgut differentiation medium, liquid IO maturation medium, liquid differentiation medium, and / or liquid organoid maturation medium, wherein the rotating chamber is a cylindrical section that rotates around its longitudinal axis, thereby suspending PSCs and / or three-dimensional PSC aggregates in the liquid culture medium, second liquid culture medium, liquid endoderm differentiation medium, liquid hindgut differentiation medium, liquid IO maturation medium, liquid differentiation medium, and / or liquid organoid maturation medium, and optionally the chamber is oriented so that its longitudinal axis is parallel to the ground.

[0109] Embodiment 103: The method according to any one of Embodiments 1 to 102, wherein the bioreactor of (a), (b), (d), (e), (f), (g), (h), and / or (i) comprises a rotating chamber containing liquid culture medium in a volume of about 5 mL to about 50 L, a second liquid culture medium, liquid endoderm differentiation culture medium, liquid hindgut differentiation culture medium, liquid IO maturation culture medium, liquid differentiation culture medium, and / or liquid organoid maturation culture medium.

[0110] Embodiment 104: The method according to any one of Embodiments 1 to 103, wherein the bioreactor of (a), (b), (d), (e), (f), (g), (h), and / or (i) comprises a rotating chamber containing liquid culture medium, a second liquid culture medium, liquid endoderm differentiation culture medium, liquid hindgut differentiation culture medium, liquid IO maturation culture medium, liquid differentiation culture medium, and / or liquid organoid maturation culture medium, wherein the rotation of the chamber is approximately 3 to 7 rpm, and optionally the rotation speed is a speed selected to maintain the PSCs, three-dimensional PSC aggregates, spheroids, and / or organoids in a stationary orbit.

[0111] Embodiment 105: The bioreactor of (a), (b), (d), (e), (f), (g), (h), and / or (i) comprises a rotating chamber containing liquid culture medium, a second liquid culture medium, liquid endoderm differentiation culture medium, liquid hindgut differentiation culture medium, liquid IO maturation culture medium, liquid differentiation culture medium, and / or liquid organoid maturation culture medium, wherein the mean shear stress on PSCs, three-dimensional PSC aggregates, spheroids, and / or organoids is approximately 5.0 dynes / cm 2 The method according to any one of embodiments 1 to 104, wherein the result is less than [amount missing].

[0112] Embodiment 106: The method according to any one of Embodiments 1 to 105, wherein the liquid culture medium, second liquid culture medium, liquid endoderm differentiation culture medium, liquid hindgut differentiation culture medium, liquid IO maturation culture medium, liquid differentiation culture medium, and / or liquid organoid maturation culture medium contains an anti-apoptotic agent.

[0113] Embodiment 107: The method according to any one of Embodiments 1 to 106, wherein the liquid culture medium, second liquid culture medium, liquid endoderm differentiation culture medium, liquid hindgut differentiation culture medium, liquid IO maturation culture medium, liquid differentiation culture medium, and / or liquid organoid maturation culture medium contain an anti-adhesion agent.

[0114] Embodiment 108: The method according to any one of Embodiments 1 to 107, wherein the liquid culture medium contains an anti-adhesion agent.

[0115] Embodiment 109: The method according to Embodiment 108, wherein the anti-adhesion agent is DSS, xanthan gum, A-205804, I-CAM1, carboxymethylcellulose, and / or Neural Organoid Basal Medium 2 (NOBM).

[0116] Embodiment 110: The method according to Embodiment 108 or 109, wherein the anti-adhesion agent is a DSS at a concentration of approximately 1 μg / mL to 1000 μg / mL relative to the liquid culture medium, second liquid culture medium, liquid endoderm differentiation culture medium, liquid hindgut differentiation culture medium, liquid IO maturation culture medium, liquid differentiation culture medium, and / or liquid organoid maturation culture medium.

[0117] Embodiment 111: A composition for three-dimensional expansion and maintenance of pluripotent stem cell (PSC) cultures, wherein the composition comprises a liquid culture medium containing recombinant human basic fibroblast growth factor (rh bFGF) and / or recombinant human transformation growth factor β (rh TGFβ), and PSCs suspended in the culture medium.

[0118] Embodiment 112: The composition according to Embodiment 111, wherein the liquid culture medium is a serum-free medium, the liquid culture medium does not contain any animal or human-derived material, and optionally the culture medium does not contain any extracellular matrix and / or basement membrane matrix.

[0119] Embodiment 113: The composition according to Embodiment 111 or 112, further comprising an anti-apoptotic agent.

[0120] Embodiment 114: The composition according to any one of Embodiments 111 to 113, wherein the PSC expresses Oct4, SSEA1, TRA 1-60, Sox 2, and / or TRA-1-81.

[0121] Embodiment 115: The composition according to any one of Embodiments 111 to 114, wherein the PSC expresses Oct4, SSEA1, TRA 1-60, Sox 2, and TRA-1-81.

[0122] Embodiment 116: The composition according to any one of Embodiments 111 to 115, further comprising an anti-adhesion agent.

[0123] Embodiment 117: The composition according to Embodiment 116, wherein the anti-adhesion agent is either or both DSS and xanthan gum.

[0124] Embodiment 118: The composition according to any one of Embodiments 111 to 117, wherein PSCs are suspended in a liquid culture medium at a density of approximately 50,000 to 1,000,000 PSCs / mL relative to the culture medium.

[0125] Embodiment 119: The composition according to any one of Embodiments 111 to 117, wherein PSCs are suspended in a liquid culture medium at a density of approximately 100,000 to 300,000 PSCs / mL relative to the culture medium.

[0126] Embodiment 120: The composition according to any one of Embodiments 111 to 119, wherein PSCs are suspended in the culture medium at a density of approximately 180,000 to 220,000 PSCs / mL relative to the culture medium.

[0127] Embodiment 121: A composition for differentiating PSCs into endoderm (DE) in three-dimensional suspension culture, wherein the composition comprises a liquid DE differentiation culture medium and PSCs suspended in the liquid DE differentiation culture medium.

[0128] Embodiment 122: The composition according to Embodiment 121, wherein the liquid DE differentiation culture medium is a serum-free medium, the liquid DE differentiation culture medium does not contain any animal or human-derived material, and optionally the liquid DE differentiation culture medium does not contain any extracellular matrix and / or basement membrane matrix.

[0129] Embodiment 123: The composition according to Embodiment 121 or 122, wherein the PSC has an average diameter of less than approximately 500 μm.

[0130] Embodiment 124: The composition according to any one of Embodiments 121 to 123, wherein the PSC has an average diameter of less than approximately 400 μm.

[0131] Embodiment 125: The composition according to any one of Embodiments 121 to 124, wherein the PSC has an average diameter of less than approximately 300 μm.

[0132] Embodiment 126: The composition according to any one of Embodiments 121 to 125, wherein the liquid DE differentiation culture medium contains nodal signaling pathway activator and / or Wnt signaling pathway activator at a concentration of about 10 to 200 ng / mL relative to the liquid DE differentiation culture medium.

[0133] Embodiment 127: The composition according to Embodiment 126, wherein the nodal signaling pathway activator or Wnt signaling pathway activator is present at a concentration of approximately 10 to 200 ng / mL relative to liquid DE differentiation culture medium.

[0134] Embodiment 128: The composition according to Embodiment 126 or 127, wherein the nodal signaling pathway activator or Wnt signaling pathway activator is present at a concentration of approximately 50 to 150 ng / mL ng / mL relative to liquid DE differentiation culture medium.

[0135] Embodiment 129: A composition according to any one of Embodiments 126 to 128, wherein the composition is a nodal signaling pathway activator or a Wnt signaling pathway activator at a concentration of approximately 100 to 200 ng / mL ng / mL relative to liquid DE differentiation culture medium.

[0136] Embodiment 130: The composition according to any one of Embodiments 126 to 129, wherein the liquid DE differentiation culture medium further comprises serum or a serum substitute at a concentration of about 0% to 20%.

[0137] Embodiment 131: The composition according to any one of Embodiments 126 to 130, wherein the liquid DE differentiation culture medium further comprises serum or a serum substitute at a concentration of about 2% to 5%.

[0138] Embodiment 132: The composition according to any one of Embodiments 111 to 131, further comprising DE differentiated from PSC.

[0139] Embodiment 133: The composition according to Embodiment 132, wherein the DE differentiated from the PSC expresses Sox17 and / or FoxA2.

[0140] Embodiment 134: The composition according to Embodiment 132 or 133, wherein the DE differentiated from the PSC expresses Sox17 and FoxA2.

[0141] 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 medium containing Wnt signaling pathway activator, FGF signaling pathway activator, and optionally FBS; and DE suspended in the liquid hindgut differentiation culture medium.

[0142] Embodiment 136: The composition according to Embodiment 135, wherein the liquid hindgut differentiation culture medium does not contain any animal or human-derived material, and optionally the culture medium does not contain any extracellular matrix and / or basement membrane matrix.

[0143] Embodiment 137: The composition according to Embodiment 135 or 136, wherein the Wnt signaling pathway activator comprises CHIR99021 and the FGF signaling pathway activator comprises FGF4.

[0144] Embodiment 138: The composition according to any one of Embodiments 135 to 137, wherein the FGF signaling pathway activator is present at a concentration of at least about 50 ng / mL relative to liquid hindgut differentiation culture medium.

[0145] Embodiment 139: The composition according to any one of Embodiments 135 to 138, wherein the concentration of FGF signaling pathway activator is at least about 500 ng / mL relative to liquid hindgut differentiation culture medium.

[0146] Embodiment 140: The composition according to any one of Embodiments 135 to 139, wherein the Wnt pathway activator is present at a concentration of at least about 0.5 μM relative to liquid hindgut differentiation culture medium.

[0147] Embodiment 141: A composition for differentiating HGS into intestinal organoids (IOs) in a three-dimensional suspension culture, wherein the composition comprises a liquid IO maturation culture medium containing EGF and HGS suspended in the liquid IO maturation culture medium.

[0148] Embodiment 142: The composition according to Embodiment 141, wherein the liquid IO mature culture medium does not contain any animal or human-derived material, and optionally, the liquid IO mature culture medium does not contain any extracellular matrix and / or basement membrane matrix.

[0149] Embodiment 143: The composition according to Embodiment 141 or 142, wherein the lumen of the HGS suspended in the liquid IO mature culture medium faces outward relative to the liquid IO mature culture medium.

[0150] Embodiment 144: The composition according to any one of Embodiments 141 to 143, wherein the concentration of EGF is at least about 25 ng / mL.

[0151] Embodiment 145: The composition according to any one of Embodiments 141 to 144, wherein the concentration of EGF is at least about 100 ng / mL.

[0152] Embodiment 146: The composition according to any one of Embodiments 141 to 145, wherein at least a portion of the HGS suspended in liquid IO mature culture medium comprises dissociated HGS single cells.

[0153] Embodiment 147: The composition according to Embodiment 146, wherein at least 80% of the HGS are dissociated HGS single cells, and optionally at least 90% of the HGS are dissociated single cells.

[0154] Embodiment 148: The composition according to Embodiment 146 or 147, wherein the concentration of dissociated HGS single cells in the liquid IO mature culture medium is in the range of approximately 0.1 × 10⁵ to 80 × 10⁵ dissociated HGS single cells / mL relative to the liquid IO mature culture medium.

[0155] Embodiment 149: The composition according to any one of Embodiments 146 to 148, wherein the concentration of dissociated HGS single cells in the liquid IO mature culture medium is in the range of approximately 20 × 10⁵ to 60 × 10⁵ dissociated HGS single cells / mL relative to the liquid IO mature culture medium.

[0156] Embodiment 150: The composition according to any one of Embodiments 141 to 149, further comprising IO differentiated from HGS.

[0157] Embodiment 151: The composition according to Embodiment 150, wherein the epithelial cells of the IO formed from the dissociated HGS single cell have polarity such that their apical surface is oriented inward towards the IO.

[0158] Embodiment 152: The composition according to any one of Embodiments 141 to 151, wherein HGS expresses CdX2.

[0159] Embodiment 153: The composition according to any one of Embodiments 141 to 152, wherein HGS expresses FOX-F1 but does not express SOX2.

[0160] Embodiment 154: The composition according to Embodiments 141 to 153, wherein the liquid IO mature culture medium further comprises noggin.

[0161] Embodiment 155: A composition comprising a liquid culture medium and three-dimensional PSC aggregates suspended in the liquid culture medium.

[0162] Embodiment 156: The composition according to Embodiment 155, wherein the liquid culture medium does not contain any animal or human-derived material, and optionally, the liquid culture medium does not contain any extracellular matrix and / or basement membrane matrix.

[0163] Embodiment 157: The composition according to Embodiment 155 or 156, wherein at least a portion of the three-dimensional PSC aggregate is dissociated as a single cell.

[0164] Embodiment 158: The composition according to any one of Embodiments 155 to 157, wherein the average diameter of the three-dimensional PSC aggregates is less than 400 μm.

[0165] Embodiment 159: The composition according to any one of Embodiments 155 to 158, wherein the average diameter of the three-dimensional PSC aggregates is less than 350 μm.

[0166] Embodiment 160: The composition according to any one of Embodiments 155 to 159, wherein the average diameter of the three-dimensional PSC aggregates is less than 300 μm.

[0167] Embodiment 161: The composition according to any one of Embodiments 155 to 160, further comprising an anti-adhesion agent.

[0168] Embodiment 162: The composition according to Embodiment 161, wherein the anti-adhesion agent is DSS, xanthan gum, A-205804, I-CAM1, carboxymethylcellulose, and / or Neural Organoid Basal Medium 2 (NOBM).

[0169] Embodiment 163: The composition according to Embodiment 161 or 162, wherein the anti-adhesion agent is concentrated at a concentration of approximately 1 μg / mL to 1000 μg / mL relative to the liquid culture medium. [Brief explanation of the drawing]

[0170] In addition to the features described herein, additional features and variations will readily become apparent from the following drawings and descriptions of exemplary embodiments. It should be understood that these drawings illustrate embodiments and are not intended to limit the scope. [Figure 1A] An embodiment of an experimental protocol for investigating the effects of various culture conditions on the maintenance and expansion of PSCs is illustrated. [Figure 1B] An illustrative embodiment of the basic principle of suspension culture operation is illustrated, in which the chamber rotates around its longitudinal axis when the longitudinal axis is oriented parallel to the ground. The illustrated rotating tank bioreactor (cell culture system) rotates continuously to maintain suspended cells, PSC aggregates, spheroids, and / or organoids by balancing gravity, thereby ideally maintaining them in a stationary orbit. As a result, cells grown in the rotating tank bioreactor are subjected to very low shear forces. [Figure 2] The results of one embodiment of a study investigating the effect of PSC inoculation density in suspension culture medium on PSC aggregate formation and cell death at various time points after culture inoculation are illustrated. [Figure 3] The results of one embodiment of a study comparing the ability of dissociation reagents to form single-cell cultures from PSC aggregates that form viable and homogeneous PSC aggregates 24 hours and 96 hours after single-cell inoculation are illustrated. [Figure 4A] Figure 4A illustrates the results of one embodiment of a study investigating how the growth stages of two-dimensional PSC cultures were used for inoculation of suspension cultures. Figure 4A illustrates one embodiment of the iPSC growth curve and two time points for taking iPSCs for inoculation of suspension cultures, where d0 refers to the day when the PSC strain would normally be passaged (80-90% confluence), and it should be noted that this day may differ for different PSC strains (usually in the range of 4-6 days after seeding). d-1 refers to the day before the PSC culture reached “ready for passage” confluence, when the monolayer is at 40-50% confluence. Figure 4B illustrates one embodiment of photographs of the starting two-dimensional PSC cultures at d-1 and d0, and the PSC aggregates resulting from the fourth day (d4) of the suspension culture inoculated with PSCs at d-1 or d0. Figure 4C illustrates one embodiment of the graph showing the size distribution of PSC aggregates at d4 in suspension cultures using either d-1 or d0 PSC inoculum. [Figure 4B]Figure 4A illustrates the results of one embodiment of a study investigating how the growth stages of two-dimensional PSC cultures were used for inoculation of suspension cultures. Figure 4A illustrates one embodiment of the iPSC growth curve and two time points for taking iPSCs for inoculation of suspension cultures, where d0 refers to the day when the PSC strain would normally be passaged (80-90% confluence), and it should be noted that this day may differ for different PSC strains (usually in the range of 4-6 days after seeding). d-1 refers to the day before the PSC culture reached “ready for passage” confluence, when the monolayer is at 40-50% confluence. Figure 4B illustrates one embodiment of photographs of the starting two-dimensional PSC cultures at d-1 and d0, and the PSC aggregates resulting from the fourth day (d4) of the suspension culture inoculated with PSCs at d-1 or d0. Figure 4C illustrates one embodiment of the graph showing the size distribution of PSC aggregates at d4 in suspension cultures using either d-1 or d0 PSC inoculum. [Figure 4C] Figure 4A illustrates the results of one embodiment of a study investigating how the growth stages of two-dimensional PSC cultures were used for inoculation of suspension cultures. Figure 4A illustrates one embodiment of the iPSC growth curve and two time points for taking iPSCs for inoculation of suspension cultures, where d0 refers to the day when the PSC strain would normally be passaged (80-90% confluence), and it should be noted that this day may differ for different PSC strains (usually in the range of 4-6 days after seeding). d-1 refers to the day before the PSC culture reached “ready for passage” confluence, when the monolayer is at 40-50% confluence. Figure 4B illustrates one embodiment of photographs of the starting two-dimensional PSC cultures at d-1 and d0, and the PSC aggregates resulting from the fourth day (d4) of the suspension culture inoculated with PSCs at d-1 or d0. Figure 4C illustrates one embodiment of the graph showing the size distribution of PSC aggregates at d4 in suspension cultures using either d-1 or d0 PSC inoculum. [Figure 5]The results of one embodiment of a study comparing how the size of PSC aggregates in suspension culture during subculturing affects the yield of PSC aggregates formed after reinoculation are illustrated. Subculturing PSC aggregates on day 3 (when the diameter of most aggregates is less than 400 μm) results in successful culture growth. Subculturing PSC aggregates on day 4 results in a much lower yield of aggregates when their diameter is greater than 400 μm. [Figure 6A] Figure 6A illustrates the results of one embodiment of a study comparing how bioreactor rotation speed affects the yield of PSC aggregates formed. Figure 6A illustrates one embodiment of PSC aggregates on day 3 (d3) of suspension culture at various bioreactor chamber rotation speeds. Figure 6B illustrates one embodiment of a graph showing the PSC aggregate size distribution at d3 for suspensions at various bioreactor chamber rotation speeds. Figure 6C illustrates one embodiment of a chart comparing the production of PSC cells in the first (P1), second (P2), and third (P3) passages of suspension cultures at various bioreactor chamber rotation speeds. [Figure 6B] Figure 6A illustrates the results of one embodiment of a study comparing how bioreactor rotation speed affects the yield of PSC aggregates formed. Figure 6A illustrates one embodiment of PSC aggregates on day 3 (d3) of suspension culture at various bioreactor chamber rotation speeds. Figure 6B illustrates one embodiment of a graph showing the PSC aggregate size distribution at d3 for suspensions at various bioreactor chamber rotation speeds. Figure 6C illustrates one embodiment of a chart comparing the production of PSC cells in the first (P1), second (P2), and third (P3) passages of suspension cultures at various bioreactor chamber rotation speeds. [Figure 6C]Figure 6A illustrates the results of one embodiment of a study comparing how bioreactor rotation speed affects the yield of PSC aggregates formed. Figure 6A illustrates one embodiment of PSC aggregates on day 3 (d3) of suspension culture at various bioreactor chamber rotation speeds. Figure 6B illustrates one embodiment of a graph showing the PSC aggregate size distribution at d3 for suspensions at various bioreactor chamber rotation speeds. Figure 6C illustrates one embodiment of a chart comparing the production of PSC cells in the first (P1), second (P2), and third (P3) passages of suspension cultures at various bioreactor chamber rotation speeds. [Figure 7] The results of one embodiment based on a study comparing the use of anti-apoptotic agents are illustrated. As shown in Figure 7, the use of anti-apoptotic agents increases cell (e.g., PSC) recovery in each passage. Specifically, the anti-apoptotic agent CEPT was found to provide higher cell recovery than the anti-apoptotic agent ROCKi. [Figure 8] This figure illustrates the results of one embodiment of a study comparing how the culture media mTeSR 1 (research medium) and mTeSR AOF (animal product-free medium) affect PSC expansion and maintenance in the first (P1), second (P2), and third (P3) passages of suspension culture. [Figure 9A] The results of one embodiment of a study comparing how culture media mTeSR 1 (research medium, Figure 9A) and mTeSR AOF (animal product-free medium, Figure 9B) affect stem cell markers: Oct4, SSEA4, and TRA 1-60 are illustrated. [Figure 9B] The results of one embodiment of a study comparing how culture media mTeSR 1 (research medium, Figure 9A) and mTeSR AOF (animal product-free medium, Figure 9B) affect stem cell markers: Oct4, SSEA4, and TRA 1-60 are illustrated. [Figure 10] This figure illustrates the results of one embodiment of a study comparing how cell lines (research grade, PSC cell line 72.3, and clinical-grade PSC cell line FF3 produced under GMP conditions) affect the production of PSC aggregates in the first (P1), second (P2), and third (P3) passages of suspension culture. [Figure 11A] This figure illustrates the results of one embodiment of a study comparing the effects of two-dimensional (2D) culture (Figure 11A) versus suspension culture (3D) (Figure 11B) on the expression of stem cell markers Oct4, SSEA4, and TRA 1-60 in research-grade PSC cell line 72.3. [Figure 11B] This figure illustrates the results of one embodiment of a study comparing the effects of two-dimensional (2D) culture (Figure 11A) versus suspension culture (3D) (Figure 11B) on the expression of stem cell markers Oct4, SSEA4, and TRA 1-60 in research-grade PSC cell line 72.3. [Figure 12A] This figure illustrates the results of one embodiment of a study comparing the effects of two-dimensional (2D) culture (Figure 12A) versus suspension culture (3D) (Figure 12B) on the expression of stem cell markers Oct4, SSEA4, and TRA 1-60 in the clinical-grade PSC cell line FF3 produced under GMP conditions. [Figure 12B] This figure illustrates the results of one embodiment of a study comparing the effects of two-dimensional (2D) culture (Figure 12A) versus suspension culture (3D) (Figure 12B) on the expression of stem cell markers Oct4, SSEA4, and TRA 1-60 in the clinical-grade PSC cell line FF3 produced under GMP conditions. [Figure 13A] The results of one embodiment based on a study investigating the formation of three-dimensional PSC aggregates from PSCs are illustrated, demonstrating that the three-dimensional PSC aggregates can retain their pluripotency. As shown in the image in Figure 13A, PSCs can form three-dimensional PSC aggregates progressing from day 1 to day 4. As shown in Figure 13B, the number of such aggregates increases from 600 on day 1 to approximately 1400 on day 4. In addition, pluripotency markers such as OCT4 and SSEA4 are evident in the three-dimensional PSC aggregates in confocal imaging. [Figure 13B]The results of one embodiment based on a study investigating the formation of three-dimensional PSC aggregates from PSCs are illustrated, demonstrating that the three-dimensional PSC aggregates can retain their pluripotency. As shown in the image in Figure 13A, PSCs can form three-dimensional PSC aggregates progressing from day 1 to day 4. As shown in Figure 13B, the number of such aggregates increases from 600 on day 1 to approximately 1400 on day 4. In addition, pluripotency markers such as OCT4 and SSEA4 are evident in the three-dimensional PSC aggregates in confocal imaging. [Figure 14A] The results of evaluating pluripotent 3D PSC aggregates across multiple strains and passages are illustrated. As shown in Figure 14A, significant formation of three-dimensional PSC aggregates was observed across passages in iPSC strain 72.3 and ESC strain H1. Figure 14B shows the increase in cell number and PSC aggregate diameter across passages for both strains. Furthermore, Figure 14B shows that the expression of pluripotency markers (e.g., OCT4 and SSEA-4) remained at least 90% across all passages and strains tested on the three-dimensional PSC aggregates, similar to the expression in conventionally grown two-dimensional PSC monolayers. [Figure 14B] The results of evaluating pluripotent 3D PSC aggregates across multiple strains and passages are illustrated. As shown in Figure 14A, significant formation of three-dimensional PSC aggregates was observed across passages in iPSC strain 72.3 and ESC strain H1. Figure 14B shows the increase in cell number and PSC aggregate diameter across passages for both strains. Furthermore, Figure 14B shows that the expression of pluripotency markers (e.g., OCT4 and SSEA-4) remained at least 90% across all passages and strains tested on the three-dimensional PSC aggregates, similar to the expression in conventionally grown two-dimensional PSC monolayers. [Figure 15]The results of evaluating the pluripotency of PSC strains grown in three-dimensional suspension culture according to the method described herein, compared to PSC strains grown in conventional 2D monolayers, are shown in Figure 15. As shown in Figure 15, the pluripotency of PSC strains (H1 and 72.3) grown in three-dimensional suspension culture was increased compared to those grown in 2D monolayers, based on higher gene expression of the pluripotency markers OCT4, SOX2, and KLF4. [Figure 16] One embodiment of an experimental protocol for matrix-free suspension culture production of HIO from hiPSCs is illustrated. [Figure 17] The results of one embodiment of a study investigating the effect of PSC acclimatization to suspension culture on HIO production at various time points are illustrated. [Figure 18A] This figure illustrates the results of one embodiment of a study comparing the efficiency of DE induction in two-dimensional (2D) culture (Figure 18A) and suspension culture (3D) (Figure 18B) by examining the expression of endoderm markers Sox 17 and FoxA2. [Figure 18B] This figure illustrates the results of one embodiment of a study comparing the efficiency of DE induction in two-dimensional (2D) culture (Figure 18A) and suspension culture (3D) (Figure 18B) by examining the expression of endoderm markers Sox 17 and FoxA2. [Figure 19] The results of one embodiment demonstrating the effect of exposure to activin A at different time points (e.g., 24 hours, 48 ​​hours, or 72 hours after passage) on DE induction efficiency in 3D suspension culture are illustrated. As shown in Figure 19, 3D PSC aggregates exposed to activin A at 48 hours after passage showed the highest DE differentiation efficiency based on the expression of DE markers FoxA2 and Sox17. [Figure 20]The results of one embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the DE stage are illustrated. As shown in Figure 20, the size of 3D PSC aggregates upon exposure to activin determines the DE induction efficiency. Furthermore, Figure 20 shows that smaller 3D PSC aggregate sizes (e.g., less than 500 μm (e.g., less than approximately 400 μm (e.g., less than approximately 350 μm (e.g., less than approximately 300 μm)))) ensure better DE induction. [Figure 21] The results of one embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the HGS stage are illustrated. The effect was demonstrated by the expression of CDX2, a marker of intestinal tissue differentiation. As shown in Figure 21, CDX2 is strongly expressed in the hindgut stage of differentiation of 3D PSC aggregates with a diameter smaller than 300 μm at DE induction. However, CDX2 expression is weaker and more sparse for 3D PSC aggregates with a diameter greater than 300 μm at DE induction. The results further confirm the importance of 3D PSC aggregate size to differentiation efficiency. [Figure 22] The results of one embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the HIO stage are illustrated. The effect was demonstrated by the expression of CDX2, a marker for intestinal tissue differentiation. As shown in Figure 22, differentiation of 3D PSC aggregates with a diameter of 300 μm or less resulted in apical-out HIO formation and substantially uniform CDX2 expression across the generated HIO. However, differentiation of 3D PSC aggregates with a diameter of at least 300 μm resulted in mixed apical-out and apical-out structures, as well as epithelial structures with weak or no CDX2 expression. [Figure 23]The results of one embodiment based on a study investigating the effect of the anti-adhesion agent dextran sulfate sodium (DSS) on the size-mediating properties of 3D PSC aggregates are illustrated. As previously discussed, 3D PSC aggregates with smaller sizes (e.g., diameters less than approximately 500 μm (e.g., less than approximately 400 μm (e.g., less than approximately 300 μm)) are preferred for intestinal tissue differentiation. The image in Figure 23 shows the effect of various concentrations of DSS on the size of 3D PSC aggregates, with a concentration of 10 μg / mL having the greatest effect in reducing the size of 3D PSC aggregates. Figure 24 further shows that the effect of DSS on reducing aggregate size was observed across different PSC strains (72.3, FF3, H1, and H1 GFP). Figure 25 further shows that a DSS concentration of 10 μg / mL had the greatest reduction in average diameter across the line, while simultaneously increasing the yield of aggregates formed. Figures 26 and 27 further investigate the effect of 10 μg / mL DSS on the average size of PSC aggregates across different strains. Specifically, Figure 26 shows that 10 μg / mL DSS is sufficient to induce smaller diameter PSC aggregates, resulting in an average reduction of approximately 200 μm in the average diameter of PSC aggregates after DSS treatment compared to the untreated control. Figure 27 also shows that this effect is consistent across different iPSC and ESC strains, resulting in a shift in the frequency of the PSC aggregate size distribution. [Figure 24]The results of one embodiment based on a study investigating the effect of the anti-adhesion agent dextran sulfate sodium (DSS) on the size-mediating properties of 3D PSC aggregates are illustrated. As previously discussed, 3D PSC aggregates with smaller sizes (e.g., diameters less than approximately 500 μm (e.g., less than approximately 400 μm (e.g., less than approximately 300 μm)) are preferred for intestinal tissue differentiation. The image in Figure 23 shows the effect of various concentrations of DSS on the size of 3D PSC aggregates, with a concentration of 10 μg / mL having the greatest effect in reducing the size of 3D PSC aggregates. Figure 24 further shows that the effect of DSS on reducing aggregate size was observed across different PSC strains (72.3, FF3, H1, and H1 GFP). Figure 25 further shows that a DSS concentration of 10 μg / mL had the greatest reduction in average diameter across the line, while simultaneously increasing the yield of aggregates formed. Figures 26 and 27 further investigate the effect of 10 μg / mL DSS on the average size of PSC aggregates across different strains. Specifically, Figure 26 shows that 10 μg / mL DSS is sufficient to induce smaller diameter PSC aggregates, resulting in an average reduction of approximately 200 μm in the average diameter of PSC aggregates after DSS treatment compared to the untreated control. Figure 27 also shows that this effect is consistent across different iPSC and ESC strains, resulting in a shift in the frequency of the PSC aggregate size distribution. [Figure 25]The results of one embodiment based on a study investigating the effect of the anti-adhesion agent dextran sulfate sodium (DSS) on the size-mediating properties of 3D PSC aggregates are illustrated. As previously discussed, 3D PSC aggregates with smaller sizes (e.g., diameters less than approximately 500 μm (e.g., less than approximately 400 μm (e.g., less than approximately 300 μm)) are preferred for intestinal tissue differentiation. The image in Figure 23 shows the effect of various concentrations of DSS on the size of 3D PSC aggregates, with a concentration of 10 μg / mL having the greatest effect in reducing the size of 3D PSC aggregates. Figure 24 further shows that the effect of DSS on reducing aggregate size was observed across different PSC strains (72.3, FF3, H1, and H1 GFP). Figure 25 further shows that a DSS concentration of 10 μg / mL had the greatest reduction in average diameter across the line, while simultaneously increasing the yield of aggregates formed. Figures 26 and 27 further investigate the effect of 10 μg / mL DSS on the average size of PSC aggregates across different strains. Specifically, Figure 26 shows that 10 μg / mL DSS is sufficient to induce smaller diameter PSC aggregates, resulting in an average reduction of approximately 200 μm in the average diameter of PSC aggregates after DSS treatment compared to the untreated control. Figure 27 also shows that this effect is consistent across different iPSC and ESC strains, resulting in a shift in the frequency of the PSC aggregate size distribution. [Figure 26]The results of one embodiment based on a study investigating the effect of the anti-adhesion agent dextran sulfate sodium (DSS) on the size-mediating properties of 3D PSC aggregates are illustrated. As previously discussed, 3D PSC aggregates with smaller sizes (e.g., diameters less than approximately 500 μm (e.g., less than approximately 400 μm (e.g., less than approximately 300 μm)) are preferred for intestinal tissue differentiation. The image in Figure 23 shows the effect of various concentrations of DSS on the size of 3D PSC aggregates, with a concentration of 10 μg / mL having the greatest effect in reducing the size of 3D PSC aggregates. Figure 24 further shows that the effect of DSS on reducing aggregate size was observed across different PSC strains (72.3, FF3, H1, and H1 GFP). Figure 25 further shows that a DSS concentration of 10 μg / mL had the greatest reduction in average diameter across the line, while simultaneously increasing the yield of aggregates formed. Figures 26 and 27 further investigate the effect of 10 μg / mL DSS on the average size of PSC aggregates across different strains. Specifically, Figure 26 shows that 10 μg / mL DSS is sufficient to induce smaller diameter PSC aggregates, resulting in an average reduction of approximately 200 μm in the average diameter of PSC aggregates after DSS treatment compared to the untreated control. Figure 27 also shows that this effect is consistent across different iPSC and ESC strains, resulting in a shift in the frequency of the PSC aggregate size distribution. [Figure 27]The results of one embodiment based on a study investigating the effect of the anti-adhesion agent dextran sulfate sodium (DSS) on the size-mediating properties of 3D PSC aggregates are illustrated. As previously discussed, 3D PSC aggregates with smaller sizes (e.g., diameters less than approximately 500 μm (e.g., less than approximately 400 μm (e.g., less than approximately 300 μm)) are preferred for intestinal tissue differentiation. The image in Figure 23 shows the effect of various concentrations of DSS on the size of 3D PSC aggregates, with a concentration of 10 μg / mL having the greatest effect in reducing the size of 3D PSC aggregates. Figure 24 further shows that the effect of DSS on reducing aggregate size was observed across different PSC strains (72.3, FF3, H1, and H1 GFP). Figure 25 further shows that a DSS concentration of 10 μg / mL had the greatest reduction in average diameter across the line, while simultaneously increasing the yield of aggregates formed. Figures 26 and 27 further investigate the effect of 10 μg / mL DSS on the average size of PSC aggregates across different strains. Specifically, Figure 26 shows that 10 μg / mL DSS is sufficient to induce smaller diameter PSC aggregates, resulting in an average reduction of approximately 200 μm in the average diameter of PSC aggregates after DSS treatment compared to the untreated control. Figure 27 also shows that this effect is consistent across different iPSC and ESC strains, resulting in a shift in the frequency of the PSC aggregate size distribution. [Figure 28] The results of one embodiment based on a study investigating the effect of DSS on the pluripotency of 3D PSC aggregates, as measured by the pluripotency markers SOX2 and OCT4, are illustrated. As shown in the confocal image, DSS had no negative effect on the pluripotency of any of the concentrations tested for 3D PSC aggregates. The study also investigated the effect of DSS on the viability of 3D PSC aggregates, as measured by the release of the survival marker lactate dehydrogenase (LDH). The study found that DSS at concentrations of 1000 μg / mL or less had no negative effect on the viability of PSC aggregates. [Figure 29]The results of one embodiment based on a study investigating the effects of various treatment regimens for applying DSS on the average size, number, and pluripotency of 3D PSC aggregates are illustrated. As shown in Figure 29, the treatment regimens tested included control (i.e., no treatment regimen), at inoculation, and overall. The results show that treatment with 10 ug / mL of DSS at inoculation is sufficient to maintain PSC aggregates below 400 μm while having no negative effect on PSC aggregate number or pluripotency gene expression. However, long-term treatment with DSS (overall) results in a decrease in OCT4 expression. [Figure 30] The results of one embodiment based on a study investigating the effect of DSS on the average size of 3D PSC aggregates over multiple passages are illustrated. As shown in Figure 30, the effect of DSS treatment on PSC aggregate size (reduction in diameter) was maintained over multiple passages. [Figure 31] The results of one embodiment based on a study investigating the effect of DSS on the differentiation tendency of 3D PSC aggregates are illustrated. The tendency is measured based on differentiation efficiency characterized by the expression of markers FOXA2 and SOX17. As shown in Figure 31, similar differentiation efficiency of 3D PSC aggregates toward differentiation into DEs is found in the presence and absence of DSS, as indicated by the expression of Sox17 and FoxA2, ​​demonstrating that DSS treatment has no negative effect on the cell tendency toward differentiation. [Figure 32] The results of one embodiment of an experiment demonstrating well-patterned HIO development in suspension cultures with either outward-facing or inward-facing epithelial cell polarity are illustrated. [Figure 33] The results of one embodiment based on well-patterned HIO development in 3D suspension culture are illustrated. The success of precisely patterned HIO development in suspension culture is confirmed by immunofluorescence staining. The markers CDX2, ZO-1, and Vim1 represent differentiation into HIO. [Figure 34A]The results of one embodiment of an experiment demonstrating in vivo maturation of HIO generated in suspension culture after transplantation under the kidney capsule of mice are illustrated. Figure 34A is a photograph of HIO 9 weeks after transplantation under the kidney capsule, one embodiment of the process. Figure 34B is a H&E staining example of HIO 9 weeks after transplantation under the kidney capsule, one embodiment of the process of the process. [Figure 34B] The results of one embodiment of an experiment demonstrating in vivo maturation of HIO generated in suspension culture after transplantation under the kidney capsule of mice are illustrated. Figure 34A is a photograph of HIO 9 weeks after transplantation under the kidney capsule, one embodiment of the process. Figure 34B is a H&E staining example of HIO 9 weeks after transplantation under the kidney capsule, one embodiment of the process of the process. [Figure 35] The results of one embodiment of an experiment demonstrating that the polarity of epithelial cells in HIO generated in suspension culture can be altered are illustrated. Dissociation of hindgut spheroids on day 7 (+dissociation) and their re-aggregation in suspension culture result in an inner apical surface (apex facing inward) in the HIO. If the DE has not dissociated on day 7 (-dissociation), the result is an outer apical surface (apex facing outward) in the HIO. [Modes for carrying out the invention]

[0171] The aspects of this disclosure generally relate to suspension culture methods for pluripotent stem cells (PSCs), differentiated cells derived from PSCs, spheroids, and organoids, and compositions thereof. In some embodiments, these methods can be carried out and / or facilitated with industrial efficiency and scalability. These methods can be carried out without the use of a basement membrane matrix (e.g., heterologous basement membrane matrix) during the maintenance and expansion of PSCs, and during the differentiation of PSCs into differentiated cells and organoids, such as endoderm (DE), hindgut spheroids (HGS), and intestinal organoids (IO). In some embodiments, the methods can be xeno-free and can be carried out in accordance with Good Manufacturing Practice (GMP). In some embodiments, the disclosed methods utilize suspension culture for each of the following stages in the production of organoids from PSCs: maintenance and expansion of PSCs, differentiation of PSCs into DEs, differentiation of DEs into spheroids (e.g., hindgut spheroids), and differentiation and maturation of spheroids into organoids (e.g., intestinal organoids). Methods and compositions for transplantation and treatment are also disclosed.

[0172] In some embodiments of the methods disclosed herein, one or more of the following aspects of current culture protocols are improved: improving the transition from two-dimensional (e.g., monolayer) culture conditions to three-dimensional (e.g., Matrigel-embedded) culture conditions to eliminate or reduce potentially significant obstacles; eliminating the need for the use of an 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 composition and concentration, and advancing the transition to xeno-free production of human organoids; reducing labor by eliminating manual plating in the Matrigel dome as seen in some current organoid culture methods; eliminating manual handling around day 14 of some current culture methods; enabling large-scale production of organoids; and reducing the resulting variability of cells, spheroids, and / or organoids.

[0173] One challenge in using organoids is the difficulty in accessing the apical or luminal surface of the epithelium typically encapsulated within the organoid. Many applications require access to the apical or luminal surface of the organoid because this is the mucosal surface that typically interacts with the external environment and therefore absorbs nutrients, interacts with GI microorganisms, and takes up drugs or toxins. The apical surface also secretes mucins, antimicrobial peptides, and enzymes that regulate the interaction between the intestinal lumen contents and the epithelium. Embodiments disclosed herein relate to a suspension culture method that enables complete control of human organoid epithelial polarity and the production of organoids with the apex facing outward.

[0174] Expansion and maintenance of PSCs in suspension culture Aspects of this disclosure relate to methods for three-dimensional expansion and maintenance of pluripotent stem cells (PSCs) in suspension culture. In some embodiments, the PSCs are artificial PSCs (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the PSCs are human (hPSCs).

[0175] In some embodiments, the PSCs are human iPSCs (hiPSCs). In some embodiments, the method includes (a) inoculating a liquid culture medium with PSCs (optionally hPSCs or hiPSCs); (b) culturing the PSC-inoculated liquid culture medium in a bioreactor such that three-dimensional PSC aggregates are formed in the liquid culture medium, wherein the culturing in the bioreactor includes suspending the PSCs in the liquid culture medium; and (c) (i) dissociating at least a portion of the three-dimensional PSC aggregates into single cells, and subculturing the PSCs by inoculating a second liquid culture medium with the dissociated three-dimensional PSC aggregates from i).

[0176] In some embodiments, the diameter of most of the formed PSC aggregates (e.g., three-dimensional PSC aggregates) is 500, 450, 400, 350, 300, 250 μm, or less, or within a range defined by any two of the aforementioned values, and optionally, the diameter of most of the PSC aggregates is 500 μm or less, 400 μm or less, 350 μm or less, or 300 μm or less.

[0177] In some embodiments, the suspension involves rotating a chamber containing the culture medium, causing the culture medium to rotate around an axis parallel to the ground.

[0178] In some embodiments, at least 80, 85, 90, 95, 98, or 99% of the PSC aggregates dissociate into single cells in (c)(i), and optionally, at least 90% of the PSC aggregates dissociate into single cells in (c)(i).

[0179] In some embodiments, PSCs can be inoculated into the culture medium in the bioreactor to form a density of 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, or at least these, or less, or within the range defined by any two of the aforementioned values. In some embodiments, the density is 50,000 to 1,000,000, 50,000 to 500,000, 100,000 to 300,000, 180,000 to 220,000, or 200,000 PSCs / mL relative to the culture medium. In some embodiments, the density is 180,000 to 220,000 or 200,000 PSCs / mL relative to the culture medium.

[0180] In some embodiments, a second liquid culture medium containing the dissociated three-dimensional PSC aggregates of (i) may be inoculated with PSCs to form a density of 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 relative to the culture medium in the bioreactor, or at least these, or less, or within the range defined by any two of the aforementioned values. In some embodiments, the density is 50,000 to 1,000,000, 50,000 to 500,000, 100,000 to 300,000, 100,000 to 200,000, 180,000 to 220,000, or 200,000 PSCs / mL relative to the culture medium. In some embodiments, the density is 180,000 to 220,000 or 200,000 PSCs / mL relative to the culture medium.

[0181] In some embodiments, the method comprises passage the PSCs two or more times by culturing the PSCs in the culture medium inoculated in (c)ii) until three-dimensional PSC aggregates are formed, and repeating (c). In some embodiments, the culture is passaged two, three, four times, or more times. In some embodiments, the PSCs maintain the expression of stem cell markers, e.g., Oct4, SSEA4, and / or TRA 1-60 (e.g., Oct4, SSEA4, and TRA 1-60). In some embodiments, after at least two, three, four times, or more passages, at least 85, 90, or 95% of the cells maintain their state as PSCs, as evidenced by the expression of stem cell markers, e.g., Oct4, SSEA4, and / or TRA 1-60 (e.g., Oct4, SSEA4, and TRA 1-60). In some embodiments, in (a) (initial inoculation of suspension culture) and / or (c)(ii) (re-inoculation of suspension culture during passage), the culture medium is inoculated at a density of approximately 100,000–220,000, approximately 180,000–220,000, or approximately 200,000 PSCs / mL relative to the culture medium. In some embodiments, passage occurs after a period of approximately 40, 48, 54, 60, 66, 72, 78, 84, 90, 96, or 168 hours after inoculation in (a) and / or (c)(ii), or at least these, or less, or within a range defined by any two of the aforementioned values, and optionally, the period is approximately 40–54, 40–84, 60–84, 66–78, or 72 hours. In some embodiments, the PSC is passaged one or more times (e.g., three times), and after one or more passages (e.g., after the third passage), the portion of the PSC expressing Oct4, SSEA1, and / or TRA 1-60 (e.g., Oct4, SSEA4, and TRA 1-60) at a level at least as high as the mean expression level of the PSC used in the initial inoculation of the suspension culture in (a) is 85, 90, 93, 95, 97, 98, or 99%, or at least one of these, or less, or within the range defined by any two of the aforementioned values.In some embodiments, the portion of the PSC expressing Oct4, SSEA1, and / or TRA 1-60 (e.g., Oct4, SSEA4, and TRA 1-60) at a level at least as high as the mean expression level of the PSC used in the inoculation in (a) is at least 95%. In some embodiments, the PSC is passaged one or more times. In some embodiments, the PSC is passaged at least three times. After one or more passages (e.g., after the third passage), the portion of the PSC expressing Oct4, SSEA1, and / or TRA 1-60 (e.g., Oct4, SSEA4, and TRA 1-60) at a level at least as high as the mean expression level of the PSC used in the initial inoculation of the suspension culture in (a) is 85, 90, 93, 95, 97, 98, or 99%, or at least one of these, or less, or within the range defined by any two of the aforementioned values. In some embodiments, at least 95% of the PSCs express Oct4, SSEA1, and / or TRA 1-60 (e.g., Oct4, SSEA4, and TRA 1-60) at a level at least as high as the mean expression level of the PSCs used in the inoculation in (a). In some embodiments, the PSCs are passaged one or more times (e.g., at least three times) and the PSCs express SOX2 and / or KLF4 (e.g., SOX2 and KLF4).

[0182] In some embodiments, the method further includes replacing a portion of the culture medium in the bioreactor after a certain period following inoculation in (a) and / or (c)(ii). In some embodiments, the period is 36, 42, 48, 54, or 60 hours, or at least these, or less than or equal to these, or a range defined by any two of the aforementioned values, and optionally, the period is 42–54 or 48 hours. In some embodiments, the portion of the culture medium to be replaced is 50, 60, 70, 80, 90, or 100% of the culture medium in the bioreactor, or at least these, or a range defined by any two of the aforementioned values, and optionally, the portion is at least 80% or 90% of the culture medium in the bioreactor.

[0183] In some embodiments, PSCs used for inoculation into suspension culture are first cultured on the surface of a substrate, for example, on the surface of a culture flask (adhesion culture), also referred to herein as 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 substrate surface. In some embodiments, the method further includes culturing PSCs on the surface of a substrate before inoculation of the suspension culture with PSCs in (a), and collecting the PSCs from the surface of the substrate for use in inoculation of the suspension culture in (a) when the PSCs are in the logarithmic growth phase and / or have a confluence of 35-55% or 40-50%. In some embodiments, collection includes dissociating the PSCs before inoculation of the suspension culture in (a). Methods for dissociating cells in adhesion culture may include, but are not limited to, chemical, enzymatic, and / or mechanical dissociation. In some embodiments, dissociation is chemical, for example, through the use of EDTA. In some embodiments, dissociation is enzymatic. In some embodiments, the enzyme used for dissociation includes proteolytic enzymes and / or collagen-degrading enzymes, such as Accutase.

[0184] Devices for cell suspension culture may include bioreactors. The use of bioreactors for suspension culture (spinning flasks and vertical wheel bioreactors) can result in the generation of variable levels of shear stress (e.g., shear stress can range from 0.1 to 10 dynes / cm² in a spinning flask, depending on the rotation speed, size and shape of the vessel, and the volume of culture medium). 2 (Variable). PSCs, such as human PSCs, are sensitive to high shear stress, which can cause unexpected cell death and differentiation. In some embodiments, bioreactors with reduced and / or low shear stress, or bioreactors without shear stress, are used, for example, compared to conventional suspension culture bioreactors (spinning flasks and vertical wheel bioreactors). In some embodiments, bioreactors with reduced and / or low shear stress, and / or without shear stress, function by continuously rotating, with upward movement from rotation being offset by gravity, thereby suspending cells in the culture medium. In some embodiments, the bioreactor includes a rotating chamber containing the suspension culture medium. In some embodiments, the chamber is a tank having the shape of a cylindrical section (e.g., shaped like a Petri dish, and optionally, the depth of the dish is greater than that of a typical Petri dish). In some embodiments, the chamber rotates around its longitudinal axis, thereby rotating the liquid culture medium contained within the chamber, thereby suspending cells and aggregates in the liquid culture medium. In some embodiments, the chamber is oriented such that its longitudinal axis is parallel to the ground, so that the cells within the chamber (e.g., individual cells, aggregates, spheroids, organoids, etc.) rise on one side of the chamber due to the rotation of the chamber and the liquid culture medium, and fall on the opposite side due to gravity.

[0185] Figure 1B illustrates one embodiment illustrating the operation of suspension culture, in which the chamber rotates around its longitudinal axis (not shown, but perpendicular to the plane of the figure and passing through the center of the chamber / tank) when the longitudinal axis is oriented parallel to the ground. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the volume of culture medium in the chamber being 5, 10, 20, 30, 40, 50 mL, or at least these, or less, or a range defined by any two of the aforementioned values. In some embodiments, the volume of culture medium in the chamber is 5 to 10 mL, optionally 10 mL. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the rotation of the chamber being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or at least these, or less, or a range defined by any two of the aforementioned values. In some embodiments, the rotation speed is 40 rpm or less. In some embodiments, the rotation speed is a speed selected to maintain the suspended cells and / or aggregates in a stationary orbit. In some embodiments, the rotation speed is 3 to 7 rpm. In some embodiments, the rotation speed of the chamber is selected such that the number of PSCs in culture at subculturing in (c) is at least 2 or 2.5 times the number of PSCs used to inoculate the culture medium. In some embodiments, the rotation speed of the chamber is selected such that the number of PSCs in culture at subculturing in (c) is at least 2 or 2.5 times the number of PSCs used to inoculate the culture medium between at least 2 or at least 3 subculturings of the PSCs. In some embodiments, the bioreactor has an average shear stress on cells and aggregates in the culture medium while the culture medium is moving (e.g., the chamber is rotating) of 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, or 0.005 dynes / cm². 2 It is configured to be less than 0.1 dynes / cm². In some embodiments, the mean shear stress on cells and aggregates in the culture medium is 0.1 dynes / cm². 2 It is less than.

[0186] The culture medium may be used for the expansion and maintenance of PSCs. In some embodiments, the culture medium is serum-free. In some embodiments, the culture medium contains recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transformation growth factor β (rh TGFβ). In some embodiments, the culture medium does not contain any animal or human-derived material. In some embodiments, the culture medium does not contain any extracellular matrix and / or basement membrane matrix (e.g., Matrigel or similar products). In some embodiments, the absence of extracellular matrix and / or basement membrane matrix allows for three-dimensional suspension of the culture. In some embodiments, any of the aforementioned culture media may be liquid culture media.

[0187] Aspects of this disclosure relate to PSCs produced by the suspension culture methods described above and elsewhere in this specification.

[0188] Aspects of this disclosure include compositions for the expansion and maintenance of pluripotent stem cell (PSC) cultures. In various embodiments, the composition comprises a liquid culture medium containing recombinant human basic fibroblast growth factor (rh bFGF) and / or recombinant human transformation growth factor β (rh TGFβ), and PSCs suspended in the liquid culture medium. In some embodiments, the liquid culture medium is serum-free, free from animal or human-derived materials, and optionally, free from any extracellular matrix and / or basement membrane matrix.

[0189] In some embodiments, the composition further comprises an anti-apoptotic agent (e.g., a ROCK inhibitor (ROCKi) and / or CEPT).

[0190] In some embodiments, the PSC expresses Oct4, SSEA1, and / or TRA 1-60. In at least one embodiment, the PSC expresses Oct4, SSEA1, and TRA 1-60. In some embodiments, the PSC further expresses Oct4, SSEA1, TRA 1-60, Sox2, and / or TRA-1-81.

[0191] In some embodiments, the composition further comprises an anti-adhesion agent. For example, the anti-adhesion agent may be DSS or xanthan gum, or both. In some embodiments, the PSCs are suspended in a liquid culture medium at a density of about 50,000 to 1,000,000 PSCs / mL relative to the culture medium. In some embodiments, the PSCs are suspended in a liquid culture medium at a density of about 100,000 to 300,000 PSCs / mL relative to the culture medium. In some embodiments, the PSCs are suspended in the culture medium at a density of about 100,000 to 220,000 PSCs / mL relative to the culture medium. In some embodiments, the PSCs are suspended in the culture medium at a density of about 180,000 to 220,000 PSCs / mL relative to the medium.

[0192] Differentiation of PSC suspension cultures into endoderm (DE) Aspects of the present disclosure include methods for differentiating PSCs into endoderm (DE) in a three-dimensional suspension culture. In some embodiments, the PSCs are artificial PSCs (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the PSCs are human (hPSCs). In some embodiments, the PSCs are human iPSCs (hiPSCs). In some embodiments, the method includes (d) culturing a liquid culture medium inoculated with PSCs in a bioreactor, wherein the culturing of the PSC-inoculated culture medium in (d) includes suspending the PSCs in the liquid culture medium; and (e) culturing the PSCs from (d) in a liquid endoderm differentiation culture medium in a bioreactor for a period sufficient to differentiate the PSCs into DE, wherein the culturing of the PSC-inoculated culture medium in (d) includes suspending the PSCs in the liquid endoderm differentiation culture medium.

[0193] In some embodiments, the culture in (d) is a period of 18, 24, 30, 36, 42, 48, or 54 hours, or at least one of these, less than or equal to one of these, or a period defined by any two of the aforementioned values, and optionally the period is 18–54 or 24–48 hours.

[0194] In some embodiments of the method, the liquid culture medium inoculated with the PSCs cultured in (d) is the PSC inoculation medium of the subcultured PSC culture of (c)(ii) as described above and elsewhere in this specification. In some embodiments, differentiation is carried out on the PCS suspension culture as described above and elsewhere in this specification, which is a continuation of any one of the methods described above and elsewhere in this specification, further comprising differentiating the PSCs of the PSC inoculation medium of (c)(ii) to DE, optionally, differentiation is culture of the PSCs of the PSC inoculation medium of (c)(ii) in a bioreactor in (d'), culture comprises suspending the PSCs in a liquid culture medium, and culture comprises 18, 24, 30, 36, 42, (e) culturing for a period of time that is 48 or 54 hours, at least those, or less than or equal to those, or within the range defined by any two of the aforementioned values, for a period of 18 to 54 or 24 to 48 hours; and (e) culturing the PSCs of (d') in an endoderm differentiation culture medium in a bioreactor for a period of time sufficient to differentiate the PSCs into DEs, wherein the culturing of the PSC-inoculated culture medium of (d') includes suspending the PSCs in liquid endoderm differentiation culture medium.

[0195] In some embodiments, the liquid culture medium and / or liquid endoderm medium are present in a concentration of 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 relative to the culture medium. The PSCs are cultured with the culture medium at a density that is either, at least, or less than or equal to any two of the aforementioned values, and optionally, 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 relative to the culture medium.

[0196] In some embodiments, the period sufficient to differentiate PSC into DE is 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours, or at least one of these, less than or equal to these, or a period defined by any two of the aforementioned values, and optionally, the period is 60–84, 66–78, or 72 hours. In some embodiments, culturing PSCs in an endoderm differentiation culture medium for a period sufficient to differentiate PSCs into DEs comprises: a first period of culturing PSCs in a culture medium containing Wnt signaling pathway activator (e.g., CHIR99021) and / or Nodal signaling pathway activator (e.g., activin A), and optionally BMP signaling pathway activator; a second period of culturing PSCs in a culture medium containing Wnt signaling pathway activator and / or Nodal signaling pathway activator, and optionally serum (e.g., FBS) or serum substitute (e.g., Knockout Replacement Serum (KRS), HAS, etc.); and a third period of culturing PSCs in a culture medium containing Wnt signaling pathway activator and / or Nodal signaling pathway activator, and optionally serum or serum substitute. In some embodiments, each of the first, second, and third periods is independently selected from periods that are 18, 20, 22, 24, 26, 28, or 30 hours, at least these, or less, or within a range defined by any two of the aforementioned values, and optionally, the first, second, and third periods are 20–28, 22–26, or 24 hours. In some embodiments, the efficiency of DE induction is 35, 40, 45, 50, 55, 60, 70, 80, 90, or 95%, at least these, or within a range defined by any two of the aforementioned values, and optionally, the efficiency of DE induction is at least 45% or at least 50%, optionally, the efficiency of DE induction is 45–55%, and optionally, the efficiency of DE induction is 80–95%. In some embodiments, the DE expresses Sox17 and FoxA2.

[0197] In some embodiments, pluripotent stem cells are differentiated into endoderm cells by contacting them with activin A, BMP signaling pathway activator, or both. In some embodiments, pluripotent stem cells are contacted with activin A at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or about those concentrations, at least those concentrations, at least about those concentrations, or less than or about those concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 10-200 ng / mL, 10-100 ng / mL, 100-200 ng / mL, or 50-150 ng / mL. In some embodiments, pluripotent stem cells are contacted with activin A at a concentration of 100 ng / mL or about 100 ng / mL. In some embodiments, pluripotent stem cells are contacted with BMP signaling pathway activator at a concentration of 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 about those concentrations, at least those concentrations, at least about those concentrations, or less than or about those concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 1-200 ng / mL, 1-100 ng / mL, 1-200 ng / mL, 1-80 ng / mL, or 1-30 ng / mL. In some embodiments, pluripotent stem cells are contacted with a BMP signaling pathway activator at a concentration of 15 ng / mL or approximately 15 ng / mL.

[0198] In some embodiments, bioreactors with reduced and / or low shear stress, or bioreactors without shear stress, are used, for example, compared to conventional suspension culture bioreactors (spinning flasks and vertical wheel bioreactors). In some embodiments, bioreactors with reduced and / or low shear stress, and / or without shear stress, function by continuously rotating, with the upward movement from the rotation being offset by gravity, thereby suspending cells in the culture medium. In some embodiments, the bioreactor includes a rotating chamber containing the suspension culture medium. In some embodiments, the chamber is a tank having the shape of a cylindrical section (for example, shaped like a Petri dish, and optionally, the depth of the dish is greater than that of a typical Petri dish). In some embodiments, the chamber rotates around its longitudinal axis, thereby rotating the liquid culture medium contained within the chamber, thereby suspending cells and aggregates in the liquid culture medium. In some embodiments, the chamber is oriented such that its longitudinal axis is parallel to the ground, so that the cells within the chamber (e.g., individual cells, aggregates, spheroids, organoids, etc.) rise on one side of the chamber due to the rotation of the chamber and the liquid culture medium, and fall on the opposite side due to gravity. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the volume of which is 5, 10, 20, 30, 40, 50 mL, or at least these, or less, or a range defined by any two of the aforementioned values. In some embodiments, the volume of which is which is 5 to 10 mL, optionally 10 mL. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the rotation of which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or at least these, or less, or a range defined by any two of the aforementioned values. In some embodiments, the rotation speed is 40 rpm or less. In some embodiments, the rotation speed is 3 to 7 rpm.In some embodiments, the rotation speed is a speed selected to maintain the suspended cells and / or aggregates in a stationary orbit. In some embodiments, the bioreactor is configured such that the mean shear stress on cells and aggregates in the culture medium while the culture medium is moving (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 mean shear stress on cells and aggregates in the culture medium is less than 0.1 dynes / cm².

[0199] In some embodiments, the culture medium does not contain any animal or human-derived material. In some embodiments, the culture medium does not contain any extracellular matrix and / or basement membrane matrix (e.g., Matrigel or similar products).

[0200] Aspects of this disclosure include compositions for differentiating PSCs into endoderm (DE) in a three-dimensional suspension culture. In some embodiments, the composition comprises a liquid DE differentiation medium and PSCs suspended in the liquid DE differentiation medium. In some embodiments, the liquid DE differentiation medium is a serum-free medium, contains no animal or human-derived material, and optionally contains no extracellular matrix and / or basement membrane matrix. In some embodiments, the PSCs have an average diameter of less than about 400 μm. In some embodiments, the PSCs have an average diameter of less than about 300 μm.

[0201] In some embodiments, the liquid DE differentiation medium contains activin A at a concentration of about 10 to 200 ng / mL relative to the liquid DE differentiation medium. In some embodiments, the liquid DE differentiation medium contains activin A at a concentration of about 50 to 150 ng / mL relative to the liquid DE differentiation medium. In some embodiments, the liquid DE differentiation medium contains activin A at a concentration of about 100 to 200 ng / mL relative to the liquid DE differentiation medium. In another embodiment, the liquid DE differentiation medium further contains FBS at a concentration of about 0% to 20%. In some embodiments, the liquid DE differentiation medium further contains FBS at a concentration of about 0.2% to 10%. In some embodiments, the liquid DE differentiation medium further contains FBS at a concentration of about 2% to 5%.

[0202] In some embodiments, the composition further comprises a DE differentiated from a PSC. The DE differentiated from a PSC may express Sox17 and / or FoxA2. In some embodiments, the DE differentiated from a PSC expresses both Sox17 and FoxA2.

[0203] Aspects of this disclosure relate to DE produced by the suspension culture method described above and elsewhere in this specification.

[0204] Differentiation of DE suspension cultures into hindgut spheroids (HGS) Aspects of this disclosure include methods for differentiating DEs into hindgut spheroids (HGS) in a three-dimensional suspension culture. In some embodiments, the DEs are derived from PSCs. In some embodiments, the PSCs are artificial PSCs (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the PSCs are human (hPSCs). In some embodiments, the PSCs are human iPSCs (hiPSCs). In some embodiments, the method includes (f) culturing the DEs in liquid hindgut differentiation culture medium in a bioreactor for a period sufficient to differentiate the DEs into HGSs, wherein the culture of the DEs includes suspending the DEs in liquid hindgut differentiation culture medium. In some embodiments, the DEs cultured in (f) are DEs prepared by any one of the methods disclosed above and elsewhere in this specification. In some embodiments, differentiation is carried out on a DE culture as described above and elsewhere in this specification, which is a continuation of any one of the methods described above and elsewhere in this specification, further comprising differentiating the DE to HGS, optionally comprising (f) culturing the DE in liquid hindgut differentiation medium in a bioreactor for a period sufficient to differentiate the DE to HGS, wherein the culturing of the DE comprises suspending the DE in liquid hindgut differentiation medium.

[0205] In some embodiments, the period sufficient to differentiate DE into HGS is 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, or 120 hours, or at least one of these, less than or equal to these, or a range defined by any two of the aforementioned values, and optionally, the period is 84–108, 90–102, or 96 hours. In some embodiments, the hindgut differentiation culture medium is replaced after a period of 18, 20, 22, 24, 26, 28, or 30 hours, or at least one of these, less than or equal to these, or a range defined by any two of the aforementioned values, and optionally, the first, second, and third periods are 20–28, 22–26, or 24 hours.

[0206] In some embodiments, the hindgut differentiation culture medium includes a Wnt signaling pathway activator, an FGF signaling pathway activator, and optionally FBS. In some embodiments, the FGF signaling pathway activator is FGF4, and optionally, the concentration is 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 approximately those, at least those, or at least approximately those, or within a range defined by any two of the aforementioned values, optionally 50–750 ng / mL, 50–100 ng / mL, or 50–500 ng / mL, or optionally 500 ng / mL. In some embodiments, the Wnt pathway activator is CHIRON 99021, and optionally, its concentration is 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, or 6 μM, or approximately those, at least those, or at least approximately those, or a range defined by any two of the aforementioned values, optionally 0.5–6 μM, 0.5–3 μM, 3–6 μM, 2–4 μM, or optionally 3 μM.

[0207] In some embodiments, bioreactors with reduced and / or low shear stress, or bioreactors without shear stress, are used, for example, compared to conventional suspension culture bioreactors (spinning flasks and vertical wheel bioreactors). In some embodiments, bioreactors with reduced and / or low shear stress, and / or without shear stress, function by continuously rotating, with the upward movement from the rotation being offset by gravity, thereby suspending cells in the culture medium. In some embodiments, the bioreactor includes a rotating chamber containing the suspension culture medium. In some embodiments, the chamber is a tank having the shape of a cylindrical section (for example, shaped like a Petri dish, and optionally, the depth of the dish is greater than that of a typical Petri dish). In some embodiments, the chamber rotates around its longitudinal axis, thereby rotating the liquid culture medium contained within the chamber, thereby suspending cells and aggregates in the liquid culture medium. In some embodiments, the chamber is oriented such that its longitudinal axis is parallel to the ground, so that the cells within the chamber (e.g., individual cells, aggregates, spheroids, organoids, etc.) rise on one side of the chamber due to the rotation of the chamber and the liquid culture medium, and fall on the opposite side due to gravity. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the volume of which is 5, 10, 20, 30, 40, 50 mL, or at least these, or less, or a range defined by any two of the aforementioned values. In some embodiments, the volume of which is which is 5 to 10 mL, optionally 10 mL. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the rotation of which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or at least these, or less, or a range defined by any two of the aforementioned values. In some embodiments, the rotation speed is 40 rpm or less. In some embodiments, the rotation speed is 3 to 7 rpm.In some embodiments, the bioreactor provides a mean shear stress of 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, or 0.005 dynes / cm² to cells, aggregates, and / or spheroids in the culture medium while the culture medium is moving (e.g., the chamber is rotating). 2 It is configured to be less than 0.1 dynes / cm². In some embodiments, the mean shear stress on cells, aggregates, and / or spheroids in the culture medium is 0.1 dynes / cm². 2 It is less than.

[0208] In some embodiments, the culture medium does not contain any animal or human-derived material. In some embodiments, the culture medium does not contain any extracellular matrix and / or basement membrane matrix (e.g., Matrigel or similar products).

[0209] Aspects of this disclosure relate to HSGS produced by the suspension culture method described above and elsewhere in this specification.

[0210] Aspects of this disclosure include compositions for differentiating DE into hindgut spheroids (HGS) in a three-dimensional suspension culture. In some embodiments, the composition comprises a liquid hindgut differentiation culture medium containing a Wnt signaling pathway activator, an FGF signaling pathway activator, and optionally FBS, and DE suspended in the liquid hindgut differentiation culture medium. In some embodiments, the liquid hindgut differentiation culture medium is free of animal or human-derived material, and optionally, the culture medium is free of any extracellular matrix and / or basement membrane matrix.

[0211] In some embodiments, the Wnt signaling pathway activator comprises CHIR99021, and the FGF signaling pathway activator comprises FGF4. In some embodiments, the FGF signaling pathway activator is at a concentration of at least about 50 ng / mL relative to liquid hindgut differentiation culture medium. In some embodiments, the FGF signaling pathway activator is at a concentration of at least about 500 ng / mL relative to liquid hindgut differentiation culture medium. In some embodiments, the Wnt pathway activator is at a concentration of at least about 0.5 μM relative to liquid hindgut differentiation culture medium.

[0212] Differentiation of HGS suspension cultures into intestinal organoids (IOs) Aspects of this disclosure include methods for differentiating HGS into intestinal organoids (IOs) in a three-dimensional suspension culture. In some embodiments, the HGS are derived from PSCs. In some embodiments, the PSCs are artificial PSCs (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the PSCs are human (hPSCs). In some embodiments, the PSCs are human iPSCs (hiPSCs). In some embodiments, the method is (g) culturing the HGS in liquid IO maturation culture medium in a bioreactor for a period sufficient to differentiate the HGS into IOs, wherein the culture of the HGS includes suspending the HGS in liquid IO maturation culture medium. In some embodiments, the HGS cultured in (g) is any one of the methods described above and elsewhere herein for producing HGS. In some embodiments, differentiation is carried out on the HGS culture described above and elsewhere in this specification, which is a continuation of any one of the methods described above and elsewhere in this specification, further comprising differentiating the HGS to IO, optionally comprising (g) culturing the HGS in liquid IO mature culture medium in a bioreactor for a period sufficient to differentiate the HGS to IO, wherein the culturing of the HGS comprises suspending the HGS in liquid IO mature culture medium.

[0213] In some embodiments, the period sufficient to differentiate HGS into IO is approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 days, or at least those, less than or equal to those, or a range defined by any two of the aforementioned values, and optionally, the period is 12–30, 12–20, 15–28, 15–20, or 20 days, and optionally, the period is at least 12, 15, or 20 days. In some embodiments, the IO maturation culture medium is replaced after a period of approximately 24, 28, 32, 36, 38, 42, 44, 46, 48, 50, 52, or 54 hours, or at least those, less than or equal to those, or a range defined by any two of the aforementioned values. In some embodiments, the first, second, and third periods are approximately 24–54, 42–54, 46–50, or 48 hours.

[0214] In some embodiments, the IO mature culture medium comprises EGF, R-spongin, noggin, gremlin 1, and / or epiregulin (EREG). In some embodiments, the concentration of EGF, R-spongin, noggin, gremlin 1, and / or epiregulin (EREG) is 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 approximately those, at least those, or at least approximately those, or a range defined by any two of the aforementioned values, optionally 25–100 ng / mL, 50–150 ng / mL, 100 ng / mL, or optionally 100 ng / mL.

[0215] In some embodiments, HGS does not dissociate before being cultured in IO maturation culture medium, and the formed IO epithelial cells have polarity in which the apical surface is oriented outward from the IO. In other words, the apical surface faces outward relative to the culture medium.

[0216] In some embodiments, bioreactors with reduced and / or low shear stress, or bioreactors without shear stress, are used, for example, compared to conventional suspension culture bioreactors (spinning flasks and vertical wheel bioreactors). In some embodiments, bioreactors with reduced and / or low shear stress, and / or without shear stress, function by continuously rotating, with the upward movement from the rotation being offset by gravity, thereby suspending cells in the culture medium. In some embodiments, the bioreactor includes a rotating chamber containing the suspension culture medium. In some embodiments, the chamber is a tank having the shape of a cylindrical section (for example, shaped like a Petri dish, and optionally, the depth of the dish is greater than that of a typical Petri dish). In some embodiments, the chamber rotates around its longitudinal axis, thereby rotating the liquid culture medium contained within the chamber, thereby suspending cells and aggregates in the liquid culture medium. In some embodiments, the chamber is oriented such that its longitudinal axis is parallel to the ground, so that the cells within the chamber (e.g., individual cells, aggregates, spheroids, organoids, etc.) rise on one side of the chamber due to the rotation of the chamber and the liquid culture medium, and fall on the opposite side due to gravity. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the volume of which is 5, 10, 20, 30, 40, 50 mL, or at least these, or less, or a range defined by any two of the aforementioned values. In some embodiments, the volume of which is which is 5 to 10 mL, optionally 10 mL. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the rotation of which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or at least these, or less, or a range defined by any two of the aforementioned values. In some embodiments, the rotation speed is 40 rpm or less. In some embodiments, the rotation speed is 3 to 7 rpm.In some embodiments, the rotation speed is a speed selected to maintain the suspended cells and / or aggregates in a stationary orbit. In some embodiments, the bioreactor is configured such that the mean shear stress on cells, aggregates, spheroids, and / or organoids in the culture medium 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 while the culture medium is moving (e.g., the chamber is rotating). In some embodiments, the mean shear stress on cells, aggregates, spheroids, and / or organoids in the culture medium is less than 0.1 dynes / cm².

[0217] In some embodiments, the culture medium does not contain any animal or human-derived material. In some embodiments, the culture medium does not contain any extracellular matrix and / or basement membrane matrix (e.g., Matrigel or similar products).

[0218] Aspects of this disclosure relate to IO produced by suspension culture methods described above and elsewhere in this specification. In some embodiments, the epithelial cells of IO have polarity in which the apical surface is oriented outward from the IO (also known as apical outward).

[0219] Aspects of this disclosure relate to compositions for differentiating HGS into intestinal organoids (IOs) in three-dimensional suspension culture. In various embodiments, the composition comprises a liquid IO maturation culture medium containing EGF and HGS suspended in the liquid IO maturation culture medium. In some embodiments, the liquid IO maturation culture medium is free of animal or human-derived materials and, optionally, is free of any extracellular matrix and / or basement membrane matrix.

[0220] In some embodiments, the lumen of the HGS suspended in the liquid IO mature culture medium faces outward relative to the liquid IO mature culture medium.

[0221] In some embodiments, 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.

[0222] In at least one embodiment, at least a portion of the HGS suspended in the liquid IO maturation culture medium comprises dissociated HGS single cells. In some embodiments, at least 80% of the HGS is dissociated HGS single cells, and optionally, at least 90% of the HGS is dissociated single cells. In at least one embodiment, the concentration of dissociated HGS single cells in the liquid IO maturation culture medium is about 0.1×10 5 ~80×10 5 within the range of cells. In another embodiment, the concentration of dissociated HGS single cells in the liquid IO maturation culture medium is about 20×10 5 ~60×10 5 dissociated HGS single cells / mL within the range of the liquid IO maturation culture medium.

[0223] In some embodiments, the composition further comprises IO differentiated from HGS. In some aspects, the epithelial cells of the IO formed from dissociated HGS single cells have a polarity such that the apical surface is oriented inside the IO. In some embodiments, the HGS expresses CdX2. In some aspects, the HGS expresses FOX-F1 but does not express SOX2.

[0224] In some embodiments, the liquid IO maturation culture medium further comprises noggin.

[0225] Modification of epithelial cell polarity in IO Aspects of this disclosure include methods for modifying the polarity of epithelial cells in IO derived from HGS in a three-dimensional suspension culture. In some embodiments, HGS is derived from PSCs. In some embodiments, PSCs are artificial PSCs (iPSCs) or embryonic stem cells (ESCs). In some embodiments, PSCs are human (hPSCs). In some embodiments, PSCs are human iPSCs (hiPSCs). In some embodiments of a method for differentiating HGS into intestinal organoids (IOs) having apical inward polarity in a three-dimensional suspension culture, the method comprises (g) culturing the HGS in liquid IO maturation medium in a bioreactor for a period sufficient to differentiate the HGS into IOs, wherein the culture of HGS comprises suspending the HGS in liquid IO maturation medium, and the method further comprises dissociating at least a portion of the HGS into single cells prior to incubation in IO maturation medium, wherein the culture of HGS comprises suspending the dissociated HGS single cells and any undissociated HGS in liquid IO maturation medium, and the epithelial cells of IOs formed from the dissociated HGS single cells have apical surface orientation toward the inside of the IO (also known as apical inward polarity). In some embodiments, the dissociated HGS cultured in (g) is any one of the methods described above and elsewhere herein for producing HGS. In some embodiments, differentiation is carried out on the HGS culture described above and elsewhere herein, which is a continuation of any one of the methods described above and elsewhere herein, the method further comprising dissociating at least a portion of the HGS into single cells before incubation in IO maturation culture medium, the culture of HGS comprising suspending the dissociated HGS single cells and any undissociated HGS in liquid IO maturation culture medium, the epithelial cells of IO formed from the dissociated HGS single cells having polarity with their apical surface oriented inward of the IO. In some embodiments, at least 80, 85, 90, 95, 98, or 99% of the HGS dissociates into single cells, and optionally, at least 90% of the HGS dissociates into single cells.In some embodiments, a certain concentration of dissociated HGS single cells is present in IO mature culture medium, where the concentration is 0.05 × 10⁻¹⁰ relative to the IO mature culture medium. 5 , 0.1 × 10 5 , 0.5 × 10 5 , 1 x 10 5 , 2×10 5 , 4×10 5 , 6×10 5 , 8×10 5 , 16×10 5 , 10×10 5 , 20×10 5 , 40×10 5 , or 80 x 10 5 The concentration is defined as 1 dissociated HGS single cell / mL, or at least 10, or less than 10, or any two of the aforementioned values, and optionally the concentration is 0.1 × 10⁶ relative to IO mature culture medium. 5 ~80×10 5 , 1 x 10 5 ~16×10 5 , or 2 × 10 5 ~6×10 5 , 10×10 5 ~80×10 5 , 20×10 5 ~60×10 5 The concentration is 40 × 10¹ dissociated HGS single cells / mL, optionally, relative to IO mature culture medium. 5 This is one dissociated HGS single cell / mL.

[0226] In some embodiments, the dissociation is chemical, enzymatic, and / or mechanical. In some embodiments, the dissociation is chemical, for example, EDTA. In some embodiments, the dissociation is enzymatic, and optionally the enzyme includes proteases and / or collagenases, and optionally the enzyme is Accutase.

[0227] In some embodiments, bioreactors with reduced and / or low shear stress, or bioreactors without shear stress, are used, for example, compared to conventional suspension culture bioreactors (spinning flasks and vertical wheel bioreactors). In some embodiments, bioreactors with reduced and / or low shear stress, and / or without shear stress, function by continuously rotating, with the upward movement from the rotation being offset by gravity, thereby suspending cells in the culture medium. In some embodiments, the bioreactor includes a rotating chamber containing the suspension culture medium. In some embodiments, the chamber is a tank having the shape of a cylindrical section (for example, shaped like a Petri dish, and optionally, the depth of the dish is greater than that of a typical Petri dish). In some embodiments, the chamber rotates around its longitudinal axis, thereby rotating the liquid culture medium contained within the chamber, thereby suspending cells and aggregates in the liquid culture medium. In some embodiments, the chamber is oriented such that its longitudinal axis is parallel to the ground, so that the cells within the chamber (e.g., individual cells, aggregates, spheroids, organoids, etc.) rise on one side of the chamber due to the rotation of the chamber and the liquid culture medium, and fall on the opposite side due to gravity. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the volume of which is 5, 10, 20, 30, 40, 50 mL, or at least these, or less, or a range defined by any two of the aforementioned values. In some embodiments, the volume of which is which is 5 to 10 mL, optionally 10 mL. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the rotation of which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or at least these, or less, or a range defined by any two of the aforementioned values. In some embodiments, the rotation speed is 40 rpm or less. In some embodiments, the rotation speed is 3 to 7 rpm.In some embodiments, the rotation speed is a speed selected to maintain the suspended cells, aggregates, spheroids, and / or organoids in a stationary orbit. In some embodiments, the bioreactor is configured such that the mean shear stress on the cells, aggregates, spheroids, and / or organoids in the culture medium 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 while the culture medium is moving (e.g., the chamber is rotating). In some embodiments, the mean shear stress on the cells, aggregates, spheroids, and / or organoids in the culture medium is less than 0.1 dynes / cm².

[0228] In some embodiments, the culture medium does not contain any animal or human-derived material. In some embodiments, the culture medium does not contain any extracellular matrix and / or basement membrane matrix (e.g., Matrigel or similar products).

[0229] Aspects of this disclosure relate to IO produced by the suspension culture method described above and elsewhere in this specification. In some embodiments, the epithelial cells of IO have polarity in which the apical surface is oriented inward of the IO (also known as apical inward). Aspects of this disclosure relate to IO, wherein the epithelial cells of IO have polarity in which the apical surface is oriented inward of the IO (also known as apical inward).

[0230] In vivo maturation of IO by subcapsular transplantation of the kidney Aspects of this disclosure relate to methods for in vivo maturation of IO by transplantation of IO under the kidney capsule of a subject (e.g., an animal). In some embodiments, the IO is derived from PSCs. In some embodiments, the PSC is an artificial PSC (iPSC) or an embryonic stem cell (ESC). In some embodiments, the PSC is human (hPSC). In some embodiments, the PSC is a human iPSC (hiPSC). In some embodiments, the method comprises transplanting IO under the kidney capsule of a non-human animal for a period of time that is optionally 6, 8, 10, 12, 14, 16, 18, or 20 weeks, or at least those, or less than or equal to any two of the aforementioned values, optionally 6–20, 6–14, or 8–12 weeks. In some embodiments, the IO is one of the IOs described above and elsewhere in this specification for producing IO. In some embodiments, the IO matures in vitro for a certain period prior to transplantation, and optionally, the period is 7, 10, 14, 16, 21, 25, or 28 days, or at least one of these, or less than or equal to any two of the aforementioned values, optionally 7–28, 14–28, or 21–28 days.

[0231] IO transplantation for intestinal treatment Aspects of this disclosure relate to methods for treating the intestines of subjects (e.g., those resulting from intestinal lesions, injuries, tissue loss, etc.) by transplanting IOs into the intestinal lumen of the subject (e.g., a patient, a human, an animal, etc.). In some embodiments, the IOs are derived from PSCs. In some embodiments, the PSCs are artificial PSCs (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the PSCs are human (hPSCs). In some embodiments, the PSCs are human iPSCs (hiPSCs). In some embodiments, the method comprises transplanting IOs into the intestinal lumen for a period of time that is optionally 6, 8, 10, 12, 14, 16, 18, or 20 weeks, or at least those, or less than or equal to any two of the aforementioned values, optionally 6–20, 6–14, or 8–12 weeks. In some embodiments, the IOs are any one of the methods described above and elsewhere in this specification for producing IOs. In some embodiments, the IO matures in vitro for a certain period prior to transplantation, and optionally, the period is 7, 10, 14, 16, 21, 25, or 28 days, or at least one of these, or less than or equal to any two of the aforementioned values, optionally 7–28, 14–28, or 21–28 days.

[0232] Production of spheroids and organoids by suspension culture One aspect of the present disclosure is a method for differentiating a DE into a spheroid, optionally comprising (f) culturing the DE in a liquid differentiation medium in a bioreactor for a period sufficient to differentiate the DE into a spheroid, wherein the culturing of the DE comprises suspending the DE in a liquid differentiation medium, and optionally the culturing is such that the spheroid is a foregut or hindgut spheroid. Differentiation media for differentiating a DE into a spheroid (e.g., foregut or hindgut) are known in the art and may be used in the manner disclosed herein.

[0233] One aspect of the present disclosure is a method for differentiating spheroids into organoids. In some embodiments, the method involves culturing spheroids in a liquid organoid maturation medium in a bioreactor for a period sufficient to differentiate the spheroids into organoids, wherein the culture of the spheroids includes suspending the spheroids in the liquid organoid maturation medium, and optionally, the organoids are selected from the group consisting of liver, pancreas, stomach, gastric antrum, gastric fundus, intestine, lung, or colon organoids. Differentiation media for differentiating spheroids (e.g., foregut or hindgut) into organoids are known in the art and may be used in the manner disclosed herein.

[0234] In some embodiments, bioreactors with reduced and / or low shear stress, or bioreactors without shear stress, are used, for example, compared to conventional suspension culture bioreactors (spinning flasks and vertical wheel bioreactors). In some embodiments, bioreactors with reduced and / or low shear stress, and / or without shear stress, function by continuously rotating, with the upward movement from the rotation being offset by gravity, thereby suspending cells in the culture medium. In some embodiments, the bioreactor includes a rotating chamber containing the suspension culture medium. In some embodiments, the chamber is a tank having the shape of a cylindrical section (for example, shaped like a Petri dish, and optionally, the depth of the dish is greater than that of a typical Petri dish). In some embodiments, the chamber rotates around its longitudinal axis, thereby rotating the liquid culture medium contained within the chamber, thereby suspending cells and aggregates in the liquid culture medium. In some embodiments, the chamber is oriented such that its longitudinal axis is parallel to the ground, so that the cells within the chamber (e.g., individual cells, aggregates, spheroids, organoids, etc.) rise on one side of the chamber due to the rotation of the chamber and the liquid culture medium, and fall on the opposite side due to gravity. In some embodiments, the bioreactor includes a rotating chamber containing culture medium, the volume of which is 5, 10, 20, 30, 40, 50 mL, or at least one of these, or less, or within a range defined by any of the aforementioned values. In some embodiments, the volume of which is which is about 5 to 50 mL. In some embodiments, the volume of which is which is about 5 to 10 mL, optionally about 10 mL. In some embodiments, the bioreactor includes a rotating chamber containing a culture medium, the rotation of the chamber being approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 rpm, or at least these, or less, or within a range defined by any two of the aforementioned values. In some embodiments, the rotation is approximately 40 rpm or less.In some embodiments, the rotation is 3 to 7 rpm. In some embodiments, the rotation speed is selected to maintain suspended cells, aggregates, spheroids, and / or organoids in a stationary orbit. In some embodiments, the bioreactor is configured such that the average shear stress on cells, aggregates, spheroids, and / or organoids in the culture medium during movement of the culture medium (e.g., rotation of the chamber) is less than 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, or 0.005 dyn / cm2. In some embodiments, the average shear stress on cells, aggregates, spheroids, and / or organoids in the culture medium is less than 0.1 dyn / cm2.

[0235] In some embodiments, the culture medium does not contain materials of animal or human origin. In some embodiments, the culture medium does not contain any extracellular matrix and / or basement membrane matrix (e.g., Matrigel or similar products).

[0236] Aspects of the present disclosure relate to spheroids produced by the suspension culture methods described above and elsewhere in this specification. Aspects of the present disclosure relate to organoids produced by the suspension culture methods described above and elsewhere in this specification.

[0237] PSCs for use in the methods disclosed herein In some embodiments of any of the methods disclosed above and elsewhere in this specification, the PSC is an induced pluripotent stem cell (iPSC) or an embryonic stem cell (ESC). In some embodiments of any of the methods disclosed above and elsewhere in this specification, the PSC is a human PSC, optionally a human iPSC (hiPSC). The pluripotent stem cells can be derived from any suitable source. In some embodiments of any of the methods disclosed above and elsewhere in this specification, the source of the pluripotent stem cells is a mammalian source, optionally human, non-human primate, rodent, pig, and bovine.

[0238] In some embodiments, PSCs, endoderm cells, spheroids, or organoids are genetically modified or edited according to methods known in the art. For example, gene editing using CRISPR nucleases such as Cas9 has been discussed in International Publications 2013 / 176772, 2014 / 093595, 2014 / 093622, 2014 / 093655, WO2014 / 093712, 2014 / 093661, 2014 / 204728, 2014 / 204729, 2015 / 071474, 2016 / 115326, 2016 / 141224, 2017 / 023803, and 2017 / 070633, each of which is expressly incorporated herein in its entirety by reference.

[0239] Cells, aggregates, spheroids, and organoids One aspect of this disclosure is a PSC or PSC aggregate prepared by any of the methods disclosed above and elsewhere in this specification. One aspect of this disclosure is a DE prepared by any of the methods disclosed above and elsewhere in this specification. One aspect of this disclosure is an HGS prepared by any of the methods disclosed above and elsewhere in this specification. One aspect of this disclosure is an IO prepared by any of the methods disclosed above and elsewhere in this specification. One aspect of this disclosure is an IO having apical inward polarity, wherein the epithelial cells of the IO have polarity with their apical surface oriented inward of the IO, and optionally, the IO is human IO (hIO). In some embodiments, the IO having apical inward polarity is prepared by any of the methods disclosed above and elsewhere in this specification.

[0240] Method and Use One aspect of this disclosure is a therapeutic method comprising transplanting IO or cells derived therefrom, prepared by any of the methods disclosed above and elsewhere in this specification, wherein the animal is optionally suffering from a GI disease condition. In some embodiments, the animal is human. One aspect of this disclosure is the use of IO or cells derived therefrom, prepared by any of the methods disclosed above and elsewhere in this specification, in the manufacture of a drug for the treatment of an animal, comprising transplanting IO or cells derived therefrom, wherein the animal is optionally suffering from a GI disease condition. In some embodiments, the animal is human.

[0241] One aspect of this disclosure is a method for screening a compound for activity, comprising contacting an IO, or cells derived therefrom, prepared by any of the methods disclosed above and elsewhere in this specification, with a compound, and measuring the response of the IO to the compound. In some embodiments, the IO is a model of intestinal disease, and evaluating the effect of a candidate compound or composition on the IO includes evaluating the effect of the candidate compound or composition on the disease. In some embodiments, the IO is produced from cells derived from a subject. In some embodiments, the cells derived from the subject are induced pluripotent stem cells. In some embodiments, the subject has an intestinal disease.

[0242] term The following detailed description refers to the accompanying drawings, which form part of it. In the drawings, unless otherwise indicated in the context, similar symbols typically identify similar components. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. The aspects of this disclosure generally described herein and illustrated in the drawings may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which will be readily understood as being expressly intended herein.

[0243] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those generally understood by a person skilled in the art who reads this disclosure in light of it. For the purposes of this disclosure, the following terms are defined below:

[0244] This disclosure uses definitive language to describe numerous embodiments. This disclosure also includes embodiments that completely or partially exclude the subject matter, such as substances or materials, methods, processes and conditions, protocols, or procedures.

[0245] The articles "a" and "an" are used herein to refer to one or more (e.g., at least one) grammatical objects of the article. For example, "an element" means one or more elements.

[0246] Throughout this specification, unless otherwise required by context, the words “comprise,” “comprises,” and “comprising” will be understood to mean encompassing the described process or element or group of processes or elements, but not to exclude any other process or element or group of processes or elements. “Consisting of” means including and being limited to what follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the enumerated elements are necessary or essential, and other elements are optional. “Consisting essentially of” means encompassing all elements enumerated after this phrase, and is limited to other elements that do not interfere with or contribute to the activity or action expressed in this disclosure with respect to the enumerated elements. Thus, the phrase “consisting essentially of” indicates that the enumerated elements are necessary or essential, but other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the enumerated elements.

[0247] As used herein, the terms “individual,” “subject,” or “patient” have their general and ordinary meanings as understood in light of this specification and mean human or non-human mammals, e.g., dogs, cats, mice, rats, cattle, sheep, pigs, goats, non-human primates, or birds, e.g., chickens, and any other vertebrates or invertebrates. The term “mammal” is used in its ordinary biological sense. This includes, specifically, primates including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, and others.

[0248] As used herein, the terms “effective dose” or “effective amount” have their general and ordinary meanings as understood in light of this specification and refer to the amount of the described composition or compound that produces an observable effect. The actual dose levels of the active ingredient in the active composition of the subject currently disclosed may be varied to administer an amount of the active composition or compound that is effective in achieving a desired response for a particular subject and / or use. The selected dose level will depend on a variety of factors, including but not limited to the activity of the composition, the formulation, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated. In some embodiments, a minimum dose is administered, and if there is no dose-limiting toxicity, the dose is increased to the minimum effective dose. This specification is intended to evaluate the determination and adjustment of effective doses, and when and how such adjustments should be made.

[0249] As used herein, the terms “function” and “functional” have their general and ordinary meanings as understood in light of this specification, and refer to biological, enzymatic, or therapeutic functions.

[0250] As used herein, the term “inhibit” has its general and ordinary meaning as understood herein and may mean the reduction or prevention of biological activity. Reduction may be a percentage that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or about those, at least those, at least about those, less than or equal to those, or about less than or equal to those, or within the range defined by any two of the aforementioned values. As used herein, the term “delay” has its general and ordinary meaning as understood herein and may mean the delay, postponement, or delay of a biological event to a later time than would otherwise be expected. The delay may be 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a percentage that is approximately one of those, at least one of those, less than or equal to those, or approximately less than or equal to those, or within the range defined by any two of the aforementioned values. The terms inhibition and delay do not necessarily imply 100% inhibition or delay. Partial inhibition or delay may be achieved.

[0251] As used herein, the term “isolated” has its general and ordinary meaning as understood in light herein, and means a substance and / or entity that (1) was separated from at least some of the constituent elements with which it was originally produced (in nature and / or in an experimental environment) and / or (2) was produced, prepared and / or manufactured by human hands. An isolated substance and / or entity may be separated from 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 equal to, about, at least, about, or less than or about 100% of the other constituent elements with which they were originally related (or a range including and / or spanning the aforementioned values). In some embodiments, the isolated drug is 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, about those, at least those, at least about those, less than those, or about less than those (or a range including and / or spanning the aforementioned values). As used herein, “isolated” substance can be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multicellular organism or tissue.

[0252] As used herein, “in vivo” is given its general and ordinary meaning as understood in light herein, and refers to the implementation of the method in living organisms, typically animals, mammals including humans, and plants, as opposed to tissue extracts or dead organisms.

[0253] As used herein, “ex vivo” is given its general and ordinary meaning as understood herein, and refers to the execution of a method in vitro with minimal alteration of natural conditions.

[0254] As used herein, “in vitro” is given its general and ordinary meaning as understood in light of this specification and refers to the execution of a method outside of biological conditions, for example, in a petri dish or test tube.

[0255] As used herein, the terms “nucleic acid” or “nucleic acid molecule” have their general and ordinary meanings as understood herein, and refer to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that occur naturally in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease activity, and exonuclease activity. Nucleic acid molecules may consist of monomers that are naturally occurring nucleotides (such as DNA and RNA), analogs of naturally occurring nucleotides (e.g., enantiomers of naturally occurring nucleotides), or combinations of both. Modified nucleotides may have changes in the sugar moiety and / or pyrimidine or purine base moiety. Sugar modifications may include, for example, the substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the functionalization of sugars as ethers or esters. Furthermore, the entire sugar moiety can be replaced with a sterically and electronically similar structure, such as aza sugars and carbocyclic sugar analogs. Examples of modifications to the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoranilideates, or phosphoramidates. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded. “Oligocyte” can be used interchangeably with nucleic acid and can refer to either double-stranded or single-stranded DNA or RNA.Nucleic acids may be contained in nucleic acid vectors or constructs (e.g., plasmids, viruses, retroviruses, lentiviruses, bacteriophages, cosmids, fosmids, phagemids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or human artificial chromosomes (HACs)) that can be used for the amplification and / or expression of nucleic acids in various biological systems. Typically, the vector or construct may also contain elements such as 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, targeted sequences, peptide purification tags, or accessory genes, or any combination thereof.

[0256] A nucleic acid or nucleic acid molecule may contain one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences may be contiguous within the same nucleic acid or nucleic acid molecule, or, for example, with extra nucleic acids between linker, repeat, or restriction enzyme sites, or with any other sequence having a length of 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, or about that length, at least that length, at least about that length, less than or equal to that length, or about that length, or any length within the range defined by any two of the aforementioned lengths. As used herein, the term “downstream” with respect to nucleic acids has its general and ordinary meaning as understood herein, and, in the case of a double-stranded nucleic acid, refers to the sequence following the 3' end of the sequence on the strand containing the coding sequence (sense strand). As used herein, the term “upstream” with respect to nucleic acids has its general and ordinary meaning as understood herein, and, in the case of a double-stranded nucleic acid, refers to the sequence following the 5' end of the sequence on the strand containing the coding sequence (sense strand). As used herein, the term “grouping” with respect to nucleic acids has its general and ordinary meaning as understood in light of this specification and refers to two or more sequences that occur in close proximity to any other sequence that is, for example, an extra nucleic acid between linkers, repeats, or restriction enzyme sites, or that is, about, at least, less than, less than, or

[0257] The nucleic acids described in this specification contain nucleobases. Primary, standard, 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.

[0258] As used herein, the terms “peptide,” “polypeptide,” and “protein” have their general and ordinary meanings as understood herein and refer to macromolecules composed of amino acids linked by peptide bonds. Many functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymes, structural, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, though not always, produced biologically by ribosome complexes using nucleic acid templates, but chemosynthesis is also available. By manipulating nucleic acid templates, peptide, polypeptide, and protein mutations can be performed, such as substitution, deletion, shortening, addition, replication, or fusion of two or more peptides, polypeptides, or proteins. These fusions of two or more peptides, polypeptides, or proteins can be joined adjacent to each other within the same molecule, or, for example, to an extra amino acid between linkers, repeats, epitopes, or tags, or to any other sequence of any length that is, about, at least, at least about, less than, or less than, or about two of the aforementioned lengths. The term “downstream” in relation to polypeptides as used herein has its general and ordinary meaning as understood herein and refers to the sequence following the C-terminus of the preceding sequence. As used herein, the term “upstream” in relation to polypeptides has its general and ordinary meaning as understood in light of this specification, and refers to the sequence preceding the N-terminus of the subsequent sequence.

[0259] The term “purity” used herein for any given substance, compound, or material has its general and ordinary meaning as understood herein and refers to the actual amount of the substance, compound, or material compared to the expected amount. For example, a 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 may be affected by undesirable impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, vehicles, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process-related components, mycoplasmas, pyrogens, bacterial endotoxins, and exogenous infectious agents. Purity can be measured using techniques such as electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin-layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-Vis spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetric analysis, or titration, or any combination thereof.

[0260] The term “yield” for any given substance, compound, or material used herein has its general and ordinary meaning as understood herein and refers to the actual total amount of the substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material may be 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total, or about that, at least that, at least about that, less than or equal to that, or about less than or equal to that, including all fractions in between. Yields may be affected by the efficiency of the reaction or process, undesirable side reactions, decomposition, the quality of inputs, compounds, or materials, or the loss of the desired substance, compound, or material at any stage of production.

[0261] As used herein, the term “intestinal organoid” has its general and ordinary meaning as understood in light of this specification and refers to a three-dimensional cellular structure that exhibits many of the characteristics of the small intestine of an organism. In some embodiments, intestinal organoids are those derived from human cells and exhibit the characteristics of the human small intestine. However, intestinal organoids from other mammals are also included. Intestinal organoids as used herein are derived from pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) or intermediates thereof (e.g., endoderm of the embryo), where the process of differentiating pluripotent stem cells into endoderm of the embryo, then into hindgut endoderm (which may be in the form of spheroids), and finally into intestinal organoids results in a cellular structure having a composition, structure, and function similar to that of a naturally occurring intestine. A significant difference between intestinal organoids as used herein and enteroids, which are cellular structures derived from adult intestinal epithelium, and other so-called organoids made from non-pluripotent adult intestinal stem cells is that intestinal organoids as used herein include both epithelium and mesenchyme. The mesenchyme plays a crucial supportive role to the epithelium, significantly enhancing the viability and robust function of intestinal organoids. The intestinal organoids used herein may exhibit a lumen with epithelial villi-like inclusions very similar to those of a normal intestine, and may also exhibit peristaltic behavior. As a result of the differentiation process from pluripotent stem cells, the intestinal organoids used herein also include specialized intestinal cell types, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. References disclosing embodiments of intestinal organoids suitable for use herein include International Publication Nos. 2011 / 140441, 2016 / 061464, 2018 / 200481, 2020 / 160371, and 2021 / 030373, each of which is incorporated herein by reference in whole.

[0262] As used herein, the term “colon organoid” has its general and ordinary meaning as understood in light of this specification and refers to a three-dimensional cellular structure that exhibits many of the characteristics of the colon of an organism. In some embodiments, colon organoids are those derived from human cells and exhibit the characteristics of the human colon. However, colon organoids from other mammals are also included. Colon organoids as used herein are derived from pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) or intermediates thereof (e.g., endoderm of the embryo), where the process of differentiating pluripotent stem cells into endoderm of the embryo, then hindgut endoderm (which may be in the form of spheroids), and finally colon organoids results in a structure having a composition, structure, and function similar to that of a naturally occurring colon. A significant difference between colon organoids as used herein and colonoids, which are cellular structures derived from adult colon epithelium, and other so-called organoids made from non-pluripotent adult colon stem cells is that colon organoids as used herein include both epithelium and mesenchyme. The mesenchyme plays a crucial supportive role to the epithelium, significantly enhancing the viability and robust function of colonic organoids. The colonic organoids used herein may exhibit a lumen with crypts but substantially lacking villous structures. As a result of the differentiation process from pluripotent stem cells, the colonic organoids used herein also include specialized colonic cell types containing numerous goblet cells (compared to intestinal organoids) and colonic enteroendocrine cells, but substantially lacking Paneth cells. International Publication No. 2018 / 106628, which discloses embodiments of colonic organoids suitable for use herein, is cited as a reference and is incorporated herein in its entirety by reference.

[0263] As used herein, the terms “fragmentation,” “fragmented,” “dissociate,” and “dissociated” have their general and ordinary meanings as understood herein and refer to the partial or complete fragmentation or dissociation of organoids or other three-dimensional multicellular structures to produce a population of single cells and viable multicellular structures, fragments, or aggregates without excessive shearing or damage to the cells, such that all or most of the dissociated organoids contain intact and healthy cells. Therefore, “fragmented,” etc., generally do not refer to non-viable intracellular components or fragments of single cells, such as free intracellular contents or non-viable vesicles, although these components may be present in embodiments of organoid compositions that have been fragmented by natural apoptosis of cells or unintentional damage during organoid dissociation. Organoid fragmentation or dissociation can be carried out in a variety of methods commonly known in the art. The fragmentation or dissociation process may be such that some of the resulting cells are found not as single cells, but as small multicellular aggregates / fragments. Populations of dissociated cells containing multicellular aggregates / fragments between single cells are intended for use herein. In some embodiments, the dissociated cell population or composition exists exclusively as multicellular aggregates / fragments. In some embodiments, the dissociated cell population or composition exists exclusively as single cells without multicellular aggregates / fragments. In some embodiments, the dissociated cell population or composition is primarily (e.g., more than 70%, 80%, or 90%) multicellular aggregates / fragments, containing relatively few single cells. In some embodiments, the dissociated cell population or composition exists as a mixture of single cells and multicellular aggregates / fragments.

[0264] As used herein, the term “enzymatic dissociation” has its general and ordinary meaning as understood herein and refers to the fragmentation or dissociation of an organoid or other three-dimensional multicellular structure using the catalytic activity of one or more enzymes. Enzymatic dissociation, a process generally known in the art, typically involves the use of a protease (e.g., trypsin) or an enzyme (e.g., hyaluronidase) specific to other molecules involved in adhesion to surfaces or intercellular junctions.

[0265] As used herein, the term “mechanical dissociation” has its general and ordinary meaning as understood in light of this specification and refers to the fragmentation or dissociation of organoids or other three-dimensional multicellular structures using mechanical force. Mechanical dissociation, a process generally known in the art, may be achieved, for example, by grinding through a narrow-diameter channel, which may be in the form of a pipette, needle, or microfluidic channel.

[0266] As used herein, the term “mucosa” has its general and ordinary meaning as understood in light of this text and refers to the innermost layer of the gastrointestinal tract. The epithelium is the innermost 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 of the intestine. The epithelium is surrounded by connective tissue called the lamina propria and a thin layer of smooth muscle.

[0267] As used herein, the term “muscular layer” has its general and ordinary meaning as understood herein and refers to the muscular layer of the gastrointestinal tract. The muscular layer regulates the peristaltic behavior of the intestine and colon and originates from the mesenchymal layer of the developing neointestinal tract.

[0268] As used herein, the term “territoriality” has its general and ordinary meaning as understood herein and refers to the qualities and characteristics that distinguish one cell type from another. In the context of the intestine and colon (and other gastrointestinal organs), both organs originate from the same endoderm of the embryo, but early identification leads to the proper development and differentiation of the two organs and the constituent cells corresponding to their functions. As a result, intestinal tissue exhibits different territoriality from colonic tissue. As shown herein, intestinal and colonic organoids used for engraftment in intestinal injury models retain their respective qualities even after incorporation into the cell layers of different organs (e.g., intestinal organoids into host colonic tissue or colonic organoids into host intestinal tissue).

[0269] As used herein, the term “intestinal barrier” has its general and ordinary meaning as understood herein, and refers to the cellular and mucosal barriers that separate the luminal contents of the gastrointestinal tract from the surrounding tissues and circulatory system while still allowing for the exchange of nutrients. This barrier is mediated by intracellular junctions between epithelial cells. During intestinal injury, this barrier can be disrupted, leading to abnormal intestinal function, potentially allowing pathogenic microorganisms or antigens to enter the body, and leakage of blood and molecules into the lumen.

[0270] As used herein, “pharmaceutically acceptable” means, in its general and ordinary sense as understood herein, a carrier, excipient, and / or stabilizer that is non-toxic or has an acceptable level of toxicity to cells or mammals to which it is exposed at the doses and concentrations used. As used herein, “pharmaceutically acceptable,” “diluent,” “excipient,” and / or “carrier” means, in its general and ordinary sense as understood herein, and is intended to include any solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent and absorption retardant, etc., that is suitable for administration to human, cat, dog, or other vertebrate hosts. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are diluents, excipients, and / or carriers that are approved by federal, state, or other regulatory authorities for use in animals, including humans and non-human mammals such as cats and dogs, or that are listed in the United States Pharmacopeia or other generally accepted pharmacopoeias. The terms diluent, excipient, and / or “carrier” may refer to a diluent, adjuvant, excipient, or vehicle used when a pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin. Water, physiological saline, and aqueous solutions of dextrose and glycerol can be used 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, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. A non-limiting example of a physiologically acceptable carrier is a pH-buffered aqueous solution.Physiologically acceptable carriers may also contain one or more of the following: antioxidants such as ascorbic acid; low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; carbohydrates such as amino acids, glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; nonionic surfactants such as TWEEN® and polyethylene glycol (PEG); and PLURONICS®. The compositions may also optionally contain small amounts of wetting agents, fillers, emulsifiers, or pH buffers. These compositions may take the form of solutions, suspensions, emulsions, or sustained-release formulations. The formulations are typically adapted to the mode of administration.

[0271] Antifreezing agents are cell composition additives used to improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Examples of antifreezing agents include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methylformamide, dimethylformamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. Antifreezing agents may be used as part of a cryopreservation medium containing other components such as nutrients to enhance cell viability after thawing (e.g., albumin, serum, bovine serum, fetal calf serum [FCS]). In these cryopreservation media, at least one antifreeze agent may be found in concentrations of 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 about those, at least those, at least about those, less than or equal to those, or about that or less, or any percentage within the range defined by any two of the aforementioned numbers.

[0272] Additional excipients having desirable properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, 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, urea, or vitamins, or any combination thereof. Some excipients may include, but are not limited to, serum, albumin, ovalbumin, antibiotics, inactivators, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof, as residues or contaminants from the manufacturing process. The amount of excipients may be found in the composition in any weight percentage within the range defined by 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 about those, at least those, at least about those, less than or about those, or about less than or about those, or any two of the aforementioned numbers.

[0273] The term “pharmaceutically acceptable salt” has its general and ordinary meaning as understood herein and includes relatively non-toxic inorganic and organic acid or base addition salts of compositions or excipients, including but not limited to analgesics, therapeutic agents, and other materials. Examples of 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, and p-toluenesulfonic acid. Examples of inorganic bases suitable for salt formation include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, such a class of 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; and trihydroxymethylaminoethane.

[0274] The appropriate formulation will vary depending on the chosen route of administration. The formulations and administration techniques for the compounds described herein are known to those skilled in the art. Multiple techniques for administering the compounds exist in the art, including, but are not limited to, enteral, oral, rectal, topical, sublingual, oral cavity, intraocular, epidural, transdermal, aerosol, and parenteral delivery (including intramuscular, subcutaneous, intra-arterial, intra-venous, intra-portal, intra-articular, intradermal, peritoneal, intrathecal, intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injection). Pharmaceutical compositions will generally be adjusted to suit a specific intended route of administration.

[0275] As used herein, “carrier” has its general and ordinary meaning as understood herein and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or uptake of a compound into cells, tissues, and / or organs of the body.

[0276] As used herein, “diluent” has its general and ordinary meaning as understood herein and refers to a component in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent can be used to increase the bulk of a potent drug whose mass is too small to manufacture and / or administer. It may also be a liquid for dissolving a drug administered by injection, ingestion, or inhalation. Common forms of diluents in the art are buffered aqueous solutions, such as phosphate-buffered saline that mimics the composition of human blood, but are not limited thereto.

[0277] As used herein, the terms “basement membrane matrix” or “extracellular matrix” have their general and ordinary meanings in light of this specification and refer to any biological or synthetic compound, substance, or composition that enhances cell adhesion and / or growth. Any extracellular matrix known in the art, as well as its mimics or derivatives, may be used for the methods disclosed herein. Some examples of extracellular matrices, or their mimics or derivatives, include, but are not limited to, cell-based feeder layers, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, polylysine, polyornithine, 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. Common basement membrane matrices used in laboratories are isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. However, these basement membrane matrices are derived from non-human animals and therefore contain heterogeneous components that hinder their use in humans. They are also undefined, can introduce variability in production, and are potentially pathogenic. Therefore, in some embodiments, methods for culturing cells may involve the use of synthetic and / or defined substitutes for these heterogeneous basement membrane matrices. The use of non-heterogeneous basement membrane matrices or their mimics or derivatives enables the production of biological products more suitable for use in humans.

[0278] As used herein, the terms “passaging” and “passaging” have their general and ordinary meanings as understood herein and refer to conventional approaches employed in biological cell culture methods to maintain viable cell populations over extended periods. Since cells are generally proliferative in cell culture, they undergo multiple cycles of mitosis until they occupy available space (which is typically the surface of a cell culture vessel (e.g., a plate, dish, or flask) immersed in culture medium). For example, cells may grow as a monolayer on the surface of a cell culture vessel or as aggregates in the culture medium. If growing cells occupy all the available space on the surface or form aggregates that are too large, they may be unable to proliferate further and may exhibit senescent behavior. To continue cell growth (this may be done to maintain cell viability and proliferative capacity and / or to increase the number of cells for downstream purposes), cells can be passaged by taking a fraction of cells and, after dissociation of aggregates, seeding this fraction on a fresh surface or in fresh culture medium. This cell fraction continues to proliferate and increase until it occupies available space on a new surface or forms aggregates again, after which this passaging can be repeated continuously.

[0279] The term "three-dimensional" as used in "three-dimensional pluripotent stem cell (PSC) aggregates," "three-dimensional suspension," "suspension culture," "three-dimensional culture," "culturing in three dimensions," "three-dimensional expansion," or "three-dimensional aggregates" refers to the ability of cells, PSCs, PSC aggregates, DEs, spheroids, and / or organoids to grow, develop, regenerate, expand in three dimensions and interact with a framework around them. Such growth, development, regeneration, expansion, and / or interactions may be facilitated by the suspension of such cells, PSCs, PSC aggregates, DEs, spheroids, and / or organoids in a framework. In some embodiments, such growth, development, regeneration, expansion, and / or interactions may be facilitated by a suspension of beads or bead-like structures that hold, or otherwise provide, a place for such cells, PSCs, PSC aggregates, DEs, spheroids, and / or organoids in a framework. The use of “three-dimensional” in the terms and / or phrases referenced above may be contrasted with the use of “two-dimensional” in “two-dimensional PSC,” “two-dimensional culture,” “to culture in two dimensions,” or “two-dimensional monolayer,” where cells, PSCs, PSC aggregates, DEs, spheroids, and / or organoids can grow, develop, regenerate, and expand in two dimensions (e.g., along a monolayer of a plate) and interact with the surrounding framework.

[0280] As used herein, the terms "w / w%" or "weight / weight%" have their general and ordinary meanings as understood herein, and refer to a percentage expressed in relation to the weight of the component or agent multiplied by 100 relative to the total weight of the composition. As used herein, the terms "v / v%" or "volume / volume%" have their general and ordinary meanings as understood herein, and refer to a percentage expressed in relation to the liquid volume of the compound, substance, component or agent multiplied by 100 relative to the total liquid volume of the composition.

[0281] stem cells As used herein, the term “totipotent stem cell” (also known as “omnipotent stem cell”) has its general and ordinary meaning as understood herein and refers to a stem cell capable of differentiating into embryonic and extraembryonic cell types. Such cells can construct a complete and viable organism. These cells are generated from the fusion of an egg and a sperm cell. Cells produced by the first few divisions of a fertilized egg are also totipotent.

[0282] As used herein, the term “embryonic stem cell (ESC)” is commonly abbreviated as ES cell and has its general and ordinary meaning as understood herein, referring to pluripotent cells derived from the inner cell mass of the blastocyst, which is an early embryo.

[0283] As used herein, the term “pluripotent stem cells (PSCs)” has its general and ordinary meaning as understood herein and encompasses any cell that can differentiate into any of the body’s nearly all cell types, namely any cell that can differentiate into any of the three germ layers (embryonic epithelium), including the endoderm (endoderm, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, genitourinary tract), and ectoderm (epidermal tissue and nervous system). PSCs may be descendants of inner cell mass cells of a preimplantation blastocyst, or they may be obtained by inducing non-pluripotent cells, such as adult somatic cells, by forcing the expression of certain genes. Pluripotent stem cells may be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including humans, rodents, pigs, and cattle.

[0284] As used herein, the term “induced pluripotent stem cell (iPSC)” has its general and ordinary meaning as understood herein, and is commonly abbreviated as iPS cell. It refers to a type of pluripotent stem cell artificially induced from normally non-pluripotent cells, such as adult somatic cells, by inducing the “forced” expression of certain genes. hiPSC refers to human iPSCs. In some methods known in the art, iPSCs can be induced by transfection of certain stem cell-related genes into non-pluripotent cells, such as adult fibroblasts. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes may include the major transcription factors Oct-3 / 4 (POU5F1) and Sox2, but other genes may also improve the efficiency of induction. After 3-4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically, and are typically isolated by morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSCs include first-generation iPSCs, second-generation iPSCs in mice, and human induced pluripotent stem cells. Some methods use retroviral systems to transform human fibroblasts into pluripotent stem cells using four critical genes: Oct3 / 4, Sox2, Klf4, and c-Myc. Other methods use lentiviral systems to transform somatic cells with OCT4, SOX2, NANOG, and LIN28.Genes whose expression is induced in iPSCs include, but are not limited to, Oct-3 / 4(POU5F1), certain members of the Sox gene family (e.g., Soxl, Sox2, Sox3, and Sox15), certain members of the Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, LIN28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tel1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fthl17, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or any combination thereof.

[0285] As used herein, the term “progenitor cell” has its general and ordinary meaning as understood herein and encompasses any cell that can be used in the methods herein, through which one or more progenitor cells acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, the progenitor cells are pluripotent or capable of becoming pluripotent. In some embodiments, the progenitor cells are subjected to treatment with an extrinsic factor (e.g., a growth factor) to acquire pluripotency. In some embodiments, the progenitor cells may be totipotent (or omnipotent) stem cells, pluripotent stem cells (artificial or unartificial), multipotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, the progenitor cells may be derived from an embryo, infant, child, or adult. In some embodiments, the progenitor cells may be somatic cells subjected to treatment such that pluripotency is conferred via genetic engineering or protein / peptide treatment. Examples of progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSCs).

[0286] In some embodiments, one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, the pluripotent stem cells are derived from embryonic stem cells, which are derived from totipotent cells of an 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 a blastocyst, which is an early stage embryo. Methods for deriving embryonic stem cells from blastocysts are well known in the art. It will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cells.

[0287] Additional stem cells that may be used in embodiments of this disclosure include, but are not limited to, those provided by or described in the National Stem Cell Bank (NSCB), the database hosted by the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF), the WTSC cell bank of the Wi Cell Research Institute, the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC), Novocell, Inc. (San Diego, California), Cellartis AB (Goteborg, Sweden), ES Cell International Pte Ltd (Singapore), Technion of the Israel Institute of Technology (Haifa, Israel), and the Stem Cell Databases hosted by Princeton University and the University of Pennsylvania. Examples of embryonic stem cells that may be used in embodiments of this 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), and WA14 (H14). Examples of 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.

[0288] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term “directed differentiation” describes the process by which less specialized cells become a specific specialized target cell type. The specialization of the specialized target cell type can be determined by any applicable method that can be used to define or modify the fate of the initial cell. Exemplary methods include, but are not limited to, genetic engineering, chemical treatment, protein treatment, and nucleic acid treatment.

[0289] In some embodiments, adenoviruses can be used to transport the four required genes, resulting in iPSCs substantially identical to embryonic stem cells. Since adenoviruses do not combine their own genes with any of the target hosts, the risk of tumor formation is eliminated. In some embodiments, non-viral-based techniques are used to generate iPSCs. In some embodiments, reprogramming can be achieved via plasmids without the use of any viral transfection system, albeit with very low efficiency. In other embodiments, iPSCs are generated using direct protein delivery, thus eliminating the need for viruses or gene modification. In some embodiments, mouse iPSC generation is possible using a similar methodology: repeated treatment of cells with a specific protein delivered to the cells via a polyarginine anchor was sufficient to induce pluripotency. In some embodiments, the expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0290] As used herein, the terms “embryonic endoderm” or “DE” have their general and ordinary meanings as understood herein and refer to the developmental cell type that gives rise to the intestines and the resulting 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, while the posterior DE forms the midgut and hindgut, which in turn form the small and large intestines and the genitourinary system. Markers of DE include SOX17 and FOXA2. During development, Wnt and FGF signaling pathways establish the regionalization between the anterior and posterior patterning of DE.

[0291] Pluripotent stem cells can be differentiated into endoderm by methods known in the art. In some embodiments, endoderm cells can be differentiated from pluripotent cells by contacting the endoderm with Nodal, activin (e.g., activin A or activin B), and / or the BMP subgroup of the TGFβ superfamily of growth factors. In some embodiments, pluripotent stem cells are differentiated into endoderm by contacting them with Nodal, activin A, activin B, BMP signaling pathway activators, or any combination thereof. In some embodiments, pluripotent stem cells are differentiated into endoderm by contacting them with activin A. In some embodiments, one or more growth factors are selected from the group consisting of Nodal, activin A, activin B, BMP signaling pathway activators, or any combination of these growth factors. In some embodiments, stem cells are contacted with activin A and BMP signaling pathway activators. In some embodiments, the PSCs are treated with 100 ng / mL of activin A for 3 days, as described above.

[0292] As used herein, the term “feeder cell” has its general and ordinary meaning as understood herein and refers to cells that support the growth of pluripotent stem cells by secreting growth factors into the culture medium or displaying them on the cell surface, etc. Feeder cells are generally adherent cells and may cease to grow. For example, feeder cells may cease to grow by irradiation (e.g., gamma rays), mitomycin-C treatment, electrical pulses, or mild chemical fixation (e.g., with formaldehyde or glutaraldehyde). However, feeder cells do not necessarily cease to grow. 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. In some embodiments, feeder cells are homogeneous or heterogeneous to the supported target stem cells, which may affect downstream applications. In some embodiments, feeder cells are mouse cells. In some embodiments, feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76 / 7 cells, human fibroblasts, human precutaneous 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 myocytes, human fetal fibroblasts, or human adult Fallopian tube epithelial cells. In some embodiments, a conditioned medium prepared from the feeder cells is used instead of, or in combination with, the feeder cell co-culture. In some embodiments, the feeder cells are not used during the proliferation of target stem cells.

[0293] The term "three-dimensional" as used in "three-dimensional pluripotent stem cell (PSC) aggregates," "three-dimensional suspension," "three-dimensional culture," "two-dimensional culture," "three-dimensional expansion," or "three-dimensional aggregates" refers to the ability of cells, PSCs, PSC aggregates, DEs, spheroids, and / or organoids to grow, develop, regenerate, expand in three dimensions and interact with the surrounding framework. Such growth, development, regeneration, expansion, and / or interactions may be facilitated by the suspension of such cells, PSCs, PSC aggregates, DEs, spheroids, and / or organoids within the framework. The use of "three-dimensional" in the terms and / or phrases referenced above may be contrasted with "two-dimensional" as used in "two-dimensional PSCs," "two-dimensional culture," "two-dimensional culture," or "two-dimensional monolayer," where cells, PSCs, PSC aggregates, DEs, spheroids, and / or organoids can grow, develop, regenerate, expand in two dimensions (e.g., along a monolayer of a plate) and interact with the surrounding framework.

[0294] Known methods for producing downstream cell types such as endoderm, foregut endoderm, ventral foregut endoderm, and / or liver lineages from pluripotent cells (e.g., iPSCs or ESCs) are applicable to the methods described herein. In some embodiments, iPSCs are used to produce endoderm, or other downstream cell types such as foregut endoderm, ventral foregut endoderm, and liver lineages. In some embodiments, human iPSCs (hiPSCs) are used to produce endoderm, or other downstream cell types such as foregut endoderm, ventral foregut endoderm, and / or liver lineages.

[0295] In some embodiments, PSCs, such as ESCs and iPSCs, undergo directed differentiation into embryonic germ cells, organ tissue progenitor cells, and then into tissues such as liver tissue or any other biological tissue. In some embodiments, directed differentiation is carried out in a stepwise manner to obtain each of the differentiated cell types, with molecules (e.g., growth factors, ligands, agonists, antagonists) being added sequentially as differentiation progresses. In some embodiments, directed differentiation is carried out in a non-stepwise manner, with molecules (e.g., growth factors, ligands, agonists, antagonists) being added simultaneously. In some embodiments, directed differentiation is achieved by selectively activating a particular signaling pathway in the PSC or any downstream cells.

[0296] In some embodiments, embryonic stem cells are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a period of time that is 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, or 300 hours, or approximately those hours, at least those hours, at least approximately those hours, less than or approximately those hours, or less than or approximately those hours, or any time within the range defined by any two of the aforementioned hours, for example, between 6 hours and 300 hours, between 24 hours and 120 hours, between 48 hours and 96 hours, between 6 hours and 72 hours, or between 24 hours and 300 hours. In some embodiments, two or more small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the two or more small molecule compounds, activators, inhibitors, or growth factors may be added simultaneously or separately.

[0297] In some embodiments, embryonic stem cells or iPSCs are concentrated at concentrations of 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 approximately these concentrations, or at least these concentrations. The patient is treated with one or more small molecule compounds, activators, inhibitors, or growth factors at concentrations of at least approximately those, less than or equal to those concentrations, or any concentration within the 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 / mL to 2000 ng / mL, 10 ng / mL to 2000 ng / mL, or 1000 ng / mL to 15000 ng / mL. In some embodiments, the concentration of one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, the concentration of one or more small molecule compounds, activators, inhibitors, or growth factors changes during the course of treatment. In some embodiments, two or more small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the concentrations of the two or more small molecule compounds, activators, inhibitors, or growth factors may differ.

[0298] In some embodiments, ESCs or iPSCs are cultured in a growth medium that supports stem cell growth. In some embodiments, the stem cell growth medium is RPMI1640, DMEM, DMEM / F12, or Advanced DMEM / F12. In some embodiments, the stem cell growth medium contains fetal bovine serum (FBS). In some embodiments, the stem cell growth medium contains FBS at concentrations of 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 about those, at least those, at least about those, less than or about those, or about less than or about those, or any percentage within the 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%. In some embodiments, the stem cell growth medium does not contain heterogeneous components. In some embodiments, the growth medium comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0299] In some embodiments, pluripotent stem cells are prepared from somatic cells. In some embodiments, pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, pluripotent stem cells are cryopreserved. In some embodiments, somatic cells are cryopreserved. In some embodiments, pluripotent stem cells are prepared from PBMCs. In some embodiments, human PSCs are prepared from human PBMCs. In some embodiments, pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, PBMCs are grown on a feeder cell matrix. In some embodiments, PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell matrix. In some embodiments, PBMCs are grown on an irradiated MEF feeder cell matrix.

[0300] In some embodiments, the endoderm (DE) can undergo further anterior endoderm patterning, foregut identification, and morphogenesis depending on FGF, Wnt, BMP, or retinoic acid, or any combination thereof. In some embodiments, human PSCs are directed to efficiently differentiate into hepatic epithelium and mesenchyme in vitro. It will be understood that molecules such as growth factors can be added at any developmental stage to promote the formation of specific types of hepatic tissue. In some embodiments, siRNA and / or shRNA targeting cellular components that associate with FGF, Wnt, BMP, or retinoic acid signaling pathways are used to inhibit or activate these pathways.

[0301] Intestinal and colon organoids and methods for their manufacture The intestinal and colonic organoids disclosed herein are produced by a differentiation process from pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) or intermediates thereof (e.g., endoderm of an embryo), and include epithelial cell types and mesenchymal cell types, along with specialized cell types of the intestine or colon. Exemplary methods for preparing intestinal or colonic organoids can be found in U.S. Patent Nos. 9,719,068 and 10,781,425, U.S. Patent Application Publication No. 2020 / 190478, and International Publications Nos. 2011 / 140441, 2016 / 061464, 2018 / 106628, 2018 / 200481, 2019 / 126626, 2020 / 160371, 2020 / 056158, 2020 / 243633, and 2021 / 030373, each of which is incorporated herein by reference in whole.

[0302] In some embodiments, intestinal and colonic organoids are differentiated by culturing endoderm cells. These endoderm cells can be differentiated from pluripotent cells by contacting the endoderm with Nodal, activin, and / or the BMP subgroup of the TGFβ superfamily of growth factors. In some embodiments, pluripotent stem cells are differentiated into endoderm by contacting them with Nodal, activin A, activin B, BMP signaling pathway activators, or any combination thereof. In some embodiments, pluripotent stem cells are differentiated into endoderm by contacting them with activin A.

[0303] The endoderm of the embryo can be further subjected to FGF / Wnt-induced posterior endoderm patterning to target hindgut specificity.

[0304] In some embodiments, to produce intestinal and colonic organoids, the endoderm is first contacted with Wnt signaling pathway activators and FGF signaling pathway activators to posteriorly transform the endoderm into hindgut endoderm. During this culture process, the hindgut endoderm grows as a monolayer, but spontaneously budding in suspension as aggregates of cells called hindgut spheroids. In some embodiments, the Wnt signaling pathway activators include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, or Wnt16, or any combination thereof. In some embodiments, the Wnt signaling pathway activator is Wnt3a. In some embodiments, the Wnt signaling pathway activator includes a glycogen synthase kinase-3 (GSK3) inhibitor that acts as a Wnt signaling pathway activator. In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, the FGF signaling pathway activator includes 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. In some embodiments, the FGF signaling pathway activator is FGF4. The produced hindgut endoderm and hindgut spheroid contain CDX2+ polarized epithelium surrounded by CDX2+ mesenchyme and lack Alb and Pdx1, which are characteristic of foregut endoderm.

[0305] Following the formation of the hindgut endoderm or hindgut spheroid, the BMP signaling pathway regulates the formation of different regional types of intestines. Inhibition of BMP signaling after the hindgut stage promotes the fate of proximal intestinal cells (duodenum / jejunum). Activation of BMP signaling after the hindgut stage promotes the fate of more distal intestinal cells (cecum / colon). In some embodiments, the hindgut endoderm is brought into contact with a BMP signaling pathway activator to differentiate it into intestinal organoids. In some embodiments, the hindgut endoderm is brought into contact with a BMP signaling pathway inhibitor to differentiate it into colonic organoids. In some embodiments, the BMP signaling pathway activator includes BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, or IDE2, or any combination thereof. In some embodiments, the BMP signaling pathway activator includes BMP2. In some embodiments, the BMP signaling pathway inhibitor includes noggin, RepSox, LY364947, LDN193189, or SB431542, or any combination thereof. In some embodiments, the BMP signaling pathway inhibitor includes noggin.

[0306] Dissociation method In some embodiments, the methods disclosed herein involve the dissociation of cell aggregates (e.g., PSC aggregates) and / or spheroids (e.g., hindgut spheroids), and / or organoids (e.g., intestinal and / or colon organoids). In some embodiments, the dissociated cell populations are prepared by chemical, enzymatic, and / or mechanical dissociation of the aggregates, spheroids, and / or organoids. In some embodiments, the dissociation is chemical, for example, EDTA. In some embodiments, enzymatic dissociation involves dissociating the aggregates, spheroids, and / or organoids with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof. In some embodiments, enzymatic dissociation involves incubating the aggregates, spheroids, and / or organoids with proteolytic enzymes and / or collagenases. In some embodiments, enzymatic dissociation utilizes Accutase. In some embodiments, mechanical dissociation involves passing aggregates, spheroids, and / or organoids through channels of progressively narrowing diameter.

[0307] In some embodiments, the concentration of the dissociated cell population in the cell suspension is 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 A concentration of any number of cells / mL, or about that many, at least that many, at least about that many, less than or equal to that many, or about that many or less, or any concentration of cells within the range defined by any two of the aforementioned concentrations, for example, 10 5 ~10 11 , 10 5 ~10 8 , 10 9 ~10 11 , or 10 6 ~10 10This is 10 cells / mL. In some embodiments, the concentration of cells that are mesenchymal cell types in the dissociated cell population is 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 A concentration of any number of cells / mL, or about that many, at least that many, at least about that many, less than or equal to that many, or about that many or less, or any concentration of cells within the range defined by any two of the aforementioned concentrations, for example, 10 5 ~10 11 , 10 5 ~10 8 , 10 9 ~10 11 , or 10 6 ~10 10 The concentration is 10 cells / mL. In some embodiments, the concentration of epithelial cell type cells in the dissociated cell population is 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 A concentration of any number of cells / mL, or about that number, at least that number, at least about that number, less than or equal to that number, or about that number or less, or any concentration of cells within the range defined by any two of the aforementioned concentrations, for example, 10 5 ~10 11 , 10 5 ~10 8 , 10 9 ~10 11 , or 10 6 ~10 10 It is the number of cells / mL.

[0308] In some embodiments, the dissociated cell population consists of multicellular fragments representing a percentage of 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 about that percentage, at least that percentage, at least about that percentage, less than that percentage, or about that percentage, or any percentage within the range defined by any two of the aforementioned percentages, for example, 30-100%, 50-100%, 75-100%, 90-100%, 30-75%, or 50-95%. In some embodiments, the dissociated cell population is in the form of a multicellular fragment of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0309] Culture and expansion of foregut endoderm cells and downstream cell types. Methods for creating liver organoids include, for example, Ouchi et al. "Modeling Steatohepatitis in Humans with Pluripotent Stem Cell-Derived Organoids" Cell Metabolism (2019) 30(2):374-384, and Shinozawa et al. "High-Fidelity Drug-Induced Liver Injury Screen Using Human Pluripotent Stem Cell Derived Organoids" The following have been previously discussed in “Organoids” Gastroenterology (2021) 160(3):831-846, International Publications 2018 / 085615, 2018 / 191673, 2018 / 226267, 2019 / 126626, 2020 / 023245, 2020 / 069285, and 2021 / 262676, each of which is expressly incorporated herein by reference in its entirety. The disclosure of liver organoid compositions and methods for preparing them is applicable to the human liver organoids (HLOs) described herein.

[0310] In some embodiments, pluripotent stem cells, embryonic endoderm, foregut endoderm, ventral foregut endoderm, or downstream hepatocyte types are brought into contact with a TGF-β pathway inhibitor. In some embodiments, the TGF-β pathway inhibitor includes one or more of A83-01, RepSox, LY365947, and SB431542. In some embodiments, the cells are not treated with the TGF pathway inhibitor. The TGF-β pathway inhibitors provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0311] In some embodiments, pluripotent stem cells, endoderm of the embryo, foregut endoderm, ventral foregut endoderm, or downstream hepatocyte type are brought into contact with an FGF pathway activator. In some embodiments, the FGF pathway activator comprises an FGF protein. In some embodiments, the FGF protein comprises a recombinant FGF protein. In some embodiments, the FGF pathway activator comprises one or more of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 (FGF19, FGF15 / FGF19), FGF16, FGF17, FGF18, FGF20, FGF21, FGF22, or FGF23. In some embodiments, the cells are not treated with the FGF pathway activator. The FGF pathway activators provided herein may be used in combination with any other growth factors, pathway activators, or pathway inhibitors provided herein.

[0312] In some embodiments, pluripotent stem cells, embryonic endoderm, foregut endoderm, ventral foregut endoderm, or downstream hepatocyte type are contacted with a Wnt pathway activator. In some embodiments, the Wnt pathway activator comprises a Wnt protein. In some embodiments, the Wnt protein comprises recombinant Wnt protein. In some embodiments, the Wnt pathway activator comprises Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, or any combination thereof. In some embodiments, the Wnt pathway activator comprises a GSK3 signaling pathway inhibitor. In some embodiments, the Wnt pathway activator includes CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloysin, indirubin, alsterpaulone, kaempaulone, lithium chloride, TDZD8, or TWS119, or any combination thereof. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, cells are not treated with the Wnt pathway activator. The Wnt pathway activators provided herein may be used in combination with any other growth factors, pathway activators, or pathway inhibitors provided herein.

[0313] In some embodiments, pluripotent stem cells, embryonic endoderm, foregut endoderm, ventral foregut endoderm, or downstream hepatocyte types are brought into contact with a VEGF pathway activator. In some embodiments, the VEGF pathway activator comprises one or more of VEGF or GS4012. In some embodiments, the cells are not treated with the VEGF pathway activator. The VEGF pathway activators provided herein may be used in combination with any other growth factors, pathway activators, or pathway inhibitors provided herein.

[0314] In some embodiments, pluripotent stem cells, embryonic endoderm, foregut endoderm, ventral foregut endoderm, or downstream hepatocyte type are brought into contact with an EGF pathway activator. In some embodiments, the EGF pathway activator comprises EGF. In some embodiments, the cells are not treated with the EGF pathway activator. The EGF pathway activators provided herein may be used in combination with any other growth factors, pathway activators, or pathway inhibitors provided herein.

[0315] In some embodiments, pluripotent stem cells, embryonic endoderm, foregut endoderm, ventral foregut endoderm, or downstream hepatocyte type are brought into contact with ascorbic acid. In some embodiments, the cells are not treated with ascorbic acid. The ascorbic acid provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0316] In some embodiments, pluripotent stem cells, endoderm of the embryo, foregut endoderm, ventral foregut endoderm, or downstream hepatocyte type are contacted with a BMP pathway activator or BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises a BMP protein. In some embodiments, the BMP protein is a recombinant BMP protein. In some embodiments, the BMP pathway activator comprises BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, or IDE2, or any combination thereof. In some embodiments, the BMP pathway inhibitor comprises noggin, RepSox, LY364947, LDN-193189, SB431542, or any combination thereof. In some embodiments, the cells are not treated with the BMP pathway activator or BMP pathway inhibitor. The BMP pathway activators or BMP pathway inhibitors provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0317] In some embodiments, pluripotent stem cells, embryonic endoderm, foregut endoderm, ventral foregut endoderm, or downstream hepatocyte types are brought into contact with a retinoic acid pathway activator. In some embodiments, the retinoic acid pathway activator includes retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, or AM580, or any combination thereof. In some embodiments, the cells are not treated with the retinoic acid pathway activator. The retinoic acid pathway activators provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0318] In some embodiments, pluripotent stem cells are converted to hepatocyte type by a “one-step” process. For example, pluripotent stem cells are directly treated with one or more molecules (e.g., activin A) that can differentiate pluripotent stem cells into DE cultures, in combination with additional molecules (e.g., FGF4, CHIR99021, RA) that can promote targeted differentiation of DE cultures.

[0319] In some embodiments, pluripotent stem cells (e.g., ESCs or iPSCs) are expanded in suspension culture as described above and elsewhere in this specification. In some embodiments, pluripotent stem cells are expanded in a cell culture containing a ROCK inhibitor (e.g., Y-27632). In some embodiments, iPSCs are differentiated into endoderm cells. In some embodiments, pluripotent stem cells are differentiated into endoderm cells by contacting them with activin A, BMP activator, or both. In some embodiments, pluripotent stem cells are contacted with activin A at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or about those concentrations, at least those concentrations, at least about those concentrations, or less than or about those concentrations, or within a range defined by any two of the aforementioned concentrations, for example, 10-200 ng / mL, 10-100 ng / mL, 100-200 ng / mL, or 50-150 ng / mL. In some embodiments, pluripotent stem cells are contacted with activin A at a concentration of 100 ng / mL or about 100 ng / mL. In some embodiments, pluripotent stem cells are exposed to BMP signaling pathway activators at concentrations of 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 about those concentrations, at least those concentrations, at least about those concentrations, or less than or about those concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 1-200 ng / mL, 1-100 ng / mL, 25-200 ng / mL, 1-80 ng / mL, or 25-100 ng / mL. In some embodiments, pluripotent stem cells are exposed to BMP signaling pathway activators at a concentration of 50 ng / mL or about 50 ng / mL.

[0320] In some embodiments of the methods provided herein, the TGF-β pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542. In some embodiments, the TGF-β pathway inhibitor is A83-01. In some embodiments, the TGF-β pathway inhibitor is provided at a concentration of 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or about those, at least those, at least about those, less than or equal to those, or about less than or equal to those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 100-1000 nM, 100-500 nM, 500-1000 nM, or 300-700 nM. In some embodiments, the TGF-β pathway inhibitor is provided at a concentration of 500 nM or about 500 nM.

[0321] In some embodiments of the methods provided herein, the FGF pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, the FGF signaling pathway activator is FGF2. In some embodiments, the FGF pathway activator is provided at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or about those, at least those, at least about those, less than or about those, or about less than or about those, or any concentration within the 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 5 ng / mL or about 5 ng / mL.

[0322] In some embodiments of the methods provided herein, the Wnt pathway activator is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML284, IQ-1, WAY262611, CHIR99021, CHIR98014, AZD2858, BIO, AR-A014418, SB216763, SB415286, aloysin, indirubin, alsterpaulon, kaempaulon, lithium chloride, TDZD 8, and TWS119. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the Wnt pathway activator is provided at concentrations of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 μM, or about those, at least those, at least about those, less than or about those, or about less than or about those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 1–8 μM, 1–3 μM, 3–8 μM, or 2–4 μM. In some embodiments, the Wnt pathway activator is provided at a concentration of 3 μM or about 3 μM.

[0323] In some embodiments of the methods provided herein, the VEGF pathway activator is selected from the group consisting of VEGF or GS4012. In some embodiments, the VEGF pathway activator is VEGF. In some embodiments, the VEGF pathway activator is provided at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL, or about those, at least those, at least about those, less than or about those, or about less than or about those, or any concentration within the 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 10 ng / mL or about 10 ng / mL.

[0324] In some embodiments of the methods provided herein, the foregut endoderm cells in step c) are cultured in a medium further comprising EGF. In some embodiments, EGF is provided at a concentration of 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 about those, at least those, at least about those, less than or about those, or about less than or about those, or any concentration within the 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. In some embodiments, EGF is provided at a concentration of 20 ng / mL or about 20 ng / mL. In some embodiments, the foregut endoderm cells in step c) are cultured in a medium without EGF.

[0325] In some embodiments of the methods provided herein, the foregut endoderm cells in step c) are cultured in a medium further comprising ascorbic acid. In some embodiments, ascorbic acid is provided at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL, or about those, at least those, at least about those, less than or about those, or about those less than or about those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 10–100 μg / mL, 10–50 μg / mL, 50–100 μg / mL, or 30–70 μg / mL. In some embodiments, ascorbic acid is provided at a concentration of 50 μg / mL or about 50 μg / mL. In some embodiments, the foregut endoderm cells in step c) are cultured in a medium that does not contain ascorbic acid.

[0326] In some embodiments of the methods provided herein, the foregut endoderm cells in step c) are cultured in a medium further comprising a ROCK inhibitor (ROCKi). In some embodiments, the ROCK inhibitor is provided at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about those, at least those, at least about those, less than or about those, or about less than or about those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 1–20 μM, 1–10 μM, 10–20 μM, or 5–1.5 μM. In some embodiments, the ROCK inhibitor is provided at a concentration of 10 μg / mL or about 10 μg / mL. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the foregut endoderm cells in step c) are cultured in a medium that does not contain the ROCK inhibitor.

[0327] Apoptotic agents and anti-adhesion agents in culture media In some embodiments of the methods provided herein, the culture media used in one or more of the aforementioned processes (e.g., liquid culture medium, second liquid culture medium, liquid endoderm differentiation medium, liquid hindgut differentiation medium, liquid IO maturation medium, liquid differentiation medium, and / or liquid organoid maturation medium) may contain an anti-apoptotic agent. Examples of anti-apoptotic agents include, but are not limited to, CEPT or ROCKi. As discussed herein, the use of anti-apoptotic agents such as CEPT may result in higher cell (e.g., PSC) recovery during passage. In some embodiments, CEPT may be present in the culture medium at concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about those, at least those, at least about those, less than or about those, or less than or about those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 1–20 μM, 1–10 μM, 10–20 μM, or 5–1.5 μM. In some embodiments, CEPT is provided at a concentration of 10 μg / mL or about 10 μg / mL.

[0328] In some embodiments of the methods provided herein, the culture medium used in one or more of the processes described above (e.g., liquid culture medium, second liquid culture medium, liquid endoderm differentiation medium, liquid hindgut differentiation medium, liquid IO maturation medium, liquid differentiation medium, and / or liquid organoid maturation medium) may include an anti-adhesion agent. In some embodiments, the anti-adhesion agent may include one or more of dextran sulfate sodium (DSS), xanthan gum, A-205804, I-CAM1, carboxymethyl cellulose, and / or Neural Organoid Basal Medium 2 (NOBM). In some embodiments, the anti-adhesion agent may include DSS. In some embodiments, the anti-adhesion agent (e.g., DSS) may be present in the culture medium at concentrations of 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 μg / mL, or about those concentrations, at least those concentrations, at least about those concentrations, or less than or about those concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 1 to 1000 μg / mL, 1 to 500 μg / mL, 5 to 100 μg / mL, or 5 to 50 μg / mL. In some embodiments, the anti-adhesion agent (e.g., DSS) is provided at a concentration of 10 μg / mL or about 10 μg / mL relative to the culture medium (e.g., liquid culture medium, second liquid culture medium, liquid endoderm differentiation culture medium, liquid hindgut differentiation culture medium, liquid IO maturation culture medium, liquid differentiation culture medium, and / or liquid organoid maturation culture medium). As discussed herein, DSS has been found to suppress (e.g., reduce) the average size of 3D PSC aggregates.

[0329] composition [Examples]

[0330] Some aspects of the embodiments discussed herein are further disclosed in the following examples, and these are not intended to limit the scope of this disclosure. Those skilled in the art will understand that many other embodiments, as described herein and in the claims, also fall within the scope of this disclosure.

[0331] Example 1. Determination of conditions for matrix-free suspension culture of PSCs. A series of experiments were conducted to determine the conditions for successful growth and maintenance of PSCs in suspension culture. Figure 1A illustrates one embodiment of an experimental protocol to investigate the effects of various culture conditions on PSC maintenance and growth. For example, cell culture medium (e.g., TeSR AOF) was obtained for inoculation in the bioreactor (column 104) (see column 102). Low-shear / no-shear bioreactors (Clinostar, CellVivo) were used in the following experiments. Figure 1B illustrates the operation of suspension culture, in which the chamber rotates around its longitudinal axis when the longitudinal axis is oriented parallel to the ground. The illustrated rotating tank bioreactor (cell culture system) rotates continuously to maintain suspended cells, PSC aggregates, spheroids, and / or organoids by balancing gravity, thereby ideally maintaining them in a stationary orbit. As a result, cells grown in the rotating tank bioreactor experience very low shear forces. The bioreactor allows for the suspension of cells in liquid culture medium. In various embodiments, the composition comprising the suspended cell culture undergoes continuous passage (column 106). Furthermore, analysis of the composition may be performed during or after one or more passages (column 108). Analysis may include, but is not limited to, determination of aggregate size distribution, flow cytometry, immunohistochemistry, PCR, and / or triphyletic differentiation.

[0332] Inoculation density Experiments were conducted to investigate the effect of inoculation density on the resulting PSC expansion in suspension culture. Figure 2 illustrates the results of one embodiment of a study investigating the effect of PSC inoculation density in suspension culture medium on PSC aggregate formation and cell death at various time points after culture inoculation. 2 million, 5 million, or 10 million PSCs were inoculated into 10 mL of suspension culture medium. The resulting PSC aggregates were examined after 1, 2, 3, or 4 days. All seeding densities resulted in PSC aggregate formation, but the 5 million and 10 million conditions resulted in significantly more cell death (not agglutinating single cells) and significantly larger aggregates (see markers 202 and 204 in Figure 2), which could lead to the formation of hypoxic cores (see markers 206 and 208 in Figure 2). As a result, a cell density of 2 million (200K cells / mL of culture medium) was used as the inoculation density in subsequent experiments discussed below.

[0333] Dissociation reagent Experiments were conducted to compare various dissociation reagents for preparing single-cell cultures. Single-cell PSC inoculum were prepared using TrypLE, GCDR, and Accutase, cultured in suspension cultures, and the resulting PSC aggregates were examined 24 and 96 hours after inoculation. Figure 3 illustrates the experimental results. TrypLE resulted in the formation of large cell aggregates 24 hours after inoculation. GCDR yielded second-best results, as it was far more difficult to produce single-cell suspensions using this reagent, and its use resulted in the formation of more heterogeneous aggregates (in size and shape). Accutase dissociation proved to be the most optimal method among those tested to obtain single-cell suspensions for the purposes of inoculation, passage, and downstream cell analysis. Therefore, Accutase was used in subsequent experiments discussed below.

[0334] Stage of PSC growth used for vaccination Experiments were conducted to compare how the growth stage of PSCs affects the yield of aggregate formation when they are inoculated into suspension cultures. PSCs grown in adherent cultures were harvested on day 0 (d0), which refers to the day when PSC strains are typically subcultured (80-90% confluence), or on day -1 (d-1), which refers to one day before the adherent PSC culture reaches approximately 40-50% confluence, the "prepared for subculture" confluence. Figure 4A illustrates the iPSC growth curve and the two time points at which the PSCs were harvested. Equal amounts of these d0 or d-1 PSCs were inoculated into suspension cultures, and the number and size of the resulting PSC aggregates were examined on day 4 (d4). Figure 4B illustrates one embodiment of photographs of the starting two-dimensional PSC adherent cultures at d-1 and d0, and the resulting PSC aggregates on day 4 (d4) of suspension culture. Figure 4C illustrates one embodiment of the graph showing the size distribution of PSC aggregates at d4 in suspension cultures using either d-1 or d0 PSC inoculum. The results show that the d-1 PSC inoculum resulted in significantly more PSC aggregates at d4 compared to the d0 PSC inoculum.

[0335] Subculturing stage of PSC suspension culture Experiments were conducted to compare how the size of PSC aggregates at subculturing affects the yield of aggregates formed after reinoculation. PSC aggregates in suspension culture were subculturised on day 3 or 4 by dissociating the PSC aggregates and reinoculating the suspension culture using the resulting single cells. The resulting subculturized cultures were examined 3 days after subculturing. Figure 5 illustrates the experimental results showing the culture at subculturing (day 3 or 4) and the resulting subculturized culture 3 days after subculturing. The results show that subculturing PSC aggregates on day 3 leads to successful culture growth when the diameter of most aggregates is less than 400 μm. In contrast, subculturing PSC aggregates on day 4 results in a much lower yield of aggregates when the diameter of most PSC aggregates is greater than 400 μm. It was concluded that subculturing of PSC aggregates in suspension culture should be performed before their diameter exceeds 350 μm to improve the maintenance of PSCs in suspension culture.

[0336] Bioreactor rotation speed Experiments were conducted to compare how the rotation speed of the bioreactor affects the yield of PSC aggregates formed (see Figures 6A-6C). The Clinostar bioreactor utilizes a cylindrical section with a chamber holding 10 mL of culture medium that rotates around its longitudinal axis. The rotation speed of the chamber can vary. Figure 6A illustrates one embodiment of PSC aggregates on day 3 (d3) of suspension culture at various bioreactor chamber rotation speeds. Figure 6B illustrates one embodiment of a graph showing the size distribution of PSC aggregates at d3 of suspension culture at various bioreactor chamber rotation speeds. Figure 6C illustrates one embodiment of a chart comparing the total cell yield and cell expansion ratio of PSC suspension culture in the first (P1), second (P2), and third (P3) passages at various bioreactor chamber rotation speeds. Almost all bioreactor chamber rotation speeds (except 80 rpm) resulted in successful PSC aggregate formation, but 5 and 40 rpm showed consistent maintenance of cell expansion over three passages.

[0337] Apoptotic agents We conducted an experiment to compare the effects of various anti-apoptotic agents on the cell recovery of PSCs across multiple passages (see Figure 7). Specifically, we compared the anti-apoptotic agents ROCKi and CEPT over two passages and measured the cumulative cell count. As shown in Figure 7, the use of anti-apoptotic agents increased cell (e.g., PSC) recovery in each passage. Furthermore, it was found that the anti-apoptotic agent CEPT provided higher cell recovery than the anti-apoptotic agent ROCKi.

[0338] Culture medium Experiments were conducted to compare how the culture media mTeSR 1 (research medium) and mTeSR AOF (animal product-free medium) affect PSC expansion and maintenance, as well as the expression of stem cell markers. Figure 8 illustrates the results of PSC suspension culture using mTeSR 1 (research medium) or mTeSR AOF (animal product-free medium) in the first (P1), second (P2), and third (P3) passages of suspension culture. Both media tested supported the success of PSC aggregate cell expansion and maintenance with similar levels of PSC expansion and aggregate formation efficiency across multiple passages. Figures 9A and 9B illustrate the effects of the culture media mTeSR 1 (research medium, Figure 9A) and mTeSR AOF (animal product-free medium, Figure 9B) on the expression of stem cell markers: Oct4, SSEA4, and TRA 1-60. Both culture media tested, mTeSR 1 and mTeSR AOF, supported the successful maintenance of stem cell properties by PSC aggregates grown in suspension culture (over 90% of cells expressed stem cell markers Oct4, SSEA4, and TRA 1-60).

[0339] PSC cell line Figure 10 illustrates the results of a study comparing how cell lines (research-grade PSC cell line 72.3 and clinical-grade PSC cell line FF3 produced under GMP) affect the production of PSC aggregates in the first (P1), second (P2), and third (P3) passages of suspension culture. Successful cell expansion and maintenance of stem cell marker expression (Oct4, SSEA4, and TRA 1-60) was observed in both cell lines grown in mTeSR AOF culture medium over three passages. Figures 11A and 11B illustrate the results of a study comparing the effects of two-dimensional (2D) culture (Figure 11A) versus suspension culture (3D) (Figure 11B) on the expression of stem cell markers Oct4, SSEA4, and TRA 1-60 in research-grade PSC cell line 72.3. The results show that over 95% of 72.3 cells maintained stem cell marker expression in mTeSR AOF suspension culture of PSC aggregates. The expression of Oct4, SSEA4, and TRA 1-60 is comparable (if not higher) to that of standard adherent 2D culture as a quiescent monolayer. Figures 12A and 12B illustrate the results of a study comparing the effects of two-dimensional (2D) culture (Figure 12A) versus suspension culture (3D) (Figure 12B) on the expression of stem cell markers Oct4, SSEA4, and TRA 1-60 in clinical-grade PSC cell line FF3 produced under GMP conditions. The results show that over 95% of FF3 cells maintained the expression of stem cell markers in mTeSR AOF suspension culture of PSC aggregates. The expression of Oct4, SSEA4, and TRA 1-60 is comparable (if not higher) to that of standard adherent 2D culture as a quiescent monolayer.

[0340] pluripotency Experiments were also conducted to evaluate pluripotency in three-dimensional culture. For example, Figures 13A and 13B illustrate the results of one embodiment based on a study investigating the formation of three-dimensional PSC aggregates from PSCs, and it was found that the three-dimensional PSC aggregates retained their pluripotency. As shown in the image in Figure 13A, PSCs can form three-dimensional PSC aggregates progressing from day 1 to day 4. As shown in Figure 13B, the number of such aggregates increased from 600 on day 1 to approximately 1400 on day 4. In addition, pluripotency markers such as OCT4 and SSEA4 were evident in the three-dimensional PSC aggregates as shown in confocal imaging. Figures 14A and 14B illustrate the results of evaluating pluripotent 3D PSC aggregates across multiple strains and passages. As shown in Figure 14A, significant formation of three-dimensional PSC aggregates was observed across passages for iPSC strain 72.3 and ESC strain H1. Figure 14B shows the increase in cell number and PSC aggregate diameter over passages for both strains. Furthermore, Figure 14B shows that the expression of pluripotency markers (e.g., OCT4 and SSEA-4) remained at least 90% across all passages and strains tested on the three-dimensional PSC aggregates, similar to the expression in conventionally grown two-dimensional PSC monolayers. Furthermore, Figure 15 illustrates the results of an evaluation comparing the pluripotency of PSC strains grown in three-dimensional suspension culture according to the method described herein with that of PSC strains grown in conventional 2D monolayers. As shown in Figure 15, the pluripotency of PSC strains (H1 and 72.3) grown in three-dimensional suspension culture is increased compared to their strains grown in 2D monolayers, based on higher gene expression of pluripotency markers OCT4, SOX2, and KLF4.

[0341] Example 2. Generation of HCl in suspension culture Figure 16 illustrates one embodiment of an experimental protocol for matrix-free suspension culture production of HIO from hiPSCs. As shown in Figure 16, the protocol may involve the formation of 3D iPSC cultures (e.g., using a bioreactor), including the formation of 3D PSC aggregates. Acclimatization to 3D PSC culture may involve single-cell dissociation. The protocol may further involve the differentiation of 3D iPSCs into 3D endoderm (DE) (e.g., via the use of activin A in the culture medium for DE induction). The protocol may further involve the differentiation of 3D DE cultures into hindgut spheroids (e.g., via the use of CHIR99021 and / or FGF4 in the culture medium). The protocol may further involve the development of HIO (e.g., via single-cell dissociation and the use of EGF).

[0342] PSC acclimatization to suspension culture Figure 17 illustrates the results of a study investigating the effect of PSC acclimation to suspension culture on HIO production at various time points. It compares HIO-differentiated cells either a) immediately after inoculation from 2D monolayer to 3D suspension culture (unacclimatized) or b) after one passage in 3D suspension culture (acclimatized). Inoculation methods and culture conditions are provided in more detail below. The results indicate that a lack of cell acclimation to 3D suspension culture (by passage and reintroduction into 3D bioreactor culture) resulted in significant cell death and failure of HIO development. One passage in suspension culture was demonstrated to be sufficient to acclimate cells to 3D suspension culture. Therefore, PSC acclimation to suspension culture is crucial for the successful formation of human intestinal organoids.

[0343] Induction of endoderm in suspension culture Figures 18A and 18B illustrate the results of a study comparing the efficiency of DE induction in two-dimensional (2D) aggregate monolayer culture (Figure 18A) and suspension culture (3D) (Figure 18B) by examining the expression of endoderm markers Sox 17 and FoxA2. DE was induced using the method described in more detail below. The success of DE induction in 3D suspension culture was confirmed by immunofluorescence staining and flow cytometry analysis. The results show comparable DE induction efficiency between 2D and 3D cultures (approximately 50% + / + Sox17 / FoxA2 cells). The experiment also showed that activin A contributes to DE differentiation efficiency when applied at specific time points. Figure 19 illustrates the results of one embodiment demonstrating the effect of exposure to activin A at different time points (e.g., 24 hours, 48 ​​hours, or 72 hours after passage) on DE induction efficiency in 3D suspension culture. As shown in Figure 19, 3D PSC aggregates exposed to activin A 48 hours after passage showed the highest DE differentiation efficiency based on the expression of FoxA2 and Sox17.

[0344] Effect of 3D PSC aggregate size on intestinal tissue differentiation Experiments were also conducted to compare the effect of 3D PSC aggregate size on intestinal tissue differentiation at various stages of the differentiation process. For example, Figure 20 shows the results of one embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the DE stage. As shown in Figure 20, the size of 3D PSC aggregates upon exposure to activin determines the efficiency of DE induction. Furthermore, Figure 20 shows that smaller sizes of 3D PSC aggregates (e.g., average aggregate diameter less than approximately 400 μm (e.g., less than approximately 300 μm)) ensure better DE induction. Figure 21 illustrates the results of one embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the HGS stage. The effect was demonstrated by the expression of CDX2, a marker of intestinal tissue differentiation. As shown in Figure 21, there is strong CDX2 expression at the hindgut stage of differentiation of 3D PSC aggregates with a diameter smaller than approximately 300 μm at DE induction. However, 3D PSC aggregates with a diameter greater than approximately 300 μm at DE induction showed weaker and more sparse CDX2 expression. The results further confirm the importance of the initial size of 3D PSC aggregates for differentiation efficiency. Figure 22 illustrates the results of one embodiment demonstrating the effect of 3D PSC aggregate size on intestinal tissue differentiation at the HIO stage. The effect was demonstrated by the expression of CDX2, a marker for intestinal tissue differentiation. As shown in Figure 22, differentiation of 3D PSC aggregates with a diameter of approximately 300 μm or less resulted in apical inward-facing HIO formation and substantially uniform CDX2 expression across the generated HIO. However, differentiation of 3D PSC aggregates with a diameter of at least 300 μm resulted in mixed apical outward-facing and apical outward-facing structures, as well as epithelial structures with weak or no CDX2 expression.

[0345] The effect of anti-adhesion agents on the size of 3D PSC aggregates Since other experiments discussed herein have shown the beneficial effect of having a smaller average size of 3D PSC aggregates, experiments were also conducted to investigate the effect of anti-adhesion agents on 3D PSC aggregates. For example, as previously discussed, 3D PSC aggregates with a smaller size (e.g., a diameter of less than approximately 400 μm (e.g., less than approximately 300 μm)) are preferable for intestinal tissue differentiation.

[0346] Specifically, Figures 23-27 illustrate the results of one embodiment based on a study investigating the effect of the anti-adhesion agent dextran sulfate sodium (DSS) on the size-mediating properties of 3D PSC aggregates. However, it is intended that other anti-adhesion agents, such as xanthan gum, may also have a similar effect on the size of 3D PSC aggregates. The image in Figure 23 shows the effect of various concentrations of DSS on the size of 3D PSC aggregates, with a concentration of 10 μg / mL having the greatest effect in reducing the size of 3D PSC aggregates. Figure 24 further shows that the effect of DSS on reducing aggregate size was observed across different PSC strains (72.3, FF3, H1, and H1 GFP). Figure 25 further shows that a DSS concentration of 10 μg / mL had the greatest reduction in the average diameter across the line, while simultaneously increasing the yield of aggregates formed. Figures 26 and 27 further investigate the effect of 10 μg / mL of DSS on the average size of PSC aggregates across different strains. Specifically, Figure 26 shows that 10 μg / mL of DSS is sufficient to induce smaller diameter PSC aggregates, resulting in an average reduction of approximately 200 μm in the average diameter of PSCs after DSS treatment compared to the untreated control. Figure 27 shows that this effect is consistent across different iPSC and ESC strains, resulting in a shift in the frequency of the PSC aggregate size distribution.

[0347] The effect of anti-adhesion agents on the pluripotency of 3D PSC aggregates The experiment also demonstrated that antifouling agents have no negative effect on pluripotency, and therefore make antifouling agents a useful means of suppressing 3D PSC aggregate size. Specifically, Figure 28 illustrates the results of one embodiment based on a study investigating the effect of DSS on the pluripotency of 3D PSC aggregates, as measured by the pluripotency markers SOX2 and OCT4. As shown in the confocal image of Figure 28, DSS had no negative effect on the pluripotency of any of the concentrations of 3D PSC aggregates tested. The study also investigated the effect of DSS on the viability of 3D PSC aggregates, as measured by the release of the survival marker lactate dehydrogenase (LDH). The study found that DSS at concentrations of 1000 μg / mL or less had no negative effect on the viability of PSC aggregates.

[0348] Comparison of treatment regimens for applying anti-adhesion agents Figure 29 illustrates the results of one embodiment based on a study investigating the effects of various treatment regimens for applying DSS on the average size, number, and pluripotency of 3D PSC aggregates. As shown in Figure 29, the treatment regimens tested included control (i.e., no treatment regimen), inoculation, and overall. The results show that treatment with 10 ug / mL of DSS at inoculation is sufficient to maintain PSC aggregates below 400 μm while having no negative effect on PSC aggregate number or pluripotency gene expression. However, long-term treatment with DSS (overall) results in a decrease in OCT4 expression.

[0349] The effect of anti-adhesion agents on controlling the size of 3D PSC aggregates maintained over multiple generations. The experiment also demonstrated that the aforementioned beneficial effects of the anti-adhesion agent in controlling the size of 3D PSC aggregates (e.g., reduction of average diameter) were maintained across passages. Figure 30 illustrates the results of one embodiment based on a study investigating the effect of DSS on the average size of 3D PSC aggregates across passages. As shown in Figure 30, the effect of DSS treatment on PSC aggregate size (reduction in diameter) was maintained across multiple passages.

[0350] The effect of anti-adhesion agents on the tendency of 3D PSC aggregates to differentiate. Figure 31 illustrates the results of one embodiment based on a study investigating the effect of DSS on the differentiation tendency of 3D PSC aggregates. The tendency is measured based on differentiation efficiency characterized by the expression of markers FOXA2 and SOX17. As shown in Figure 31, similar differentiation efficiency of 3D PSC aggregates toward differentiation into DEs is found in the presence and absence of DSS, as indicated by the expression of Sox17 and FoxA2, ​​demonstrating that DSS treatment has no negative effect on the cell tendency toward differentiation.

[0351] HIO generation in suspension culture Figure 32 illustrates the results of an experiment demonstrating well-patterned HIO development in suspension cultures with either apical outward or apical inward epithelial polarity (see below for further details). Induction of HIO from DE was performed using the method described in more detail below. Successful development of precisely patterned HIO in suspension culture, confirmed by immunofluorescence staining for CDx2 and E-cadherin. Figure 33 illustrates the results of one embodiment based on well-patterned HIO development in 3D suspension culture. Successful development of precisely patterned HIO in suspension culture is confirmed by immunofluorescence staining. Markers CDX2, ZO-1, and Vim1 represent differentiation to HIO. Figures 34A and 34B illustrate the results of an experiment demonstrating in vivo maturation of HIO developed in suspension culture after subcapsular transplantation of mice. Figure 34A is a photograph of one embodiment of HIO 9 weeks after subcapsular transplantation. Figure 34B shows one embodiment of H&E staining of HTO 9 weeks after subcapsular transplantation. HIO generated using the described suspension culture method showed efficient engraftment and proper maturation after subcapsular transplantation.

[0352] Modification of HIO epithelial cell polarity in suspension culture Figure 35 illustrates the results of an experiment demonstrating that the polarity of epithelial cells in HIOs generated in suspension culture can be altered. The method used for HIO generation is described in more detail below. Dissociation of hindgut spheroids on day 7 (+dissociation) and their re-aggregation in suspension culture results in an inner apical surface (apex facing inward) of the HIO. If the DEs have not dissociated on day 7 (-dissociation), the result is an outer apical surface (apex facing outward) of the HIO. These results indicate that dissociation of hindgut spheroids on day 7 and their re-aggregation in suspension culture result in a reversal of epithelial polarity from apex facing outward to apex facing inward.

[0353] Materials and methods The following is an exemplary procedure used in Experiment 2.

[0354] Bioreactor inoculation for iPSC maintenance In some embodiments, bioreactor inoculation for iPSC maintenance may be initiated after one or more passagings (e.g., day 3 of a 5-day passaging, day 4 of a 5-day passaging, day 5 of a 5-day passaging). In some embodiments, cells may be maintained in 2D (e.g., monolayer) culture until they are deemed suitable for inoculation.

[0355] In some embodiments, inoculation may begin with aspirating the culture medium and adding a dissociation reagent (e.g., TrypLE, GCDR, and / or Accutase) to each well of a multiwell plate containing the sample to be inoculated. The contents of the multiwell plate may be incubated until a sufficient number of cells have detached from the plate.

[0356] In some embodiments, inoculation may further include collecting cells and adding them to a conical tube. A culture medium may be added to the tube and gently titrated to further decompose the colonies into a single-cell suspension.

[0357] In some embodiments, the culture may settle, the supernatant may be discarded, and the medium may be re-added. The cells may be gently resuspended in the medium to prepare a single-cell suspension.

[0358] In some embodiments, an anti-adhesion agent such as CEPT or ROCKi in a 10 μM (1:1000 dilution) solution may be added to the suspension to prevent cell death. The cells are then counted to determine the amount required to obtain an appropriate number or range of cells. In some embodiments, the appropriate number may be about 0.5 × 10^6 million cells, 1 × 10^6 million cells, 1.5 × 10^6 million cells, 2 × 10^6 million cells, 2.5 × 10^6 million cells, 3 × 10^6 million cells, 3.5 × 10^6 million cells, 4 × 10^6 million cells, 4.5 × 10^6 million cells, or 5 × 10^6 million cells. In some embodiments, the appropriate range of cells may be a range formed by any two of the aforementioned cell numbers.

[0359] Next, a composition containing culture medium, cells, and any of the aforementioned components (such as anti-adhesion agents and dissociation agents) may be added to a bioreactor (e.g., Clinostar) to fill the internal chamber. In some embodiments, depending on the amount of ROCKi added with the cells, some amount of anti-adhesion agent, such as 10 μM CEPT or ROCKi, may be added to the system. The bioreactor may be rotated (e.g., at about 5 RPM) to allow the cells to aggregate (e.g., for about 24 hours).

[0360] Medium exchange for any application In some embodiments, changes in composition within the bioreactor may be visualized to ensure that the sample is free from any abnormalities (e.g., large aggregates, contamination, etc.). In some embodiments, the bioreactor may be positioned upright to allow cell aggregates to settle at the bottom of the bioreactor. In some embodiments, culture medium may be slowly added to the bioreactor while ensuring that air bubbles are removed from the system and excess medium outside the bioreactor system is aspirated and removed. In some embodiments, the bioreactor may be rotated horizontally to disperse cells throughout the inner chamber. The bioreactor speed may be adjusted to reduce or eliminate the possibility of aggregates colliding with the walls of the inner chamber.

[0361] Passaging of iPSC aggregates in the ClinoStar system In some embodiments, the dissociation agent (e.g., TrypLE, GCDR, and / or Accutase) and culture medium may be preheated (e.g., to about 37°C). The contents of the bioreactor may be visualized to assess the quality and size of the aggregates.

[0362] In some embodiments, the aggregates can be divided before reaching a given size (for example, before the aggregates reach about 500 μm, 450 μm, 400 μm, 350 μm, or 300 μm).

[0363] The aggregates can be continued to be cultured according to the aforementioned process until they are deemed ready for subculturing.

[0364] In some embodiments, subculturing may be initiated by allowing aggregates to settle (for example, by arranging the bioreactor vertically), and then by exposing the inside of the bioreactor chamber (for example, by turning the bioreactor on its side and lifting the lid).

[0365] In some embodiments, cell aggregates may be gently transferred to one or more wells of a multiwell plate for size quantification.

[0366] In some embodiments, cell aggregates may be imaged for BF image analysis (as described herein).

[0367] In some embodiments, the aggregates can be transferred to a conical tube to remove and discard excess culture medium.

[0368] In some embodiments, a dissociating agent (e.g., 1 mL of Accutase) may be added to the aggregates. In some embodiments, the aggregates may be incubated in a warm bead bath for a period of time, ensuring that the aggregates are mixed. The aggregates may be visualized periodically to assess the progress of dissociation. After the aggregates have dissociated, a certain amount of culture medium (e.g., about 5 mL) may be added (e.g., via a conical tube). The culture may settle, and the supernatant may be removed. The cells may be resuspended in the culture medium, and a certain amount of anti-adhesion agent (e.g., 10 μM of CEPT or ROCKi) may be added. The sample may be gently titrated to obtain a single-cell suspension. Cells and magnification may be counted and recorded.

[0369] In some embodiments, cells may be inoculated into a novel bioreactor using the method described herein.

[0370] iPSC culture and maintenance (e.g., using the ClinoStar system) In some embodiments, the process may be initiated by preparing a bioreactor and adding cells to the bioreactor, as previously discussed. The culture schedule may be as shown in Table 1 below. If the aggregates are larger than the specified size (e.g., 500 μm, 450 μm, 400 μm, 350 μm, or 300 μm), subculturing may be performed before reaching d4.

[0371] [Table 1]

[0372] Endoderm (DE) formation In some embodiments, the process may begin with inoculating a suitable number of cells into a bioreactor to allow growth over one or more passages. The sample may be divided into d3 cells and reinoculated back into the bioreactor to allow for 3D adjustment.

[0373] In some embodiments, the process may further include preparing a culture medium based on Table 2 shown below. DE induction may be initiated approximately 48 hours after the second inoculation.

[0374] In D0, D0 medium may be added according to the medium exchange protocol. The starting material may be imaged. In D1, D1 medium may be added according to the medium exchange protocol. The material may be imaged to ensure small changes are observed.

[0375] In D2, the D2 medium may be added according to the medium exchange protocol. The material may be imaged to ensure that even small changes are observed.

[0376] In D3, a small subset of the material may be obtained. In some embodiments, the subset may be fixed in PFA for a period of time (e.g., about 30 minutes to 1 hour). In some embodiments, the subset may be fixed from PFA to PBS+ / + and left to cool. The material may be imaged to ensure the expression of Sox17+ / FoxA2+ aggregates.

[0377] In some embodiments, a subset of cells may be obtained to perform DE FACS. The subset may be tested to ensure that 80% of the Oct4 population is Sox17+ / FoxA2+.

[0378] In some embodiments, the remaining material can be used to advance the process to promote mid-hindgut formation.

[0379] [Table 2]

[0380] Mid-hindgut generation In some embodiments, the process for mid-hindgut formation may involve preparing d3-d6 media according to Table 3. The media may be changed and imaged daily.

[0381] [Table 3]

[0382] HiO mature In some embodiments, the process for HiO maturation may begin with mid-hindgut dissociation. On D7, aggregates at the bottom of the bioreactor can be collected (e.g., using a cut tip). The aggregates can be transferred to a conical tube.

[0383] In some embodiments, the remaining culture medium may be aspirated after the aggregates have settled again. In some embodiments, a preheated dissociating agent (e.g., TrypLE, GCDR, and / or Accutase) may be added, and the tube holding the sample may be placed in a warm bead bath. The aggregates may be periodically resuspended until they are visually broken down.

[0384] In some embodiments, a culture medium containing serum may be added to the mixture and gently transferred by pipette. The sample may then be spun. The liquid may be aspirated. The aggregates may be resuspended in HiO medium, as shown in Table 4 below. The process may further involve counting and recording the amount produced.

[0385] In some embodiments, an appropriate amount of cells (e.g., about 4 × 10^6 cells) may be added to a new bioreactor in HiO medium containing an anti-adhesion agent (e.g., 1:1000 CEPT or ROCKi). The bioreactor may be rotated overnight for aggregate formation (e.g., at about 5 RPM).

[0386] In some embodiments, the HiO maturation process may involve periodically changing the culture medium (e.g., every other day or when it turns yellow) using the culture medium shown in Table 4.

[0387] In some embodiments, if the sample aggregates become excessively dense, the aggregates may be transferred to separate bioreactors. Indicators of an excessively dense culture may include, but are not limited to, rapid acidification of the medium, cell drop due to shear stress, and a visually very high number of HiO.

[0388] The samples are monitored daily to ensure continued growth until approximately day 34.

[0389] [Table 4]

[0390] Aggregate size BF analysis In some embodiments, the process for aggregate size BF analysis may begin by allowing the aggregates to settle (e.g., by positioning the bioreactor vertically). After the aggregates have settled, the process may involve exposing the inside of the bioreactor (e.g., by turning the bioreactor on its side and flattening it, and removing the front panel). The process may further involve gently transferring the cell aggregates into one or more wells of a multiwell plate (e.g., using a pipette tip) for size quantification. The sample may be imaged, and the images may be saved (e.g., on Leica software) and opened (e.g., on FIJI).

[0391] In some embodiments, the image format may be converted, and color imbalances may be removed or adjusted via thresholding. In some embodiments, a mask may be applied or adjusted.

[0392] In some embodiments, the process may further involve opening software for size calculation (e.g., Microsoft Excel) and calculating the diameter based on area measurements. Buckets may be created for each aggregate size range in increments (e.g., about 100 μm). Appropriate visualizations (e.g., bar graphs) may be created.

[0393] In at least some of the embodiments described above, one or more elements used in one embodiment may be interchangeably used in another embodiment, unless such substitution is not technically feasible. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter as defined by the appended claims.

[0394] With regard to substantially all use of plural and / or singular terms herein, those skilled in the art can paraphrase from plural to singular and / or singular to plural as appropriate to the context and / or use. For clarity, various singular / plural substitutions can be clearly described herein.

[0395] It will be understood by those skilled in the art that, in general, the terms used herein, and especially in the appended claims (e.g., the text of the appended claims), are generally intended to be “non-limiting” terms (for example, the term “including” should be interpreted as “including but not limited,” the term “having” should be interpreted as “having at least,” and the term “including” should be interpreted as “including but not limited”). If there is an intended specific number of claims to be introduced, such intent will be explicitly stated in the claims, and if such statement is not present, such intent is not present. For example, for the sake of understanding, the appended claims below may include the use of the introductory phrases “at least one” and “one or more” to introduce the claims. However, the use of such phrases should not be interpreted as implying that the introduction of a claim detail by the indefinite article "a" or "an" limits any particular claim containing such introduced claim detail to only one embodiment containing such detail (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"). The same applies to the use of definite articles used to introduce a claim description. In addition, even if a particular number of claim details being introduced is explicitly detailed, a person skilled in the art will recognize that such detail should be interpreted as meaning at least the number detailed (for example, the explicit detail "two details" without other modifying phrases means at least two details or two or more details).Furthermore, where a convention similar to "at least one of A, B, and C, etc." is used, such a construction is generally intended to mean what a person skilled in the art would understand the convention to be (for example, "a system having at least one of A, B, and C" includes, but is not limited to, A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or a system having A, B, and C together, etc.). Where a convention similar to "at least one of A, B, or C, etc." is used, such a construction is generally intended to mean what a person skilled in the art would understand the convention to be (for example, "a system having at least one of A, B, or C" includes, but is not limited to, A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or a system having A, B, and C together, etc.). It will be further understood by those skilled in the art that substantially any disjunctive word and / or phrase representing two or more alternative terms should be understood as construing the possibility of including one of the terms, either of the terms, or both of the terms, regardless of the modes for carrying out the invention, claims, or drawings. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B".

[0396] In addition, if any feature or aspect of the present disclosure is described by the Markush Group, a person skilled in the art will recognize that the present disclosure may also be described by any individual member or subgroup of a member of the Markush Group.

[0397] For all purposes, including the standpoint of documenting the scope in a manner that can be understood by those skilled in the art, all scopes disclosed herein also encompass all possible subscopes and combinations thereof. It is readily apparent that any enumerated scope can be adequately described and made possible to decompose the same scope into at least two, three, four, five, ten, etc. As a non-limiting example, each scope considered herein can be readily decomposed into a lower third, a middle third, an upper third, etc. As can be understood by those skilled in the art, all words such as “maximum,” “at least,” “greater than,” and “less than” include the number described and refer to a scope that can be later decomposed into subscopes as considered herein. Finally, as can be understood by those skilled in the art, a scope includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0398] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be obvious to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to limit, and the true scope and spirit are set forth in the following claims.

[0399] All references cited herein, including but not limited to published and unpublished applications, patents, and documents, are incorporated into this Specified in their entirety by reference and become part of this Specified. To the extent that any publications and patents or patent applications incorporated by reference conflict with the disclosures contained herein, this Specified is intended to take precedence over and / or supersede such conflicting material.

Claims

1. (a) Inoculate the liquid culture medium with PSCs. (b) Culturing the liquid culture medium inoculated with the PSCs in a bioreactor such that three-dimensional PSC aggregates are formed in the liquid culture medium, wherein the cultivation in the bioreactor includes suspending the PSCs in the liquid culture medium. (c) (i) Dissociating at least a portion of the three-dimensional PSC aggregate into single cells, (ii) A method for subculturing PSCs by inoculating the dissociated three-dimensional PSC aggregates from (i) together with PSCs into a second liquid culture medium.

2. The method according to claim 1, wherein the liquid culture medium and the second liquid culture medium do not contain any animal or human-derived material, and optionally, the liquid culture medium and the second liquid culture medium do not contain any extracellular matrix and / or basement membrane matrix.

3. The method according to claim 1 or 2, wherein the PSCs are subcultured when the diameter of most of the formed three-dimensional PSC aggregates is 500 μm or less.

4. The method according to any one of claims 1 to 3, wherein the PSCs are subcultured when the diameter of most of the formed three-dimensional PSC aggregates is 400 μm or less.

5. The method according to any one of claims 1 to 4, wherein the PSCs are subcultured when the diameter of most of the formed three-dimensional PSC aggregates is 300 μm or less.

6. The method according to any one of claims 1 to 5, wherein at least 80% of the three-dimensional PSC aggregate dissociates into single cells.

7. The method according to any one of claims 1 to 6, wherein, optionally, at least 90% of the PSC aggregates dissociate into single cells.

8. The method according to claim 1, wherein the method comprises subculturing the PSCs two or more times by culturing the PSCs in a second inoculated liquid culture medium until additional three-dimensional PSC aggregates are formed.

9. The method according to any one of claims 1 to 8, wherein the liquid culture medium of (a) and / or the second liquid culture medium of (c)(ii) are inoculated at a density of about 100,000 to 220,000 PSCs / mL.

10. The method according to any one of claims 1 to 9, wherein the liquid culture medium of (a) and / or the second liquid culture medium of (c)(ii) are inoculated at a density of about 180,000 to 220,000 PSCs / mL.

11. The method according to any one of claims 1 to 10, wherein the passage occurs after a period of approximately 40 to 168 hours after the inoculation in (a).

12. The method according to any one of claims 1 to 11, wherein the passage occurs after a period of approximately 40 to 84 hours after the inoculation in (a).

13. The method according to any one of claims 1 to 12, wherein the passage occurs after a period of approximately 66 to 78 hours after the inoculation in (a).

14. The method according to any one of claims 1 to 13, further comprising replacing a portion of the culture medium in the bioreactor in (a) after a period of about 36 to 60 hours following the inoculation in (a) and / or (c)(ii).

15. The method according to any one of claims 1 to 14, further comprising replacing a portion of the culture medium in the bioreactor in (a) after a period of about 42 to 54 hours following the inoculation in (a) and / or (c)(ii).

16. The method according to any one of claims 1 to 15, further comprising replacing a portion of the liquid culture medium in the bioreactor in (a) after a certain period following inoculation in (a) and / or (c)(ii), wherein the portion of the replaced liquid culture medium is at least 50% of the liquid culture medium in the bioreactor in (a).

17. The above method, prior to the inoculation in (a), Culturing the PSCs on the surface of the substrate, The method according to any one of claims 1 to 16, wherein the PSCs are in the logarithmic growth phase and / or have a confluence of 35 to 55%, and the method further comprises, optionally, collecting the PSCs from the surface of the substrate for use in the inoculation of (a), wherein the collection includes dissociating the PSCs before the inoculation of (a).

18. The method according to claim 17, wherein the PSCs are collected from the surface of the substrate for use in the inoculation of (a) when the PSCs are in the logarithmic growth phase and / or have a confluence of 40-50%.

19. The method according to any one of claims 1 to 18, wherein the dissociation is chemical, enzymatic, and / or mechanical dissociation.

20. The method according to any one of claims 1 to 19, wherein the dissociation is enzymatic, and optionally the enzyme comprises a protease and / or a collagenase, and optionally the enzyme is Accutase.

21. The method according to any one of claims 1 to 20, wherein the bioreactor of (a) and / or (b) includes a rotating chamber containing the liquid culture medium, the rotational speed of the rotating chamber is selected such that the number of PSCs in the liquid culture medium in (c) is at least twice or 2.5 times the number of PSCs used to inoculate the liquid culture medium, and optionally, the number of PSCs in the liquid culture medium in (c) is at least twice or 2.5 times the number of PSCs used to inoculate the liquid culture medium for at least two passages of the PSCs.

22. The method according to any one of claims 1 to 21, wherein the liquid culture medium is a serum-free medium, and optionally the medium comprises recombinant human basic fibroblast growth factor (rh bFGF) and / or recombinant human transforming growth factor β (rh TGFβ).

23. The method according to any one of claims 1 to 22, wherein, after one or more passages, at least 85% of the PSCs express Oct4, SSEA1, and TRA 1-60 at a level at least the same as the average expression level of the PSCs used in the inoculation in (a).

24. The method according to claim 23, wherein, after one or more passages, at least 95% of the portion of the PSC expressing Oct4, SSEA1, and TRA 1-60 at a level at least the same as the average expression level of the PSC used in the inoculation in (a) is the same as the portion of the PSC expressing Oct4, SSEA1, and TRA 1-60.

25. The method according to any one of claims 1 to 24, wherein the PSC expresses SOX2 and KLF4.

26. A method for differentiating PSCs into endoderm (DE) in three-dimensional suspension culture, wherein the method is (d) Culturing a liquid culture medium inoculated with PSCs in a bioreactor, (d) The cultivation of the liquid culture medium inoculated with the PSC includes suspending the PSC in the liquid culture medium, (e) A method comprising culturing the PSCs of (d) in a liquid endoderm differentiation culture medium in a bioreactor for a period of time sufficient to differentiate the PSCs into DEs, wherein the culturing of the PSCs of (d) in the liquid endoderm differentiation culture medium includes suspending the PSCs in the liquid endoderm differentiation culture medium.

27. The method according to claim 26, wherein the liquid culture medium or the liquid endoderm differentiation culture does not contain any material of animal or human origin, and optionally the culture medium does not contain any extracellular matrix and / or basement membrane matrix.

28. The method according to claim 26 or 27, wherein the culture in (d) is for a period of about 18 to 54 hours.

29. The method according to any one of claims 26 to 28, wherein the culture in (d) is for a period of about 24 to 48 hours.

30. The method according to any one of claims 26 to 29, wherein the liquid culture medium inoculated with the PSC cultured in (d) is the PSC inoculated culture medium of (c)(ii) according to any one of claims 1 to 24.

31. The method according to any one of claims 26 to 30, wherein the period sufficient to differentiate the PSC into DE is a period of about 48 to 96 hours.

32. The method according to any one of claims 26 to 31, wherein the period sufficient to differentiate the PSC into DE is a period of about 60 to 84 hours.

33. The method according to any one of claims 26 to 32, wherein the period sufficient to differentiate the PSC into DE is a period of about 66 to 78 hours.

34. The method according to any one of claims 26 to 33, wherein culturing the PSCs in the liquid embryonic endoderm differentiation culture medium for a period sufficient to differentiate the PSCs into DEs comprises: a first period of culturing the PSCs in a culture medium containing nodal signaling pathway activator and / or Wnt signaling pathway activator; a second period of culturing the PSCs in a culture medium containing nodal signaling pathway activator and / or Wnt signaling pathway activator and serum or a serum substitute; and a third period of culturing the PSCs in a culture medium containing nodal signaling pathway activator and / or Wnt signaling pathway activator and serum or a serum substitute.

35. The method according to claim 34, wherein the culture medium in which the PSCs are cultured for the first period further comprises a BMP activator.

36. The culture medium in which the PSCs are cultured for the second period, and the culture medium in which the PSCs are cultured for the third period, The method according to claim 34 or 35, comprising the nodal signaling pathway activator and / or the Wnt signaling pathway activator and serum, wherein optionally the serum is FBS.

37. The culture medium in which the PSCs are cultured for the second period, and the culture medium in which the PSCs are cultured for the third period, The method according to claim 34 or 35, comprising the nodal signaling pathway activator and / or the Wnt signaling pathway activator and a serum substitute, wherein the serum substitute is optionally a knockout serum substitute (KSR).

38. The method according to any one of claims 34 to 37, wherein each of the first, second, and third periods is approximately 18 to 30 hours.

39. The method according to any one of claims 34 to 38, wherein each of the first, second, and third periods is approximately 20 to 28 hours.

40. The method according to any one of claims 26 to 39, wherein the efficiency of DE induction is at least about 35%.

41. The method according to any one of claims 26 to 40, wherein the efficiency of the DE induction is at least about 45 to 55%.

42. The method according to any one of claims 26 to 41, wherein the DE expresses Sox17 and FoxA2.

43. A method for differentiating endoderm (DE) into hindgut spheroid (HGS) in three-dimensional suspension culture, wherein the method is (f) A method comprising culturing the DE in a liquid hindgut differentiation culture medium in a bioreactor for a period sufficient to differentiate the DE into HGS, wherein the culturing of the DE includes suspending the DE in the liquid hindgut differentiation culture medium.

44. The method according to claim 43, wherein the liquid hindgut differentiation culture medium does not contain any animal or human-derived material, and optionally, the liquid hindgut differentiation culture medium does not contain any extracellular matrix and / or basement membrane matrix.

45. The method according to claim 43, wherein the DE cultured in (f) is the DE according to any one of claims 25 to 39.

46. The method according to any one of claims 43 to 45, wherein the period sufficient to differentiate the DE into HGS is a period of about 60 to 120 hours.

47. The method according to any one of claims 43 to 46, wherein the period sufficient to differentiate the DE into HGS is a period of about 84 to 108 hours.

48. The method according to any one of claims 43 to 47, wherein the period sufficient to differentiate the DE into HGS is a period of about 90 to 102 hours.

49. The method according to any one of claims 43 to 48, wherein the liquid hindgut differentiation culture medium is replaced after a period of about 20 to 28 hours.

50. The method according to any one of claims 43 to 49, wherein the liquid hindgut differentiation culture medium is replaced after a period of about 22 to 26 hours.

51. The method according to any one of claims 43 to 50, wherein the liquid hindgut differentiation culture medium comprises Wnt signaling pathway activator, FGF signaling pathway activator, and optionally FBS.

52. The method according to claim 51, wherein the Wnt signaling pathway activator comprises CHIR99021.

53. The method according to claim 51 or 52, wherein the FGF signaling pathway activator comprises FGF4.

54. The method according to any one of claims 51 to 53, wherein the FGF signaling pathway activator is FGF4 at a concentration of about 50 to 750 ng / mL.

55. The method according to any one of claims 51 to 54, wherein the Wnt pathway activator is CHIRON 99021 at a concentration of approximately 0.5 to 6 μM.

56. A method for differentiating hindgut spheroids (HGS) into intestinal organoids (IO) in three-dimensional suspension culture, wherein the method is (g) A method comprising culturing the HGS in a liquid IO maturation culture medium in a bioreactor 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 medium.

57. The method according to claim 56, wherein the liquid IO mature culture medium does not contain any animal or human-derived material, and optionally the culture medium does not contain any extracellular matrix and / or basement membrane matrix.

58. The method according to claim 56 or 57, wherein the HGS cultured in (g) is the HGS according to any one of claims 40 to 52.

59. The method according to any one of claims 56 to 58, wherein the period sufficient to differentiate the HGS into IO is a period of about 12 to 30 days.

60. The method according to any one of claims 56 to 59, wherein the period sufficient to differentiate the HGS into IO is a period of about 15 to 28 days.

61. The method according to any one of claims 56 to 60, wherein the IO maturation culture medium is replaced after a period of about 24 to 54 hours.

62. The method according to any one of claims 56 to 61, wherein the IO maturation culture medium is replaced after a period of about 46 to 50 hours.

63. The method according to any one of claims 56 to 62, wherein the IO maturation culture medium comprises one or more of EGF, R-spongin, noggin, gremlin 1, and / or epiregulin (EREG).

64. The method according to claim 63, wherein the concentrations of EGF, R-sponging, noggin, gremlin 1, and / or EREG are about 25 to 150 ng / mL.

65. The method according to claim 63 or 64, wherein the concentrations of EGF R-sponging, noggin, gremlin 1, and / or EREG are about 50 to 100 ng / mL.

66. The method according to any one of claims 56 to 65, wherein the HGS does not dissociate before being cultured in the IO maturation culture medium, and the epithelial cells of the formed IO have polarity such that their apical surface is oriented outward from the IO.

67. A method for differentiating hindgut spheroids (HGS) into intestinal organoids (IO) having an inwardly polar apical end in three-dimensional suspension culture, wherein the method comprises the method according to any one of claims 56 to 66. The method further comprises dissociating at least a portion of the HGS into HGS single cells before incubation in the IO maturation culture medium, The culture of HGS comprises suspending dissociated HGS single cells and any undissociated HGS in the liquid IO maturation culture medium, A method wherein the epithelial cells of the IO formed from the dissociated HGS single cell have polarity such that the apical surface is oriented inward toward the IO.

68. The method according to claim 67, wherein the liquid IO mature culture medium does not contain any animal or human-derived material, and optionally, the liquid IO mature culture medium does not contain any extracellular matrix and / or basement membrane matrix.

69. The method according to claim 67 or 68, wherein at least 80% of the HGS dissociates into single cells.

70. The method according to any one of claims 67 to 69, wherein at least 90% of the HGS dissociates into single cells.

71. A certain concentration of dissociated HGS single cells is present in the IO maturation culture medium, and the concentration is approximately 0.05 × 10⁻¹⁶ relative to the IO maturation culture medium. ５ ~80 x 10 ５ The method according to any one of claims 67 to 70, wherein the amount is 1 dissociated HGS single cell / mL.

72. Dissociated HGS single cells at a certain concentration are present in the IO maturation culture medium, and the concentration is approximately 10 × 10 in relation to the IO maturation culture medium. ５ ~80 x 10 ５ The method according to any one of claims 67 to 71, wherein the amount is 1 dissociated HGS single cell / mL.

73. Dissociated HGS single cells at a certain concentration are present in the IO maturation culture medium, and the concentration is approximately 20 × 10⁻¹⁰ of the IO maturation culture medium. ５ ~60 x 10 ５ The method according to any one of claims 67 to 72, wherein the amount is 1 dissociated HGS single cell / mL.

74. The method according to any one of claims 67 to 73, wherein the dissociation is chemical, enzymatic, and / or mechanical dissociation.

75. The method according to any one of claims 67 to 74, wherein the dissociation is enzymatic, and optionally the enzyme comprises a protease and / or a collagenase, and optionally the enzyme is Accutase.

76. The method according to any one of claims 67 to 75, further comprising transplanting the I / O.

77. The method according to claim 76, wherein transplanting the IO into the subject is optionally performed by transplanting the IO under the kidney capsule of a non-human animal for a period of about 6 to 20 weeks.

78. The method according to claim 77, wherein the IO is implanted under the kidney capsule of a non-human animal for a period of about 12 to 20 weeks.

79. The method according to claim 77 or 78, wherein the IO is implanted under the kidney capsule of a non-human animal for a period of about 16 to 20 weeks.

80. The method according to claim 76, wherein transplanting the IO into the subject includes transplanting the IO into the intestinal lumen of the subject for the treatment of the subject's intestines.

81. The method according to any one of claims 76 to 80, wherein the IO matures in vitro for a certain period prior to transplantation, and optionally the period is about 7 to 28 days.

82. The method according to any one of claims 76 to 81, wherein the IO matures in vitro for a period of approximately 14 to 28 days prior to transplantation.

83. The method according to any one of claims 76 to 82, wherein the IO matures in vitro for a period of approximately 21 to 28 days prior to transplantation.

84. The method further includes differentiating the DE into spheroids, and optionally, the differentiation is performed. (h) The method according to any one of claims 26 to 42, comprising culturing the DE in a liquid differentiation medium in a bioreactor for a period of time sufficient to differentiate the DE into spheroids, wherein the culturing of the DE comprises suspending the DE in the liquid differentiation medium, and optionally the spheroids being foregut or hindgut spheroids.

85. The method according to claim 84, wherein the liquid differentiation culture medium does not contain any animal or human-derived material, and optionally, the liquid differentiation culture medium does not contain any extracellular matrix and / or basement membrane matrix.

86. The method described above is (i) culturing the spheroids in a liquid organoid maturation culture medium in a bioreactor for a period of time sufficient to differentiate the spheroids into organoids, further comprising culturing the spheroids by suspending them in the liquid organoid maturation culture medium, The method according to claim 84 or 85, wherein the organoid is optionally selected from the group consisting of liver, pancreas, stomach, gastric antrum, gastric fundus, intestine, lung, or colon organoid.

87. The method according to any one of claims 84 to 86, wherein the liquid organoid maturation culture medium does not contain any animal or human-derived material, and optionally, the liquid organoid maturation culture medium does not contain any extracellular matrix and / or basement membrane matrix.

88. The method according to any one of claims 1 to 87, wherein the PSC is an artificial PSC (iPSC) or an embryonic stem cell (ESC).

89. The method according to any one of claims 1 to 88, wherein the PSC is a human PSC, or optionally a human iPSC (hiPSC).

90. PSC or three-dimensional PSC aggregates prepared by the method described in any one of claims 1 to 25 or 88 to 89.

91. DE prepared by the method described in any one of claims 26 to 42 and 88 to 89.

92. HGS prepared by the method described in any one of claims 43 to 552 and 88 to 89.

93. I / O manufactured by the method described in any one of claims 56 to 89.

94. An IO having an inward polarity at its apical end, wherein the epithelial cells of the IO have a polarity in which their apical surface is oriented inward towards the IO, and optionally, the IO is a human IO (hIO).

95. An IO having an inwardly polar apex, manufactured by the method described in any one of claims 67 to 89.

96. A spheroid prepared by the method described in any one of claims 84, 85, or 89.

97. An organoid prepared by the method described in any one of claims 86 to 89.

98. A treatment method comprising transplanting IO or cells derived therefrom as described in any one of claims 93 to 95 into an animal, wherein the animal is optionally suffering from a GI disease state, and optionally the animal is a human.

99. A method for screening compounds for activity, comprising contacting an IO or a cell derived therefrom as described in any one of claims 93 to 95 with the compound, and measuring the response of the IO to the compound.

100. A method for screening a compound for its activity, comprising contacting the organoid described in claim 97 or a cell derived therefrom with the compound, and measuring the response of the organoid to the compound.

101. The method according to any one of claims 1 to 100, wherein the method does not include any heterogeneous material, and optionally the organoid is clinical grade and suitable for transplantation in humans.

102. The bioreactor of (a), (b), (d), (e), (f), (g), (h), and / or (i) comprises a rotating chamber containing the liquid culture medium, the second liquid culture medium, the liquid endoderm differentiation culture medium, the liquid hindgut differentiation culture medium, the liquid IO maturation culture medium, the liquid differentiation culture medium, and / or the liquid organoid maturation culture medium, The rotating chamber is a cylindrical section that rotates around its longitudinal axis, thereby suspending the PSCs and / or three-dimensional PSC aggregates in the liquid culture medium, the second liquid culture medium, the liquid endoderm differentiation culture medium, the liquid hindgut differentiation culture medium, the liquid IO maturation culture medium, the liquid differentiation culture medium, and / or liquid organoid maturation culture medium. The method according to any one of claims 1 to 101, wherein the chamber is optionally oriented such that its longitudinal axis is parallel to the ground.

103. The method according to any one of claims 1 to 102, wherein the bioreactor of (a), (b), (d), (e), (f), (g), (h), and / or (i) comprises a rotating chamber containing the liquid culture medium in a volume of about 5 mL to about 50 L, the second liquid culture medium, the liquid endoderm differentiation culture medium, the liquid hindgut differentiation culture medium, the liquid IO maturation culture medium, the liquid differentiation culture medium, and / or the liquid organoid maturation culture medium.

104. The bioreactor of (a), (b), (d), (e), (f), (g), (h), and / or (i) comprises a rotating chamber containing the liquid culture medium, the second liquid culture medium, the liquid endoderm differentiation culture medium, the liquid hindgut differentiation culture medium, the liquid IO maturation culture medium, the liquid differentiation culture medium, and / or the liquid organoid maturation culture medium, The rotation of the chamber is approximately 3 to 7 rpm. The method according to any one of claims 1 to 103, wherein the rotational speed is optionally selected to maintain the PSC, the three-dimensional PSC aggregate, the spheroid, and / or the organoid in a stationary orbit.

105. The bioreactor of (a), (b), (d), (e), (f), (g), (h), and / or (i) comprises a rotating chamber containing the liquid culture medium, the second liquid culture medium, the liquid endoderm differentiation culture medium, the liquid hindgut differentiation culture medium, the liquid IO maturation culture medium, the liquid differentiation culture medium, and / or the liquid organoid maturation culture medium, The average shear stress for the PSC, the three-dimensional PSC aggregate, the spheroid, and / or the organoid is approximately 5.0 dynes / cm². ２ The method according to any one of claims 1 to 104, wherein the result is less than [amount missing].

106. The method according to any one of claims 1 to 105, wherein the liquid culture medium, the second liquid culture medium, the liquid endoderm differentiation culture medium, the liquid hindgut differentiation culture medium, the liquid IO maturation culture medium, the liquid differentiation culture medium, and / or the liquid organoid maturation culture medium contains an anti-apoptotic agent.

107. The method according to any one of claims 1 to 106, wherein the liquid culture medium, the second liquid culture medium, the liquid endoderm differentiation culture medium, the liquid hindgut differentiation culture medium, the liquid IO maturation culture medium, the liquid differentiation culture medium, and / or the liquid organoid maturation culture medium contains an anti-adhesion agent.

108. The method according to any one of claims 1 to 107, wherein the liquid culture medium contains an anti-adhesion agent.

109. The method according to claim 108, wherein the anti-adhesion agent is DSS, xanthan gum, A-205804, 1-CAM1, carboxymethylcellulose, and / or Neural Organoid Basal Medium 2 (NOBM).

110. The method according to claim 108 or 109, wherein the anti-adhesion agent is a DSS at a concentration of about 1 μg / mL to 1000 μg / mL relative to the liquid culture medium, the second liquid culture medium, the liquid endoderm differentiation culture medium, the liquid hindgut differentiation culture medium, the liquid IO maturation culture medium, the liquid differentiation culture medium, and / or the liquid organoid maturation culture medium.

111. A composition for three-dimensional expansion and maintenance of pluripotent stem cell (PSC) cultures, wherein the composition comprises A liquid culture medium containing recombinant human basic fibroblast growth factor (rh bFGF) and / or recombinant human transforming growth factor β (rh TGFβ), A composition comprising PSC suspended in the culture medium.

112. The aforementioned liquid culture medium is a serum-free medium. The aforementioned liquid culture medium does not contain any material of animal or human origin. The composition according to claim 111, wherein the culture medium optionally contains no extracellular matrix and / or basement membrane matrix.

113. The composition according to claim 111 or 112, further comprising an anti-apoptotic agent.

114. The composition according to any one of claims 111 to 113, wherein the PSC expresses Oct4, SSEA1, TRA 1-60, Sox 2, and / or TRA-1-81.

115. The composition according to any one of claims 111 to 114, wherein the PSC expresses Oct4, SSEA1, TRA 1-60, Sox 2, and TRA-1-81.

116. The composition according to any one of claims 111 to 115, further comprising an anti-adhesion agent.

117. The composition according to claim 116, wherein the anti-adhesion agent is one or both of DSS and xanthan gum.

118. The composition according to any one of claims 111 to 117, wherein the PSCs are suspended in the liquid culture medium at a density of about 50,000 to 1,000,000 PSCs / mL relative to the culture medium.

119. The composition according to any one of claims 111 to 117, wherein the PSCs are suspended in the liquid culture medium at a density of approximately 100,000 to 300,000 PSCs / mL relative to the culture medium.

120. The composition according to any one of claims 111 to 119, wherein the PSCs are suspended in the culture medium at a density of approximately 180,000 to 220,000 PSCs / mL relative to the culture medium.

121. A composition for differentiating PSCs into endoderm (DE) in three-dimensional suspension culture, wherein the composition comprises Liquid DE differentiation culture medium and A composition comprising PSC suspended in the liquid DE differentiation culture medium.

122. The composition according to claim 121, wherein the liquid DE differentiation culture medium is a serum-free medium, the liquid DE differentiation culture medium does not contain any animal or human-derived material, and optionally the liquid DE differentiation culture medium does not contain any extracellular matrix and / or basement membrane matrix.

123. The composition according to claim 121 or 122, wherein the PSC has an average diameter of less than approximately 500 μm.

124. The composition according to any one of claims 121 to 123, wherein the PSC has an average diameter of less than about 400 μm.

125. The composition according to any one of claims 121 to 124, wherein the PSC has an average diameter of less than approximately 300 μm.

126. The composition according to any one of claims 121 to 125, wherein the liquid DE differentiation culture medium contains nodal signaling pathway activator and / or Wnt signaling pathway activator at a concentration of about 10 to 200 ng / mL relative to the liquid DE differentiation culture medium.

127. The composition according to claim 126, wherein the nodal signaling pathway activator or the Wnt signaling pathway activator is present at a concentration of approximately 10 to 200 ng / mL relative to liquid DE differentiation culture medium.

128. The composition according to claim 126 or 127, wherein the nodal signaling pathway activator or the Wnt signaling pathway activator is present at a concentration of approximately 50 to 150 ng / mL ng / mL relative to liquid DE differentiation culture medium.

129. The composition according to any one of claims 126 to 128, wherein the nodal signaling pathway activator or the Wnt signaling pathway activator is present in a concentration of approximately 100 to 200 ng / mL relative to liquid DE differentiation culture medium.

130. The composition according to any one of claims 126 to 129, wherein the liquid DE differentiation culture medium further comprises serum or a serum substitute at a concentration of about 0% to 20%.

131. The composition according to any one of claims 126 to 130, wherein the liquid DE differentiation culture medium further comprises serum or a serum substitute at a concentration of about 2% to 5%.

132. The composition according to any one of claims 111 to 131, further comprising DE differentiated from the PSC.

133. The composition according to claim 132, wherein the DE differentiated from the PSC expresses Sox17 and / or FoxA2.

134. The composition according to claim 132 or 133, wherein the DE differentiated from the PSC expresses Sox17 and FoxA2.

135. A composition for differentiating DE into hindgut spheroids (HGS) in three-dimensional suspension culture, wherein the composition comprises A liquid hindgut differentiation culture medium containing Wnt signaling pathway activator, FGF signaling pathway activator, and optionally FBS, A composition comprising DE suspended in the aforementioned liquid hindgut differentiation culture medium.

136. The composition according to claim 135, wherein the liquid hindgut differentiation culture medium does not contain any animal or human-derived material, and optionally the culture medium does not contain any extracellular matrix and / or basement membrane matrix.

137. The composition according to claim 135 or 136, wherein the Wnt signaling pathway activator comprises CHIR99021, and the FGF signaling pathway activator comprises FGF4.

138. The composition according to any one of claims 135 to 137, wherein the FGF signaling pathway activator is present at a concentration of at least about 50 ng / mL relative to the liquid hindgut differentiation culture medium.

139. The composition according to any one of claims 135 to 138, wherein the FGF signaling pathway activator is present at a concentration of at least about 500 ng / mL relative to the liquid hindgut differentiation culture medium.

140. The composition according to any one of claims 135 to 139, wherein the concentration of the Wnt pathway activator is at least about 0.5 μM relative to the liquid hindgut differentiation culture medium.

141. A composition for differentiating HGS into intestinal organoids (IOs) in three-dimensional suspension culture, wherein the composition comprises Liquid IO maturation culture medium containing EGF, A composition comprising HGS suspended in the aforementioned liquid IO mature culture medium.

142. The composition according to claim 141, wherein the liquid IO maturation culture medium does not contain any animal or human-derived material, and optionally, the liquid IO maturation culture medium does not contain any extracellular matrix and / or basement membrane matrix.

143. The composition according to claim 141 or 142, wherein the lumen of the HGS suspended in the liquid IO mature culture medium faces outward relative to the liquid IO mature culture medium.

144. The composition according to any one of claims 141 to 143, wherein the concentration of EGF is at least about 25 ng / mL.

145. The composition according to any one of claims 141 to 144, wherein the concentration of the EGF is at least about 100 ng / mL.

146. The composition according to any one of claims 141 to 145, wherein at least a portion of the HGS suspended in the liquid IO mature culture medium comprises dissociated HGS single cells.

147. The composition according to claim 146, wherein at least 80% of the HGS are dissociated single HGS cells, and optionally, at least 90% of the HGS are dissociated single cells.

148. The concentration of the dissociated HGS single cells in the liquid IO maturation culture medium is about 0.1×10 ５ to 80×10 ５ dissociated HGS single cells / mL within the range of the composition according to claim 146 or 147.

149. The concentration of the dissociated HGS single cells in the liquid IO mature culture medium is approximately 20 × 10⁻¹⁰ of the liquid IO mature culture medium. ５ ~60 x 10 ５ The composition according to any one of claims 146 to 148, wherein the dissociated HGS single cell / mL is within the range of 1.

150. The composition according to any one of claims 141 to 149, further comprising IO differentiated from the HGS.

151. The composition according to claim 150, wherein the epithelial cells of the IO formed from the dissociated HGS single cells have polarity such that their apical surfaces are oriented inward toward the IO.

152. The composition according to any one of claims 141 to 151, wherein the HGS expresses CdX2.

153. The composition according to any one of claims 141 to 152, wherein the HGS expresses FOX-F1 but does not express SOX2.

154. The composition according to claims 141 to 153, wherein the liquid IO maturation culture medium further comprises noggin.

155. Liquid culture medium and A composition comprising three-dimensional PSC aggregates suspended in the aforementioned liquid culture medium.

156. The composition according to claim 155, wherein the liquid culture medium does not contain any material of animal or human origin, and optionally, the liquid culture medium does not contain any extracellular matrix and / or basement membrane matrix.

157. The composition according to claim 155 or 156, wherein at least a portion of the three-dimensional PSC aggregate is dissociated as a single cell.

158. The composition according to any one of claims 155 to 157, wherein the average size of the diameter of the three-dimensional PSC aggregates is less than 400 μm.

159. The composition according to any one of claims 155 to 158, wherein the average size of the diameter of the three-dimensional PSC aggregates is less than 350 μm.

160. The composition according to any one of claims 155 to 159, wherein the average size of the diameter of the three-dimensional PSC aggregates is less than 300 μm.

161. The composition according to any one of claims 155 to 160, further comprising an anti-adhesion agent.

162. The composition according to claim 161, wherein the anti-adhesion agent is DSS, xanthan gum, A-205804, 1-CAM1, carboxymethylcellulose, and / or Neural Organoid Basal Medium 2 (NOBM).

163. The composition according to claim 161 or 162, wherein the anti-adhesion agent is concentrated at a concentration of about 1 μg / mL to 1000 μg / mL relative to the liquid culture medium.

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