Clinical-grade organoids

Abstract

This specification discloses improved methods for expanding cell populations and for producing organoids from pluripotent stem cells, as well as compositions and their uses, compositions comprising amino acid supplementation liquid components that can be used to carry out the Method, and kits comprising components for carrying out the Method and / or for using the products of the Method. These methods can be carried out without requiring heterogeneous and undefined basement membrane matrices for cell culture, thereby enabling a Good Manufacturing Practice (GMP) compliant approach to producing organoids for various uses, such as clinical screening and therapy, including methods for treating liver-related diseases or disorders and methods for screening candidate compounds or compositions for treating them.

Description

[Technical Field]

[0001] (Statement regarding federally supported research and development) This invention was made with government support under UH3 DK119982-02 and DP2 DK128799-01, awarded by the National Institutes of Health. The government has certain rights to this invention.

[0002] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 63 / 493,269, entitled “CLINICAL-GRADE ORGANOIDS,” filed on March 30, 2023, under Section 119(e) of the U.S. Patent Act, which is incorporated in its entirety by reference.

[0003] (Field of Invention) The aspects of this disclosure generally relate to organoid compositions and methods for preparing such compositions. These methods can be carried out without using heterologous basement membrane matrices during organoid differentiation and culture. Methods for cryopreserving organoids for long-term storage and subsequent use are also disclosed herein. [Background technology]

[0004] The culture of pluripotent stem cells and related downstream cell types, including organoid compositions, generally involves the use of a basement membrane matrix (also called the extracellular matrix) that provides a biological niche containing extracellular proteins and growth factors that support cell growth. A common basement membrane matrix used in biological studies is the extracellular matrix produced by mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. However, this basement membrane matrix is ​​composed of heterologous mouse components, is undefined (i.e., exhibits batch-to-batch variability), and may harbor pathogens, making it unsuitable for human clinical purposes.

[0005] Therefore, there is a need for the ability to produce an organoid composition without using a basement membrane matrix and other compounds that are not derived from a heterogeneous source for organoid production.

Summary of the Invention

[0006] Embodiments of the present disclosure include a method for expanding posterior foregut cells and / or posterior foregut endoderm cells, the method comprising: a) dissociating a monolayer of foregut endoderm cells into posterior foregut cells and / or posterior foregut endoderm cells; b) seeding the posterior foregut cells and / or posterior foregut endoderm cells onto a tissue culture surface; and c) culturing the posterior foregut cells and / or posterior foregut endoderm cells together with a TGF-β pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator.

[0007] In some embodiments, the monolayer of foregut endoderm cells can be dissociated into posterior foregut cells and / or posterior foregut endoderm cells using enzymatic dissociation and / or mechanical dissociation. In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells are seeded onto the surface of a tissue culture vessel at a cell density of 1×10 5 , 5 , 6 , 6 , 6 ,

[0008] , 5 , 5 , 5 , 2 , 6 , 6 , , 、2×10 5 、3×10 5 、4×10 5 、5×10 5 、6×10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、2×10 6 、3×10 6 、4×10 6 、or 5×10 6 cells / cm 2 、or at about those cell densities, or at any cell density within a range defined by any two of the aforementioned cell densities, onto the surface of a tissue culture vessel.

[0008] In some embodiments, the tissue culture surface is coated with a basement membrane matrix or its components. In some embodiments, the basement membrane matrix or its components does not contain non-human animal components so that the basement membrane matrix or its components are heterogeneous to humans, optionally, the basement membrane matrix or its components are not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally, the basement membrane matrix or its components are not Matrigel®, Cultrex®, or Geltrex®. In some embodiments, the basement membrane matrix or its components include human laminin, collagen IV, entactin, perlecan, fibrin, and / or hydrogel.

[0009] In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells may be cultured until three-dimensional (3D) spheroids can spontaneously form, optionally comprising structures having a single lumen and / or not containing hematopoietic tissue and acquired immune cells. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells may be cultured for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days.

[0010] In some embodiments, the TGF-β pathway inhibitor may be A83-01, RepSox, LY365947, and SB431542, optionally selected from A83-01. In some embodiments, the TGF-β pathway inhibitor may be provided at concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or about those concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally provided at a concentration of 500 nM or about 500 nM. In some embodiments, the FGF pathway activator may be selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23, optionally selected from FGF2. In some embodiments, the FGF pathway activator may be provided at concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or about those concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally provided at a concentration of 5 ng / mL or about 5 ng / mL. In some embodiments, the Wnt pathway activator may be 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, TDZD8, and TWS119, with CHIR99021 being optionally selected.In some embodiments, the Wnt pathway activator may be 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations. Optionally, the Wnt pathway activator may be provided at a concentration of 3 μM or about 3 μM. In some embodiments, the VEGF pathway activator may be selected from the group consisting of VEGF or GS4012, optionally from VEGF. In some embodiments, the VEGF pathway activator may be provided at concentrations 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations. Optionally, the VEGF pathway activator may be provided at a concentration of 10 ng / mL or about 10 ng / mL.

[0011] In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells in step c) may be cultured in a medium further containing EGF or in a medium without EGF. In some embodiments, EGF may be provided at concentrations 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally, EGF may be provided at a concentration of 20 ng / mL or about 20 ng / mL. In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells in step c) may be cultured in a medium further containing ascorbic acid or in a medium without ascorbic acid. In some embodiments, ascorbic acid may be provided at concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL, or about those concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations. Optionally, ascorbic acid may be provided at a concentration of 50 μg / mL or about 50 μg / mL.

[0012] In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells in step c) may be cultured in a medium further comprising a ROCK inhibitor, or in a medium not comprising a ROCK inhibitor, optionally the ROCK inhibitor being Y-27632. In some embodiments, the ROCK inhibitor may be provided 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally the ROCK inhibitor may be provided at a concentration of 10 μM or about 10 μM.

[0013] In some embodiments, the cells in step c) may be passaged one or more times. In some embodiments, the cells in step c) may be passaged until the posterior foregut cells and / or posterior foregut endoderm cells cease to spontaneously form spheroids. In some embodiments, the cells in step c) may be passaged three or fewer times.

[0014] In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells can be cultured and differentiated into liver organoids. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells can be cultured until three-dimensional (3D) spheroids spontaneously form, optionally comprising structures having a single lumen and / or not containing hematopoietic tissue and acquired immune cells, and posterior foregut cells and / or posterior foregut endoderm cells can be collected from the spheroids, optionally further comprising dissociating the spheroids into individual posterior foregut cells and / or posterior foregut endoderm cells and / or clumps of posterior foregut cells and / or posterior foregut endoderm cells before the differentiation step. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells can be collected from the foregut endoderm cell monolayer by dissociating the foregut endoderm cell monolayer into individual posterior foregut cells and / or posterior foregut endoderm cells and / or clumps of posterior foregut cells before the differentiation process.

[0015] Further embodiments of the present disclosure include a method for differentiating posterior foregut cells and / or posterior foregut endoderm cells into liver organoids, the method comprising: i) optionally in the form of spheroids, optionally in the form of individual cells or cell clusters dissociated from spheroids, optionally the spheroids comprising structures having a single lumen and / or the spheroids not containing hematopoietic tissue and acquired immune cells, and / or optionally aggregated cells in a microwell or other device as described herein, contacting a retinoic acid pathway activator; and ii) contacting the cells from step i) with a culture medium, optionally the culture medium being a hepatocyte culture medium, for a certain period of time, thereby differentiating the posterior foregut cells and / or posterior foregut endoderm cells into liver organoids.

[0016] In some embodiments, the culture medium may be supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid. In some embodiments, the cMET tyrosine kinase receptor agonist may be selected from hepatocyte growth factor (HGF), PG-001, phosgonimeton, televalefim, recombinant InlB321 protein, and agonist c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokine may be selected from IL-6, oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokine (CLC). In some embodiments, the corticosteroid may be selected from the group consisting of dexamethasone, beclomethasone, betamethasone, fluocortone, halomethasone, and mometasone. In some embodiments, the culture medium may be supplemented with HGF, OSM, and dexamethasone. In some embodiments, the culture medium may be supplemented with dexamethasone.

[0017] In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells may include posterior foregut cells and / or posterior foregut endoderm cells produced by the method described above. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells may be in the form of spheroids or in the form of individual posterior foregut cells and / or posterior foregut endoderm cells and / or clumps of posterior foregut cells and / or posterior foregut endoderm cells derived from dissociating spheroids, and optionally, the spheroids include structures having a single lumen and / or the spheroids do not contain hematopoietic tissue and acquired immune cells.

[0018] In some embodiments, the retinoic acid pathway activator may be selected from retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580, optionally from retinoic acid. In some embodiments, the retinoic acid pathway activator may be provided at any concentration within the range defined by 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or any two of the aforementioned concentrations, and optionally, the retinoic acid pathway activator may be provided at a concentration of 2.0 μM or about 2.0 μM. In some embodiments, HGF may be provided at concentrations 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, HGF may be provided at a concentration of 10 ng / mL or about 10 ng / mL. In some embodiments, OSM may be provided at concentrations 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, OSM may be provided at a concentration of 20 ng / mL or about 20 ng / mL. In some embodiments, dexamethasone may be provided at concentrations of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or about those concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations. Optionally, dexamethasone may be provided at a concentration of 100 nM or about 100 nM. In some embodiments, the cells of step i) and / or step ii) are not brought into contact with EGF.

[0019] In some embodiments, the cells of step ii) may be cultured in a growth medium supplemented with non-essential amino acids, essential amino acids, and glycine. In some embodiments, the supplemented growth medium includes 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of non-essential amino acids by total volume, or a range defined by any two of the aforementioned values. Optionally, the supplemented growth medium may contain approximately 4–10%, 6–12%, 10–16%, 12–15%, 13–19%, or approximately 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% of non-essential amino acids by total volume. In some embodiments, the supplemented growth medium contains 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of essential amino acids by total volume, or a range defined by any two of the aforementioned values. Optionally, the supplemented growth medium contains approximately 4–10%, 6–12%, 10–16%, 12–15%, 13–19%, or approximately 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% of essential amino acids by total volume. In some embodiments, the supplemented glycine may be provided at concentrations of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg / mL, or about those concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations. Optionally, the supplemented glycine may be provided at concentrations of 18–22 mg / mL or 20 mg / mL, or about 18–22 mg / mL or 20 mg / mL.

[0020] In some embodiments, the cells of step ii) may be further contacted with a low / first concentration of bilirubin, and the liver organoids formed are mature liver organoids. In some embodiments, the low / first concentration of bilirubin may be human fetal physiological concentrations of bilirubin. In some embodiments, the low / first concentration of bilirubin may be a) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, or 3.0 mg / L, or about those, or less than those, or about less than those, or any concentration within the range defined by any two of the aforementioned concentrations, e.g., 0.1-3 mg / L, 0.5-2.0 mg / L, 0.5-1.5 mg / L, 0 The concentration may be 0.3 to 2.5 mg / L, or 0.5 to 1.75 mg / L, or b) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / L, approximately these, less than these, or approximately less than these, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg / L, 0.1 to 0.5 mg / L, 0.5 to 1 mg / L, 0.3 to 0.7 mg / L, or 0.4 to 0.6 mg / L.

[0021] In some embodiments, mature liver organoids exhibit luminal projections similar to bile canaliculi, and / or structures having a single lumen and generally a spherical shape, and / or mature liver organoids do not contain hematopoietic tissue and adaptive immune cells. In some embodiments, mature liver organoids may express reduced levels of AFP, CDX2, NANOG, or any combination thereof, compared to liver organoids not exposed to a low / first dose of bilirubin. In some embodiments, mature liver organoids may express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, compared to liver organoids not exposed to a low / first dose of bilirubin. In some embodiments, mature liver organoids may express CYP2E1, CYP7A1, PROX1, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, mature liver organoids may exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids that have not been exposed to a low / first dose of bilirubin.

[0022] In some embodiments, the cells of step ii) may be further contacted with a high / second concentration of bilirubin, and the liver organoids formed are hyperbilirubinemia liver organoids. In some embodiments, the high / second concentration of bilirubin may be a) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / L, about those, greater than those, or about greater than those, or any concentration within the range defined by any two of the aforementioned concentrations, e.g., 2-20 mg / L, 2-10 mg / L, 10-20 mg / L, 5-15 mg / L, or 8 The concentration may be ~12 mg / L, or b) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / L, approximately those, greater than those, or approximately greater than those, or any concentration within the range defined by any two of the aforementioned concentrations, e.g., 4-20 mg / L, 2-10 mg / L, 10-20 mg / L, 5-15 mg / L, or 8-12 mg / L. In some embodiments, hyperbilirubinergic liver organoids may express elevated levels of UGT1A1 or NRF2, or both, compared to liver organoids not treated with high / secondary concentrations of bilirubin.

[0023] In some embodiments, the liver organoid may contain a gene or mRNA encoding a functional L-gulonolactone oxidase (GULO) protein and / or a functional GULO protein, or both, and the liver organoid may synthesize ascorbic acid. In some embodiments, the functional GULO protein may be murine GULO (mGULO). In some embodiments, the gene encoding the functional GULO protein may be conditionally expressed using a tetracycline induction system of optional choice. In some embodiments, the liver organoid may be manipulated with the gene encoding the functional GULO protein using CRISPR. In some embodiments, the gene or mRNA encoding the functional GULO protein, or both, may be introduced into the liver organoid by transfection. In some embodiments, the liver organoid containing the functional GULO protein may express increased levels of NRF2 compared to the liver organoid without the functional GULO protein. In some embodiments, liver organoids containing functional GULO protein may express reduced levels of IL1B, IL6, or TNFa, or any combination thereof, compared to liver organoids without functional GULO protein, when optionally cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid. In some embodiments, liver organoids containing functional GULO protein may exhibit reduced caspase-3 activity compared to liver organoids without functional GULO protein, when optionally cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid. In some embodiments, liver organoids containing functional GULO protein may express increased levels of ALB compared to liver organoids without functional GULO protein. In some embodiments, liver organoids containing functional GULO protein may resemble periportal liver tissue and express periportal liver markers.In some embodiments, the periportal markers may include FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, liver organoids containing functional GULO protein may exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids not containing functional GULO protein. In some embodiments, liver organoids containing functional GULO protein may exhibit increased bilirubin conjugation activity compared to liver organoids not containing functional GULO protein. In some embodiments, liver organoids containing functional GULO protein may exhibit increased viability in culture compared to liver organoids not containing functional GULO protein. In some embodiments, the liver organoids are differentiated from pluripotent stem cells that contain functional GULO protein and / or a gene or mRNA encoding functional GULO protein, or both, thereby enabling the pluripotent stem cells to synthesize ascorbic acid. In some embodiments, the liver organoids contain an inactive UGT1A1 gene, and the liver organoids can be used as a model for Crigler-Nadjar syndrome.

[0024] In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells may be aggregated in a microwell or other device (e.g., Aggrewell) prior to step i), and agglutination of posterior foregut cells and / or posterior foregut endoderm cells may result in liver organoids of a more uniform size. In some embodiments, the cells of step i) and / or step ii) are not cultured with the basement membrane matrix or its components, optionally, the cells of step i) and / or step ii) are not cultured with the basement membrane matrix or its components which are heterologous to humans, optionally, the cells of step i) and / or step ii) are not cultured with the basement membrane matrix or its components isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally, the cells of step i) and / or step ii) are not contacted with Matrigel®, Cultrex®, or Geltrex®. In some embodiments, the cells of step i) and / or step ii), and / or liver organoids formed therefrom, may be cultured in a static or non-static bioreactor, optionally a rotating bioreactor, optionally a 3D bioreactor, or optionally a 3D rotating bioreactor. In some embodiments, after culturing in a static or non-static bioreactor, the liver organoids may be dissociated into single cells, which can then be reconstituted and / or expanded through further culture steps in a static or non-static bioreactor, optionally a 3D bioreactor, or optionally a 3D rotating bioreactor. In some embodiments, the method further includes cryopreserving the liver organoids. In some embodiments, cryopreserving the liver organoids includes slow freezing or vitrification cryopreservation, and optionally, the liver organoids may be cryopreserved with chroman 1, emricasan, polyamine, and trans-ISRIB (CEPT).

[0025] In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells are derived from pluripotent stem cells, optionally embryonic stem cells, or induced pluripotent stem cells. In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells are derived from subjects, optionally subjects with liver-related disease or impairment. In some embodiments, the method can be used in a process compliant with good manufacturing practice (GMP). Embodiments of this disclosure further include posterior foregut cells and / or posterior foregut endoderm cells or liver organoids produced by any of the methods described above.

[0026] Further embodiments of the present disclosure include an in vitro composition comprising pluripotent stem cells, embryonic endoderm, foregut endoderm, abdominal foregut endoderm, and / or downstream hepatocyte types, as well as at least one exogenous tissue culture surface, at least one exogenous TGF-β pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator.

[0027] In some embodiments, the composition comprises posterior foregut cells and / or posterior foregut endoderm cells, wherein the posterior foregut cells and / or posterior foregut endoderm cells are dissociated posterior foregut cells and / or posterior foregut endoderm cells. In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells comprise a surface area of ​​1 × 10⁻⁶ of the tissue culture surface. 5 , 2×10 5 , 3 x 10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1 x 10 6 , 2×10 6 , 3 x 10 6 , 4×10 6 , or 5 x 10 6 cells / cm 2The above cell densities may be any cell density within the range defined by exactly or approximately those densities, or by any two of the aforementioned cell densities. In some embodiments, the tissue culture surface may be coated with a basement membrane matrix or its components. In some embodiments, the basement membrane matrix or its components may not contain non-human animal components so as to be heterogeneous to humans, and optionally, the basement membrane matrix or its components may not be isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, and optionally, the basement membrane matrix or its components may not be Matrigel®, Cultrex®, or Geltrex®. In some embodiments, the basement membrane matrix or its components may include human laminin, collagen IV, entactin, perlecan, fibrin, and / or hydrogel.

[0028] In some embodiments, at least a portion of posterior foregut cells and / or posterior foregut endoderm cells may be spontaneously formed three-dimensional (3D) spheroids, which optionally include structures having a single lumen, and / or mature liver organoids may not contain hematopoietic tissue and adaptive immune cells. In some embodiments, the TGF-β pathway inhibitor may be selected from A83-01, RepSox, LY365947, and SB431542, which optionally include or are A83-01. In some embodiments, the TGF-β pathway inhibitor may be at concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or about those concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the TGF-β pathway inhibitor may be at a concentration of 500 nM or about 500 nM. In some embodiments, the FGF pathway activator may be 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, and optionally, the FGF pathway activator may include or be FGF2. In some embodiments, the FGF pathway activator may be at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or about those concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the FGF pathway activator may be at a concentration of 5 ng / mL or about 5 ng / mL.In some embodiments, the Wnt pathway activator may be 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, alster paulon, kaem paulon, lithium chloride, TDZD8, and TWS119, and optionally, the Wnt pathway activator may include CHIR99021 or be CHIR99021. In some embodiments, the Wnt pathway activator may be 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 at approximately those concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the Wnt pathway activator may be at a concentration of 3 μM or approximately 3 μM. In some embodiments, the VEGF pathway activator may be selected from VEGF or GS4012, and optionally, the VEGF pathway activator may contain VEGF or be VEGF. In some embodiments, the VEGF pathway activator may be 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 concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the VEGF pathway activator may be at a concentration of 10 ng / mL or about 10 ng / mL. In some embodiments, the composition may further contain exogenous EGF, or the composition may not contain exogenous EGF. In some embodiments, EGF may be at concentrations 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 concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, and optionally, EGF may be at a concentration of 20 ng / mL or about 20 ng / mL.In some embodiments, the composition further comprises exogenous and / or genetically modified ascorbic acid, or the composition does not contain exogenous and / or genetically modified ascorbic acid. In some embodiments, the ascorbic acid may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL, or at about those concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the ascorbic acid may be at a concentration of 50 μg / mL or about 50 μg / mL. In some embodiments, the composition may further comprise a ROCK inhibitor, or may be cultured in a medium that does not comprise a ROCK inhibitor, and optionally, the ROCK inhibitor may comprise or be Y-27632. In some embodiments, the ROCK inhibitor may be 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 concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the ROCK inhibitor may be at a concentration of 10 μM or about 10 μM.

[0029] In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells, embryonic endoderm, abdominal foregut endoderm, and / or downstream hepatocytes can be differentiated from stem cells. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells, embryonic endoderm, abdominal foregut endoderm, and / or downstream hepatocytes can be differentiated from induced pluripotent stem cells. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells, embryonic endoderm, abdominal foregut endoderm, and / or downstream hepatocytes have been passaged less than four times. In some embodiments, the cells include or are essentially derived from posterior foregut cells and / or posterior foregut endoderm cells. In some embodiments, the TGF-β pathway inhibitor may be A83-01, the FGF pathway activator may be FGF2, the Wnt pathway activator may be CHIR99021, the VEGF pathway activator may be VEGF, and the ROCK inhibitor may be Y-27632.

[0030] Further embodiments of the present disclosure include liver organoids produced by any of the methods described above.

[0031] Further embodiments of the present disclosure include an in vitro composition comprising a) posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids, and b) a culture medium, optionally comprising hepatocyte culture medium and optionally supplemented with a cMET tyrosine kinase receptor agonist, IL-6 family cytokines, and corticosteroids, wherein the composition optionally further comprises c) a retinoic acid pathway activator. In some embodiments, the cMET tyrosine kinase receptor agonist may be selected from hepatocyte growth factor (HGF), PG-001, phosgonimeton, telebarefim, recombinant InlB321 protein, and agonist c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokines may be selected from IL-6, oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokines (CLC). In some embodiments, the corticosteroid may be selected from dexamethasone, beclomethasone, betamethasone, fluocortone, halomethasone, and mometasone. In some embodiments, the culture medium may be supplemented with HGF, OSM, and dexamethasone. In some embodiments, the culture medium may be supplemented with dexamethasone. In some embodiments, the retinoic acid pathway activator may be selected from retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580, and optionally, the retinoic acid pathway activator may contain retinoic acid or be retinoic acid.In some embodiments, the retinoic acid pathway activator may be at a concentration of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the retinoic acid pathway activator may be at a concentration of 2.0 μM or approximately 2.0 μM. In some embodiments, HGF may be 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 concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, and optionally, HGF may be at a concentration of 10 ng / mL or about 10 ng / mL. In some embodiments, OSM may be 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 concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, and optionally, OSM may be at a concentration of 20 ng / mL or about 20 ng / mL. In some embodiments, dexamethasone may be 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or at concentrations of about those concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, dexamethasone may be at a concentration of 100 nM or about 100 nM. In some embodiments, the composition does not contain exogenous EGF.

[0032] In some embodiments, the composition further comprises a low concentration of exogenous bilirubin, which optionally may be a human fetal physiological concentration of bilirubin or near such a concentration. In some embodiments, the bilirubin may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, or 3.0 mg / L, or about those, or less than those, or about less than those, or any concentration within the range defined by any two of the aforementioned concentrations, e.g., 0.1-3 mg / L, 0.5-2.0 mg / L, 0.5-1.5 mg / L, 0 The concentration may be 0.3 to 2.5 mg / L, or 0.5 to 1.75 mg / L, or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / L, or about these, less than these, or about less than these, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg / L, 0.1 to 0.5 mg / L, 0.5 to 1 mg / L, 0.3 to 0.7 mg / L, or 0.4 to 0.6 mg / L. In some embodiments, the composition comprises mature liver organoids, which exhibit luminal projections similar to bile canaliculi and / or structures having a single lumen and generally a spherical shape, and / or mature liver organoids do not contain hematopoietic tissue and acquired immune cells. In some embodiments, mature liver organoids may express reduced levels of AFP, CDX2, NANOG, or any combination thereof, compared to liver organoids not exposed to low doses of bilirubin. In some embodiments, mature liver organoids may express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, compared to liver organoids not exposed to low doses of bilirubin. In some embodiments, mature liver organoids may express CYP2E1, CYP7A1, PROX1, MRP3, MRP3, or OATP2, or any combination thereof.In some embodiments, mature liver organoids may exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids that have not been exposed to low doses of bilirubin.

[0033] Further embodiments of the present disclosure include an in vitro composition comprising a mature liver organoid, wherein the cells of the mature liver organoid are in contact with a low dose of bilirubin, optionally the low dose of bilirubin being exogenously supplied, the mature liver organoid exhibits luminal projections similar to bile canaliculi, and / or structures having a single lumen and generally spherical shape, and / or the mature liver organoid does not contain hematopoietic tissue and adaptive immune cells. In some embodiments, the mature liver organoid may express reduced levels of AFP, CDX2, NANOG, or any combination thereof, compared to liver organoids whose cells were not in contact with a low dose of bilirubin. In some embodiments, the mature liver organoid may express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, compared to liver organoids whose cells were not in contact with a low dose of bilirubin. In some embodiments, mature liver organoids may express CYP2E1, CYP7A1, PROX1, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, mature liver organoids may exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids that were not exposed to low doses of bilirubin.

[0034] In some embodiments, the composition further comprises hyperbilirubinemia liver organoids, the hyperbilirubinemia liver organoid cells having been in contact with a high and / or second concentration of bilirubin. In some embodiments, the high / second concentration of bilirubin was 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / L, about those, greater than those, or about greater than those, or any concentration within the range defined by any two of the aforementioned concentrations, e.g., 2-20 mg / L, 2-10 mg / L, 10-20 mg / L, 5-15 mg / L, or The concentration was 8–12 mg / L, or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / L, approximately those, greater than those, or approximately greater than those, or any concentration within the range defined by any two of the aforementioned concentrations, e.g., 4–20 mg / L, 2–10 mg / L, 10–20 mg / L, 5–15 mg / L, or 8–12 mg / L. In some embodiments, hyperbilirubinemia liver organoids may express elevated levels of UGT1A1 or NRF2, or both, compared to liver organoids not treated with high / secondary concentrations of bilirubin. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids contain a gene or mRNA encoding a functional L-gulonolactone oxidase (GULO) protein and / or a functional GULO protein, or both, and the posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids are capable of synthesizing ascorbic acid. In some embodiments, the functional GULO protein is mouse GULO (mGULO). In some embodiments, the gene encoding the functional GULO protein may be conditionally expressed using a tetracycline induction system of optional choice.In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids may be engineered using CRISPR to contain a gene encoding a functional GULO protein. In some embodiments, the gene or mRNA encoding a functional GULO protein, or both, is introduced into the liver organoid by transfection. In some embodiments, liver organoids and / or mature liver organoids containing a functional GULO protein may express increased levels of NRF2 compared to liver organoids and / or mature liver organoids not containing a functional GULO protein. In some embodiments, liver organoids and / or mature liver organoids containing a functional GULO protein may express reduced levels of IL1B, IL6, or TNFa, or any combination thereof, compared to liver organoids and / or mature liver organoids not containing a functional GULO protein. In some embodiments, liver organoids and / or mature liver organoids containing a functional GULO protein may exhibit reduced caspase-3 activity compared to liver organoids and / or mature liver organoids not containing a functional GULO protein. In some embodiments, liver organoids and / or mature liver organoids containing functional GULO protein may express increased levels of ALB compared to liver organoids and / or mature liver organoids that do not contain functional GULO protein. In some embodiments, liver organoids and / or mature liver organoids containing functional GULO protein may resemble periportal liver tissue and express periportal liver markers. In some embodiments, the periportal markers may include FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, liver organoids and / or mature liver organoids containing functional GULO protein may exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids and / or mature liver organoids that do not contain functional GULO protein.In some embodiments, liver organoids and / or mature liver organoids containing functional GULO protein may exhibit increased bilirubin conjugation activity compared to liver organoids and / or mature liver organoids not containing functional GULO protein. In some embodiments, liver organoids and / or mature liver organoids containing functional GULO protein may exhibit increased viability in culture compared to liver organoids and / or mature liver organoids not containing functional GULO protein. In some embodiments, the liver organoids and / or mature liver organoids are differentiated from pluripotent stem cells that contain functional GULO protein and / or a gene or mRNA encoding functional GULO protein, or both, thereby enabling the pluripotent stem cells to synthesize ascorbic acid.

[0035] Further embodiments of the present disclosure include methods for administering the aforementioned liver organoids or compositions to subjects requiring such administration, and methods for administering one or more of the aforementioned liver organoids or compositions to subjects requiring treatment for liver-related diseases or disorders.

[0036] In some embodiments, the liver organoids are produced from cells derived from the subject, and optionally, the cells derived from the subject are induced pluripotent stem cells. In some embodiments, administration includes transplanting the liver organoids or composition into the subject. In some embodiments, liver-related diseases or disorders include one or more of the following: liver dysfunction and / or liver failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic disorders, autoimmune liver diseases, Wilson's disease, metabolic-related fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Nadjar syndrome, urea cycle disorders, Wolmann disease, liver cancer, hepatoblastoma, metabolic dysfunction-associated liver disease (MASLD), MetALD, metabolic dysfunction-associated steatohepatitis (MASH), drug-induced liver injury (DILI), glycogen storage disorders, hemorrhagic disorders, hepatic cysts, acetaminophen-induced acute liver injury, and / or alcohol-related liver diseases. In some embodiments, hepatic dysfunction and / or hepatic failure includes hyperammonemia and / or hyperbilirubinemia, or metabolic disorders include nonalcoholic fatty liver disease (NAFLD), or nonalcoholic fatty liver disease (NAFLD) includes metabolic dysfunction-associated steatohepatitis (MASH), or hepatitis includes hepatitis A, B, C, D, E, G, TT, and / or autoimmune hepatitis. In some embodiments, the subject may have reduced serum bilirubin and / or ammonia levels, and / or increased serum proteinalbumin after transplantation. In some embodiments, the subject may have improved symptoms of biliary stenosis and / or liver regeneration after transplantation. In some embodiments, the subject may have increased survival rates after transplantation. In some embodiments, the liver organoid engrafts in the subject's liver.In some embodiments, liver organoids are treated with amino acid (AA) supplementation. In some embodiments, liver organoids are treated with amino acid (AA) supplementation for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days or more prior to transplantation. In some embodiments, liver-related disease or disorder includes acetaminophen acute liver injury. In some embodiments, the method can be used in a process compliant with Good Manufacturing Practices (GMP).

[0037] Further embodiments of the present disclosure include a screening method comprising contacting the aforementioned liver organoids with a candidate compound or composition and evaluating the effect of the candidate compound or composition on the liver organoids. In some embodiments, the liver organoids may be models of liver-related diseases or disorders, and determining the effect of the candidate compound or composition on the liver organoids includes evaluating the effect of the candidate compound or composition on liver-related diseases or disorders. In some embodiments, the liver organoids are produced from cells derived from a subject, and optionally, the cells derived from the subject are induced pluripotent stem cells. In some embodiments, the subject has a liver-related disease or disorder. In some embodiments, the method can be used in a process compliant with Good Manufacturing Practices (GMP).

[0038] Further embodiments of this disclosure include compositions comprising amino acid supplementation liquid components as described in Table 3. Further embodiments include compositions comprising growth factor cocktails as described in the embodiments of Table 1 or Table 2.

[0039] Further embodiments of the present disclosure include a solution of non-essential amino acids in exactly or about 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 11 vol%, 12 vol%, 13 vol%, 14 vol%, or 15 vol% (containing exactly or about 890 mg / L alanine, 1320 mg / L asparagine, 1330 mg / L aspartic acid, 750 mg / L glycine, 105 mg / L serine, 1150 mg / L proline, and 1470 mg / L glutamic acid), or a solution of essential amino acids in exactly or about 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 11 vol%, 12 vol%, 13 vol%, 14 vol%, or 15 vol% (containing exactly or about 6320 mg / L arginine, 1200 mg / L cysteine, histidine, etc.). (Contains 2100 mg / L of phosphate, 2620 mg / L of isoleucine, 2620 mg / L of leucine, 3625 mg / L of lysine, 755 mg / L of methionine, 1650 mg / L of phenylalanine, 2380 mg / L of threonine, 510 mg / L of tryptophan, 1800 mg / L of tyrosine, and 2340 mg / L of valine), and exactly or approximately 65% ​​by volume, 66% by volume, 6 7% by volume, 68% by volume, 69% by volume, 70% by volume, 71% by volume, 72% by volume, 73% by volume, 74% by volume, 75% by volume, 76% by volume, 77% by volume, 78% by volume, 79% by volume, 80% by volume, 81% by volume, 82% by volume, 83% by volume, 84% by volume, 85% by volume, 86% by volume, 87% by volume, 88% by volume, 89% by volume, or 90% by volume of hepatocyte culture medium (hepatocyte A composition comprising an amino acid supplement liquid component containing culture medium (HCM), and further supplemented with exactly or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg / mL of glycine.In some embodiments, the composition contains exactly or about 14% non-essential amino acid solution (containing exactly or about 890 mg / L alanine, 1320 mg / L asparagine, 1330 mg / L aspartic acid, 750 mg / L glycine, 105 mg / L serine, 1150 mg / L proline, and 1470 mg / L glutamic acid), and exactly or about 6 volume% essential amino acid solution (containing exactly or about 6320 mg / L arginine, 1200 mg / L cysteine, and 210 mg / L histidine). The composition comprises an amino acid supplement liquid component containing exactly or about 80 volume% hepatocyte culture medium (HCM), further supplemented with exactly or about 20 g / L of glycine. In some embodiments, the pH is about pH 6-8, or pH 6.5-7.5, or pH exactly or about pH 7.0. In some embodiments, the composition further comprises hepatocyte growth factor (HGF), oncostatin M, dexamethasone, and / or ascorbic acid. In some embodiments, the composition further comprises hepatocyte lineage-determined cells differentiated from endoderm cells using retinoic acid. In some embodiments, hepatic lineage-determined cells are characterized as liver organoids.

[0040] In some embodiments, liver organoids may be characterized by secreting increased levels of albumin and urea compared to liver organoids in HCM without amino acid supplementation. In some embodiments, liver organoids may be characterized by expressing increased levels of liver maturation-related gene expression compared to liver organoids in HCM without amino acid supplementation. In some embodiments, liver organoids may be characterized by expressing reduced levels of vimentin compared to liver organoids in HCM without amino acid supplementation.

[0041] In some embodiments, the composition does not contain non-human animal components such that the basement membrane matrix or its components are heterogeneous to humans. In some embodiments, the composition does not contain mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, Matrigel®, Cultrex®, and / or Geltrex®.

[0042] Further embodiments of the present disclosure include in vitro hyperbilirubinemia liver organoids comprising naturally occurring and / or engineered mutations in the UDP glucuronosyltransferase family 1 member A1 (UGT1A1) gene. In some embodiments, the hyperbilirubinemia liver organoids were produced by exposing progenitor cells, progenitor liver organoids, and / or progenitor mature liver organoids to exogenous bilirubin for at least two rounds. In some embodiments, the hyperbilirubinemia liver organoids were clonally induced and / or derived from iPSCs.

[0043] Further embodiments of the present disclosure include cryopreserved compositions comprising liver organoids, chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT), and / or cryopreserved compositions comprising mature liver organoids, chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT), and / or cryopreserved compositions comprising hyperbilirubinergic liver organoids, chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT).

[0044] Further embodiments of the present disclosure include kits comprising means for carrying out any of the methods described above, and / or kits comprising compositions or means for producing any of the compositions described above, and / or kits for producing any of the liver organoids described above. Further embodiments of the present disclosure include the use of methods, compositions, or kits as pharmaceuticals, means for the treatment and / or prevention of diseases, means for diagnosis, and / or medical research. [Brief explanation of the drawing]

[0045] 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] This diagram shows a schematic embodiment of the subsequent steps for cryopreservation and selective selection of liver organoids. [Figure 1B] Embodiments of fluorescence microscopy images of liver organoids, frozen using slow freezing and vitrification cryopreservation approaches and thawed (compared to unfrozen organoids) to observe the abundance of live cells (labeled with calcein AM) and dead cells (labeled with ethidium homodimer-1), are shown. [Figure 1C] This document describes an embodiment of quantifying albumin secretion by liver organoids (compared to unfrozen organoids) that were frozen and thawed using slow freezing and vitrification cryopreservation approaches. [Figure 2A1] This document describes an embodiment of RT-qPCR quantification of ALB, CYP3A4, PCK1, and G6PC expression in liver organoids cultured with amino acid (AA) supplementation medium added at various time points in the culture process. [Figure 2A2] This document describes an embodiment of RT-qPCR quantification of ALB, CYP3A4, PCK1, and G6PC expression in liver organoids cultured with amino acid (AA) supplementation medium added at various time points in the culture process. [Figure 2A3]This document describes an embodiment of RT-qPCR quantification of ALB, CYP3A4, PCK1, and G6PC expression in liver organoids cultured with amino acid (AA) supplementation medium added at various time points in the culture process. [Figure 2A4] This document describes an embodiment of RT-qPCR quantification of ALB, CYP3A4, PCK1, and G6PC expression in liver organoids cultured with amino acid (AA) supplementation medium added at various time points in the culture process. [Figure 2B1] This describes an embodiment of quantifying albumin secretion and urea production in liver organoids cultured with AA supplementation medium added at various points in the culture process. [Figure 2B2] This describes an embodiment of quantifying albumin secretion and urea production in liver organoids cultured with AA supplementation medium added at various points in the culture process. [Figure 2C] A schematic embodiment is shown for detecting PCK1 activity and other activities in liver organoids grown in AA supplementation medium using a luciferase reporter approach with either live cells or cell lysates. [Figure 2D] Embodiments of bright-field microscopy images of organoids grown in AA supplement medium and standard hepatocyte culture medium (HCM) are shown. [Figure 2E1] This document describes embodiments for quantifying PCK1 expression, as measured by a luciferase reporter, in liver organoids grown in AA supplementation medium compared to liver organoids grown in standard hepatocyte culture medium, in both live cell assays and cell lysate assays. [Figure 2E2] This document describes embodiments for quantifying PCK1 expression, as measured by a luciferase reporter, in liver organoids grown in AA supplementation medium compared to liver organoids grown in standard hepatocyte culture medium, in both live cell assays and cell lysate assays. [Figure 3A1] This diagram illustrates a schematic embodiment of two-dimensional (2D) hepatocyte differentiation from pluripotent stem cells and culture using AA supplementation medium added at various points in the culture process. [Figure 3A2] This diagram illustrates a schematic embodiment of two-dimensional (2D) hepatocyte differentiation from pluripotent stem cells and culture using AA supplementation medium added at various points in the culture process. [Figure 3B] This shows a bright-field image of 2D hepatocyte cultures grown in AA supplement medium added at various points in the culture process. [Figure 3C] This document describes an embodiment of quantifying lactate production in 2D hepatocytes grown in AA supplementation medium compared to 2D hepatocytes grown in standard hepatocyte culture medium. [Figure 3D] This document describes a method for quantifying albumin secretion in 2D hepatocytes grown in AA supplementation medium added at various culture points, compared to 2D hepatocytes grown in standard hepatocyte culture medium. [Figure 3E] This document describes an embodiment of quantifying albumin secretion in 2D hepatocytes grown for an extended period in AA supplement medium, where the rate of albumin secretion is normalized to the rate of hepatocytes grown in standard hepatocyte culture medium. [Figure 3F1] This document describes the RT-qPCR quantification of gene expression for ALB, E-cadherin, CYP3A4, G6PC, PKM, and PCK1 in 2D hepatocytes grown in AA supplement medium added at various culture points, compared to 2D hepatocytes grown in standard hepatocyte culture medium. [Figure 3F2] This document describes the RT-qPCR quantification of gene expression for ALB, E-cadherin, CYP3A4, G6PC, PKM, and PCK1 in 2D hepatocytes grown in AA supplement medium added at various culture points, compared to 2D hepatocytes grown in standard hepatocyte culture medium. [Figure 3F3] This document describes the RT-qPCR quantification of gene expression for ALB, E-cadherin, CYP3A4, G6PC, PKM, and PCK1 in 2D hepatocytes grown in AA supplement medium added at various culture points, compared to 2D hepatocytes grown in standard hepatocyte culture medium. [Figure 3F4]This document describes the RT-qPCR quantification of gene expression for ALB, E-cadherin, CYP3A4, G6PC, PKM, and PCK1 in 2D hepatocytes grown in AA supplement medium added at various culture points, compared to 2D hepatocytes grown in standard hepatocyte culture medium. [Figure 3F5] This document describes the RT-qPCR quantification of gene expression for ALB, E-cadherin, CYP3A4, G6PC, PKM, and PCK1 in 2D hepatocytes grown in AA supplement medium added at various culture points, compared to 2D hepatocytes grown in standard hepatocyte culture medium. [Figure 3F6] This document describes the RT-qPCR quantification of gene expression for ALB, E-cadherin, CYP3A4, G6PC, PKM, and PCK1 in 2D hepatocytes grown in AA supplement medium added at various culture points, compared to 2D hepatocytes grown in standard hepatocyte culture medium. [Figure 3G] Embodiments of fluorescence and bright-field microscopy images of 2D hepatocytes grown in AA supplementation medium with or without insulin starvation, manipulated to express mScarlet under a PCK1 reporter, compared to 2D hepatocytes grown in standard hepatocyte culture medium with or without insulin starvation, are shown. [Figure 3H] This document illustrates fluorescence microscopy images detecting the expression of EpCAM (epithelial cells), vimentin (mesenchymal cells), and DAPI or HNF4α (nuclei) in 2D hepatocytes grown in AA supplement medium compared with 2D hepatocytes grown in standard hepatocyte culture medium. [Figure 4A] A schematic embodiment for culturing liver organoids without using Matrigel® or other basement membrane matrices containing heterogeneous components is shown. [Figure 4B] This exhibits a bright-field image embodiment showing the spontaneous formation of 3D spheroids from a foregut cell monolayer, where the spheroids may transition to mature liver organoids without the use of Matrigel® or other basement membrane matrices containing heterogeneous components. [Figure 4C]Embodiments of bright-field and fluorescence microscopy images showing that organoids grown under conditions without Matrigel® exhibit normal organoid morphology are presented. [Figure 4D] This document presents embodiments for quantifying albumin secretion compared between organoids grown under conditions without Matrigel®, organoids grown under Matrigel® conditions according to a previous protocol, and 2D hepatocyte cultures. [Figure 4E] This document describes embodiments of RT-qPCR quantification of ALB, AFP, HNF4α, RBP4, and AAT expression between organoids grown under conditions without Matrigel®, organoids grown under Matrigel® conditions according to a previous protocol, and 2D hepatocyte culture. [Figure 5A] This diagram illustrates a schematic embodiment of the passage and expansion of foregut cells after pluripotent stem cell differentiation to scale organoid production. [Figure 5B] This document shows bright-field microscopy images illustrating the growth of foregut cells (days 1-7) and spontaneous spheroid formation when plated on laminin or Matrigel® coated plates. [Figure 5C] The images show embodiments of bright-field microscopy illustrating further growth of foregut cells (days 8-10) and complete spheroid formation, with cells plated on laminin-coated plates appearing to result in more efficient spheroid formation compared to cells plated on Matrigel®-coated plates. [Figure 5D] This document shows embodiments of bright-field microscopy images illustrating the growth of foregut cells (days 1-5) and spontaneous spheroid formation when plated on laminin-coated plates at different seeding densities. [Figure 5E] This embodiment of bright-field microscopy images shows that when spheroids are collected from initial spontaneous formation from foregut cells, additional spheroids arise from additional culture. [Figure 5F1]This document describes a method for obtaining bright-field microscopy images and quantifying the total number of latent cells through multiple passages of foregut cells from early differentiation of pluripotent stem cells. Spheroids were formed during passages 1-3, but not from foregut cells in passage 4. [Figure 5F2] This document describes a method for obtaining bright-field microscopy images and quantifying the total number of latent cells through multiple passages of foregut cells from early differentiation of pluripotent stem cells. Spheroids were formed during passages 1-3, but not from foregut cells in passage 4. [Figure 5G1] This document describes the RT-qPCR quantification of CDX2, FOXA2, AFP, VIM, SOX17, HNF4α, and ALB expression in foregut cells from passages 1-4. [Figure 5G2] This document describes the RT-qPCR quantification of CDX2, FOXA2, AFP, VIM, SOX17, HNF4α, and ALB expression in foregut cells from passages 1-4. [Figure 5G3] This document describes the RT-qPCR quantification of CDX2, FOXA2, AFP, VIM, SOX17, HNF4α, and ALB expression in foregut cells from passages 1-4. [Figure 5G4] This document describes the RT-qPCR quantification of CDX2, FOXA2, AFP, VIM, SOX17, HNF4α, and ALB expression in foregut cells from passages 1-4. [Figure 5G5] This document describes the RT-qPCR quantification of CDX2, FOXA2, AFP, VIM, SOX17, HNF4α, and ALB expression in foregut cells from passages 1-4. [Figure 5G6] This document describes the RT-qPCR quantification of CDX2, FOXA2, AFP, VIM, SOX17, HNF4α, and ALB expression in foregut cells from passages 1-4. [Figure 5G7] This document describes the RT-qPCR quantification of CDX2, FOXA2, AFP, VIM, SOX17, HNF4α, and ALB expression in foregut cells from passages 1-4. [Figure 5H]A schematic embodiment of liver organoid formation, starting from pluripotent stem cells and including foregut cell passage for scaling, is shown. [Figure 5I] A schematic embodiment of liver organoid formation is shown, including starting with pluripotent stem cells, passaging of foregut cells for scaling, and the use of a device to aggregate the foregut cells to improve the uniformity of organoid size and shape. [Figure 6A] This diagram shows a schematic embodiment of 3D rotational culture of liver organoids grown under conditions that do not contain Matrigel®. [Figure 6B] This image shows a bright-field embodiment demonstrating that liver organoids can be grown using 3D rotation culture under conditions that do not include Matrigel®. [Figure 6C1] This document describes the RT-qPCR quantification of AFP, HNF4α, FOXA2, ALB, CDX2, and VIM in liver organoids grown by 3D rotation culture, compared to foregut cells from passage 1. [Figure 6C2] This document describes the RT-qPCR quantification of AFP, HNF4α, FOXA2, ALB, CDX2, and VIM in liver organoids grown by 3D rotation culture, compared to foregut cells from passage 1. [Figure 6C3] This document describes the RT-qPCR quantification of AFP, HNF4α, FOXA2, ALB, CDX2, and VIM in liver organoids grown by 3D rotation culture, compared to foregut cells from passage 1. [Figure 6C4] This document describes the RT-qPCR quantification of AFP, HNF4α, FOXA2, ALB, CDX2, and VIM in liver organoids grown by 3D rotation culture, compared to foregut cells from passage 1. [Figure 6C5] This document describes the RT-qPCR quantification of AFP, HNF4α, FOXA2, ALB, CDX2, and VIM in liver organoids grown by 3D rotation culture, compared to foregut cells from passage 1. [Figure 6C6]This document describes the RT-qPCR quantification of AFP, HNF4α, FOXA2, ALB, CDX2, and VIM in liver organoids grown by 3D rotation culture, compared to foregut cells from passage 1. [Figure 7A] This diagram illustrates a schematic representation of HLO generation and maturation by low doses of bilirubin. [Figure 7B] Bright-field images of HLO treated with a low dose of bilirubin (1 mg / L) compared to the control, and embodiments of the luminal contour using ImageJ are shown, with arrows indicating luminal projections similar to bile canaliculi found in the human liver. [Figure 7C1] This document illustrates an embodiment for comparing the lumen size and roundness of a control and a 1 mg / L bilirubin-treated HLO. [Figure 7C2] This document illustrates an embodiment for comparing the lumen size and roundness of a control and a 1 mg / L bilirubin-treated HLO. [Figure 7D1] This document describes the RT-qPCR assays performed on immature and mature marker genes (ALB, NANOG, SLC4A2, HO-1, AFP, and CDX2) in control organoids and organoids treated with 1 mg / L bilirubin, compared to human liver samples. [Figure 7D2] This document describes the RT-qPCR assays performed on immature and mature marker genes (ALB, NANOG, SLC4A2, HO-1, AFP, and CDX2) in control organoids and organoids treated with 1 mg / L bilirubin, compared to human liver samples. [Figure 7E1] This document describes embodiments of CYP3A4 and CYP1A2 activity assays in response to rifampicin and omeprazole in a control and 1 mg / L bilirubin-treated HLO (RLU (relative light unit): relative luminescence, CTG: CellTiter-Glo assay). [Figure 7E2] This document describes embodiments of CYP3A4 and CYP1A2 activity assays in response to rifampicin and omeprazole in a control and 1 mg / L bilirubin-treated HLO (RLU (relative light unit): relative luminescence, CTG: CellTiter-Glo assay). [Figure 7F] This figure shows an embodiment of immunofluorescence of mature liver enzymes and transport proteins in 1 mg / L bilirubin-treated liver organoids. Figure 7F shows the detection of CYP2E1 and MRP3. Figure 7G shows the detection of CYP7A1 and MRP1. Figure 7H shows the detection of PROX1 and OATP2. [Figure 7G] This figure shows an embodiment of immunofluorescence of mature liver enzymes and transport proteins in 1 mg / L bilirubin-treated liver organoids. Figure 7F shows the detection of CYP2E1 and MRP3. Figure 7G shows the detection of CYP7A1 and MRP1. Figure 7H shows the detection of PROX1 and OATP2. [Figure 7H] This figure shows an embodiment of immunofluorescence of mature liver enzymes and transport proteins in 1 mg / L bilirubin-treated liver organoids. Figure 7F shows the detection of CYP2E1 and MRP3. Figure 7G shows the detection of CYP7A1 and MRP1. Figure 7H shows the detection of PROX1 and OATP2. [Figure 8A] This shows an embodiment of a bright-field image of HLO with ascorbic acid depletion on day 15 compared to a control. [Figure 8B] This document illustrates an exemplary workflow for generating mGULO iPSC. [Figure 8C1] This document presents a linear map of the synthetic mGULO-mCherry gene designed for mGULO expression under the doxycycline-activated TetOn system in hiPSCs, and an embodiment of the vector map of the pAAVS1-Ndi-CRISPRi(Gen1) plasmid used to clone the mGULO gene into hiPSCs. [Figure 8C2] This document presents a linear map of the synthetic mGULO-mCherry gene designed for mGULO expression under the doxycycline-activated TetOn system in hiPSCs, and an embodiment of the vector map of the pAAVS1-Ndi-CRISPRi(Gen1) plasmid used to clone the mGULO gene into hiPSCs. [Figure 8D]A schematic embodiment for generating HLO using iPSCs modified to express the TetOn mGULO gene is shown. [Figure 8E] This document presents bright-field and fluorescence images of mCherry expression in doxycycline (Dox)-treated mGULO HLO compared to control HLO. [Figure 8F] This document shows examples of bright-field and fluorescence images of mCherry expression in ascorbic acid-depleted mGULO HLO with and without Dox treatment on day 18. [Figure 8G1] This document describes an ELISA method for comparing mGULO protein expression and cellular antioxidant concentrations in mGULO HLO treated with Dox (10 or 100 ng / mL) compared to a control HLO. [Figure 8G2] This document describes an ELISA method for comparing mGULO protein expression and cellular antioxidant concentrations in mGULO HLO treated with Dox (10 or 100 ng / mL) compared to a control HLO. [Figure 8H] This document describes the RT-qPCR assay of inflammation and detoxification marker genes (NRF2, 1L1B, IL6, and TNFa) in ascorbic acid-depleted Dox-treated mGULO HLO compared to ascorbic acid-depleted controls or mGULO HLO. [Figure 8I] This document describes an embodiment of a caspase-3 activity assay for ascorbic acid-depleted Dox-treated mGULOHLO compared to an ascorbic acid-depleted control or mGULOHLO. [Figure 8J] This example shows a heatmap from RNA-seq demonstrating that Dox-treated mGULO HLO expresses periportal markers compared to control HLO. [Figure 8K] This paper demonstrates functionally classified embodiments of gene upregulation, showing that the paraportal pathway is over-presented in Dox-treated mGULO HLO. [Figure 8L]Bright-field images and ImageJ-based embodiments of tubular contours of Dox-treated mGULO and control HLO with and without 1 mg / L bilirubin treatment are shown, with arrows indicating tubular projections similar to bile canaliculi found in human liver. [Figure 8M1] This example illustrates a comparison of lumen size and roundness between Dox-treated mGULO HLO or control HLO with or without 1 mg / L bilirubin treatment. [Figure 8M2] This example illustrates a comparison of lumen size and roundness between Dox-treated mGULO HLO or control HLO with or without 1 mg / L bilirubin treatment. [Figure 8N] This document describes an embodiment for quantifying albumin expression in Dox-treated mGULO HLO or control HLO with or without 1 mg / L bilirubin treatment. [Figure 8O] Embodiments of bright-field images of mGULO HLO treated with bilirubin and Dox at various concentrations (0, 10, 100, or 1000 ng / mL) are shown. [Figure 8P1] This document describes embodiments of CYP3A4 and CYP1A2 activity assays in control or Dox-treated mGULO HLO containing 1 mg / L bilirubin, in response to rifampicin and omeprazole. [Figure 8P2] This document describes embodiments of CYP3A4 and CYP1A2 activity assays in control or Dox-treated mGULO HLO containing 1 mg / L bilirubin, in response to rifampicin and omeprazole. [Figure 8Q] This document describes an embodiment of the UnaG assay that shows loss of fluorescence indicating bilirubin conjugation even in the presence of dark yellow bilirubin. [Figure 8R] This document describes an embodiment of the UnaG assay for mGULO organoids treated with Dox, compared to a control. [Figure 8S] This document describes an embodiment for quantifying the total percentage of viable organoids and organoids containing conjugated bilirubin in Dox-treated mGULO organoids compared to a control. [Figure 9A]A schematic diagram illustrating an embodiment for the generation of HLO and treatment with bilirubin at various concentrations is shown. [Figure 9B] The following are embodiments of bright-field images of HLO treated with bilirubin (0-10 mg / L) after 1 and 4 days. [Figure 9C] This document describes the RT-qPCR of the UGT1A1 and NRF2 genes in organoids treated with various concentrations of bilirubin, compared to untreated organoids. [Figure 9D] This document presents a profile of a patient with Crigler-Najjar Syndrome (CNS) who has produced CNS iPSCs. DNA sequencing of the patient revealed a nonsense mutation c.858C>A(p.Cys280X) in the UGT1A1 gene. [Figure 9E] This document presents a fluorescence image embodiment showing that CNS iPSCs derived from patients with Crigler-Nadjar syndrome express the standard pluripotency markers Sox2 and Oct4. [Figure 9F] This document presents bright-field imaging embodiments demonstrating that CNS iPSCs can be differentiated into definitive endoderm (DE) and liver organoid (hLO) according to a standard protocol. [Figure 9G] This document presents a fluorescence image demonstrating that liver organoids produced from CNS iPSCs express the proliferation marker Ki67, the liver-specific marker AFP, and the epithelial marker ECAD. [Figure 9H] Bright-field images of CNS HLOs treated with bilirubin (10 mg / L) and a control (0 mg / L bilirubin) at 1 and 4 days post-treatment are shown, demonstrating that these HLOs are susceptible to bilirubin toxicity. [Figure 9I] This image shows a bright-field image of CNS HLO transfected with CNS HLO and UGT1A1 mRNA 10 days after treatment with bilirubin (10 mg / L). [Figure 9J]Figure 9I shows an embodiment of a bilirubin assay for measuring unconjugated bilirubin (UCB) and conjugated bilirubin (CB) in HLO. [Figure 9K] This document describes an embodiment of a bilirubin assay for measuring unconjugated (UCB) and conjugated (CB) bilirubin in mGULO HLO treated with 10 mg / L bilirubin and Dox (0, 10, 100, or 1000 ng / mL). [Figure 10A] The following are embodiments of bright-field images of liver organoids treated with 10 mg / L bilirubin and the glucocorticoid agonist hydrocortisone (HCl or 5 μM) or dexamethasone (Dex1 or 5 μM). [Figure 10B] Figure 10A shows an embodiment of a bilirubin assay for measuring unconjugated and conjugated bilirubin in liver organoids. [Figure 10C] The image shows a bright-field image of liver organoids treated with 10 mg / L bilirubin and the glucocorticoid antagonist ketoconazole (KCZ, 1 or 5 μM) or mifepristone (Mif, 1 or 5 μM). [Figure 10D] Figure 10C shows an embodiment of a bilirubin assay for measuring unconjugated and conjugated bilirubin in liver organoids. [Figure 10E] This document describes the RT-qPCR of UGT1A1 and NRF2 genes in organoids treated with 10 mg / L bilirubin compared to organoids treated with 10 mg / L bilirubin and hydrocortisone, dexamethasone, ketoconazole, or mifepristone. [Figure 10F1] This document shows an embodiment of comparing enrichment pathways obtained from RNA sequencing between organoids treated with 10 mg / L bilirubin and 1 μM mifepristone, compared to a control, as well as a GSEA plot comparing enriched ROS and xenobiotic metabolism. [Figure 10F2]This document shows an embodiment of comparing enrichment pathways obtained from RNA sequencing between organoids treated with 10 mg / L bilirubin and 1 μM mifepristone, compared to a control, as well as a GSEA plot comparing enriched ROS and xenobiotic metabolism. [Figure 10G] This diagram illustrates an embodiment of a Venn diagram showing genes that are differentially expressed in ROS and xenobiotic metabolism. [Figure 10H1] Embodiments of ChIP-PCR and CH1P-qPCR on organoids treated with 10 mg / L bilirubin and either 1 μM mifepristone (Mife) or 1 μM dexamethasone (Dex) are shown. [Figure 10H2] Embodiments of ChIP-PCR and CH1P-qPCR on organoids treated with 10 mg / L bilirubin and either 1 μM mifepristone (Mife) or 1 μM dexamethasone (Dex) are shown. [Figure 11A1] This document describes an embodiment of a workflow for orthotopic transplantation of HLOs in rodents. [Figure 11A2] This document describes an embodiment of a workflow for orthotopic transplantation of HLOs in rodents. [Figure 11B] This document describes an example of albumin ELISA on serum collected from Gunn rats transplanted with mGULO HLO or pseudo-gang at different time points after transplantation. [Figure 11C] Figure 11B shows an example of a bilirubin assay in Gunn rats after transplantation. [Figure 11D1] Figure 11B shows an example of AST and ALT assays performed on Gunn rats after transplantation. [Figure 11D2] Figure 11B shows an example of AST and ALT assays performed on Gunn rats after transplantation. [Figure 12A] This document describes exemplary process embodiments for subjecting HLOs induced by conventional methods to expansion culture in a 3D bioreactor under conditions with or without Matrigel®. [Figure 12B]This document illustrates the HLO growth after 15 days in 3D bioreactor culture compared to conventional static culture. [Figure 12C] This invention demonstrates an embodiment that obtains a large HLO in 3D bioreactor culture, even in the group without the addition of Matrigel®. [Figure 13] Embodiments showing similar expression levels of E-cadherin, vimentin, and Prox1 demonstrate that HLO obtained from 3D bioreactor culture has comparable properties to that obtained from static culture. [Figure 14A] This invention describes an embodiment in which HLOs induced by a conventional method are dissociated into single cells, and then subculturing and HLO reconstitution are carried out using a 3D bioreactor. [Figure 14B] Embodiments are shown that induce a uniform HLO after 6 days via HLO reconstruction in a 3D bioreactor, both with and without Matrigel®. [Figure 15A] This diagram shows a schematic embodiment of a model for acetaminophen-assisted acute liver injury rescue using HLO transplantation. [Figure 15B] An embodiment of the HLO image used for transplantation is shown. [Figure 15C] This shows an embodiment of the Kaplan-Meier survival curve for acute liver injury rescue by HLO transplantation. [Modes for carrying out the invention]

[0046] 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 are expressly intended herein.

[0047] The following description of various embodiments is illustrative and descriptive only and should not be construed as limiting or restricting in any way. Other embodiments, features, purposes, and advantages of this teaching will be apparent from the description and accompanying drawings, as well as from the claims.

[0048] 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.

[0049] It should be understood that the use of subheadings in this specification is for structural purposes only and should not be interpreted as limiting the application of the features described herein to the various embodiments. All features described herein are applicable and usable in all of the various embodiments discussed herein, and all features described herein can be used in any intended combination, regardless of the specific exemplary embodiments described herein. Furthermore, it should be noted that exemplary descriptions of specific features are used primarily for informational purposes and are never used to limit the design, sub-features, and functions of the specifically described features.

[0050] overview The development of a method for producing human liver organoids from pluripotent stem cells has made it possible to study liver function that closely resembles that of a natural liver. Compared to methods involving combinations of primary hepatocytes or differentiation from adult pluripotent cells, differentiation of pluripotent stem cells results in liver organoids with rich cell type diversity, including hepatocytes, stellate cells, Kupffer cells, and other epithelial and mesenchymal cell lineages that constitute a normal liver.

[0051] However, because the maintenance of pluripotent stem cells and the formation of downstream intermediates are sensitive, previous culture methods generally involved the use of basement membrane matrices that mimic niches that promote cell proliferation. Common basement membrane matrices used in laboratory settings are isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. While these matrices offer great utility in cell culture, their presence hinders their subsequent use in humans because they contain heterologous animal components and may potentially be pathogenic. Therefore, there is an ongoing process to develop cell culture methods that do not involve the use of heterologous basement membrane matrices.

[0052] This specification provides a method for culturing organoids, such as liver organoids, without using Matrigel® or other heterogeneous basement membrane matrices. Other improvements for culturing liver organoids, such as supplementing the growth medium and approaches for scaling cells beyond the limitations of laboratory culture for large-scale production, are also disclosed.

[0053] Furthermore, it is disclosed herein that liver-related diseases and disorders may be modeled using human liver organoids (HLOs), such as those produced according to the methods disclosed herein. For example, HLOs produced according to the methods disclosed herein may be used to model hyperbilirubinemia by treating them with various concentrations of bilirubin. {Once a claim is agreed / confirmed, it will be updated with any further independent claim embodiment.}

[0054] The incidence of liver disease is increasing at an accelerating rate. Neonatal hyperbilirubinemia (NH) is one condition that worsens the health of newborns. Neonatal hyperbilirubinemia (NH) affects 60% of all newborns and accounts for 114,000 deaths worldwide annually. Currently, the only treatment for NH involves 12 hours of phototherapy or exchange transfusion, but this can cause other complications. Therefore, efficient and scalable model systems for these liver diseases are now needed to understand the molecular mechanisms behind liver disease and to develop potential therapies.

[0055] Currently, there are two main model organisms for modeling NH: Gunn rats and UGT1A1 knockout mice. However, these models lack key human proteins (OATP family) and epigenetic regulation involved in bilirubin metabolism. Recent studies have revealed that many aspects of UGT1A1 regulation are poorly translated across species. The genetic regulation of the UGT1A1 gene, the rate-limiting enzyme in bilirubin metabolism, differs significantly in humans. UGT-deficient Gunn rats are extremely difficult to maintain and require special housing facilities. In addition, UGT1 KO mouse models exhibit lethal hyperbilirubinemia and survive for only a few weeks. Many treatments have been attempted using these models, but most have failed.

[0056] Breeding model organisms such as mice and rats require months of work and planning, and the probability of obtaining the desired genotype is relatively low. Furthermore, model organisms exhibit high variability in response to biochemical perturbations over generations. These rodents also risk losing the desired genotype when reared over long periods, and require complex training and procedures to model diseases and evaluate the effectiveness of treatments. Therefore, there is a great need to develop more balanced models to understand the dynamics of bilirubin metabolism.

[0057] HLOs, for example, those produced according to the methods disclosed herein, are easy to handle and exhibit very low batch-to-batch variability. Even large batches of HLOs can be produced within a few weeks. These properties have been used to test several drugs over short periods and identify key pathways involved in bilirubin metabolism. Therefore, liver organoids are a useful model for studying diseases and disorders associated with bilirubin metabolism dysfunction, such as jaundice, Crigler-Nadjar syndrome, Gilbert's syndrome, Dubin-Johnson syndrome, or Rotor syndrome.

[0058] These HLOs can be derived from patient-derived induced pluripotent stem cells (iPSCs), which can be healthy or diseased, and have the same genetic content as the respective patient. HLOs express most liver markers expressed in the prenatal stage of development. Furthermore, HLOs are clones and therefore respond similarly to external stimuli and biochemical perturbations. These HLOs are highly scalable and easy to handle, enabling a screening approach for testing a vast number of drugs and small molecules.

[0059] Abnormal bilirubin metabolism leads to disease, but bilirubin is an important metabolite during early fetal development and acts as a metabolic hormone that plays an antioxidant role in adulthood. HLO models prepared by previous methods that do not involve the use of bilirubin resemble immature tissues and express fetal and intestinal markers. Therefore, as disclosed herein, HLO maturation was induced by using low doses of bilirubin (mimicking normal physiological levels) during the culture process disclosed herein. Modulation of the glucocorticoid receptor pathway in these HLOs, such as by treatment with mifepristone and ketoconazole, improved bilirubin conjugation and metabolism.

[0060] Vitamin C is also necessary for proper fetal development and is involved in the formation of the periportal zone of the liver. L-gulonolactone oxidase (GULO) is a naturally occurring enzyme that synthesizes vitamin C, but this enzyme does not function in humans and some other animals, such as guinea pigs, and requires exogenous vitamin C supplementation (typically through diet). As shown in guinea pig animal models, vitamin C deficiency causes significant metabolic disorders.

[0061] Compared to model organisms, genetic modification of iPSC cell lines is far easier and can be easily maintained for longer periods before differentiation into organoids. This was utilized to generate iPSC-derived organoids expressing functional L-gulonolactone oxidase (GULO), such as mouse GULO (mGULO), (e.g., those produced according to the methods disclosed herein). When iPSCs and organoids are of human origin, the expression of functional L-gulonolactone enables ascorbic acid synthesis, which is normally inactive in humans. These mGULO organoids showed increased bilirubin conjugation efficiency and improved viability when treated with bilirubin. Ascorbic acid production in mGULO organoids reduces oxidative stress in the organoids, drives the expression of NRF2, a key regulator of the cellular detoxification pathway, and subsequently promotes the expression of UGT1A1, which catalyzes bilirubin conjugation. These mGULO organoids are otherwise genetically identical to those from which they originate and encompass aspects of human bilirubin metabolism. Therefore, these organoids can be used as model systems to elucidate the mechanistic development of liver diseases and disorders such as NH and to develop therapeutic measures for them.

[0062] Definition of Terms 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 are expressly intended herein.

[0063] 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:

[0064] 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.

[0065] 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.

[0066] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by approximately 10% relative to the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length being referenced.

[0067] The use of the term “or” in the claims is used to mean “and / or” unless it is expressly indicated that it refers only to substitutes, or unless the substitutes are mutually exclusive; however, this disclosure supports the definitions of substitutes only and “and / or” as they refer. For example, “x, y, and / or z” could mean “x” only, “y” only, “z” only, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically intended that x, y, or z may be specifically excluded from the embodiments. As used herein, “another” may mean at least two or more.

[0068] The term "one" means more than one.

[0069] As used herein, the term “plural” may mean two, three, four, five, six, seven, eight, nine, ten or more.

[0070] As used herein, the term "set" means one or more. For example, a set of items includes one or more items.

[0071] As used herein, the phrase “at least one of” means, when used with a list of items, that one or more different combinations of the enumerated items may be used, and only one of the items in the list may be required. An item may be a specific object, thing, process, action, or category. In other words, “at least one of” means that any combination of items or several items may be used from the list, but not all items in the list may be required. For example, but not limited to, “at least one of item A, item B, or item C” means item A, item A and item B, item B, item A, item B, and item C, item B and item C, or item A and C. In some cases, “at least one of item A, item B, or item C” means, but not limited to, two item A, one item B, and ten item C, four item B and seven item C, or any other appropriate combination.

[0072] As used herein, “substantially” means sufficient to function for the intended purpose. Therefore, the term “substantially” allows for slight, insignificant variations from absolute or perfect conditions, dimensions, measurements, results, etc., that are expected by those skilled in the art but do not significantly affect the overall performance. Where used in relation to numerical values ​​or parameters or features that can be expressed numerically, “substantially” means within 10 percent.

[0073] 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.

[0074] Throughout this specification, references to “one embodiment,” “embodiment,” “specific embodiment,” “related embodiment,” “a particular embodiment,” “additional embodiment,” or “further embodiment,” or any combination thereof, mean that the specific features, structures, or characteristics described in relation to an embodiment are included in at least one embodiment of this disclosure. Therefore, occurrences of the aforementioned phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, specific features, structures, or characteristics may be combined in any preferred manner in various embodiments.

[0075] 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.

[0076] As used herein, terms such as “treatment,” “to treat,” and “to cure” in relation to a disease or condition may refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. For example, treatment may include implementing a protocol that may involve administering one or more drugs to a patient to alleviate the signs or symptoms of a disease. Desired effects of treatment include a reduction in the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. Mitigation may occur before the signs or symptoms of the disease or condition appear, and after they appear. Therefore, “to treat” or “treatment” may include “prevention” or “prevention” of a disease or undesirable condition. In addition, “to treat” or “treatment” may include protocols that do not require complete mitigation of signs or symptoms, do not require a cure, and specifically have only a minor effect on the patient.

[0077] As used herein, “treatment” can encompass any treatment of a disease in a subject, particularly in humans, and includes (a) preventing the onset of the disease in a subject who is susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., halting its development; and (c) reducing the disease, i.e., causing disease regression and / or reducing one or more disease symptoms. “Treatment” can also encompass the delivery of drugs or administration of therapies to provide pharmacological effects, even in the absence of a disease or condition.

[0078] As used throughout this application, the terms “therapeutic effective” or “therapeutic effective dose” may refer to any amount effective in achieving a desired and / or beneficial effect, and / or any amount that promotes or enhances the well-being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the frequency or severity of one or more signs or symptoms of a disease. An effective dose may be administered in one or more doses. In this method, a therapeutic effective dose is an amount appropriate for treating the indication. Treating the indication means achieving any desired effect, such as inhibiting, improving, stabilizing, reversing, slowing or delaying the progression of a disease, improving quality of life, or extending lifespan. Such achievement may be measured by any preferred method, such as measuring tumor size or blood cell count, or any other preferred measurement.

[0079] 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.

[0080] As used herein, the term “disease state” may generally refer to a condition affecting the structure or function of an organism. A disease state may include, for example, stages in disease progression.

[0081] As used herein, the term “evaluate” may include any form of measurement, including determining whether or not an element is present. The terms “determine,” “measure,” “evaluate,” “assessing,” and “assay” may be used interchangeably and may include quantitative and / or qualitative determinations.

[0082] As used herein, the terms “modulated” or “modulation,” “regulated” or “regulation,” and “differentially regulated” may refer to both upregulation (i.e., activation or stimulation, e.g., by stimulation or enhancement) and downregulation (i.e., inhibition or suppression, e.g., by antagonism, reduction, or inhibition), unless otherwise specified or as is evident from the context of a particular use.

[0083] 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.

[0084] As used herein, the terms “marker” or “biomarker” may refer to any measurable substance taken as a sample from an object whose presence indicates a certain phenomenon. Non-limiting examples of such phenomena include disease states, conditions, or exposure to compounds or environmental conditions. In various embodiments described herein, biomarkers may be used for diagnostic purposes (e.g., to diagnose disease states, health states, asymptomatic states, symptomatic states, etc.). The term “biomarker” may be used interchangeably with the term “marker.” The terms “marker” or “biomarker” may include biomolecules such as nucleic acids, peptides, proteins, and hormones whose presence or concentration can be detected and correlated with known conditions, such as disease states. It may also be used to refer to differentially expressed genes whose expression patterns can be used as part of a predictive, prognostic, or diagnostic process in healthy or diseased states, or in methods for identifying useful therapeutic or prophylactic therapies.

[0085] As used herein, the term “cellular phenotype” may refer to any determinable, observable, and / or measurable characteristic associated with a population of cells.

[0086] As used herein, “Model” may include one or more in vitro or in vivo disease models, and a Model may also include algorithms, one or more mathematical techniques, one or more machine learning algorithms, or a combination thereof. A Model may be used in a process and / or applied to an assay according to the various embodiments disclosed herein.

[0087] As used herein, “process” may include one or more steps having one or more features of one or more models disclosed herein.

[0088] As used herein, the terms “function” and “functional” have their general and ordinary meanings as understood in light of this specification and may refer to biological, enzymatic, or therapeutic functions.

[0089] As used herein, the term “inhibit” has its general and ordinary meaning as understood herein and may mean a reduction or prevention of biological activity. A 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 about those, or about less than or about 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 a 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.

[0090] 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.

[0091] As used herein, “in vivo” is given its general and ordinary meaning as understood herein, and refers to the execution of a method in living organisms, typically animals, mammals including humans, and plants, as opposed to tissue extracts or dead organisms.

[0092] As used herein, “ex vivo” is given its general and ordinary meaning as understood herein, and refers to the execution of a method outside of a living organism with little alteration of natural conditions.

[0093] 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.

[0094] 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 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.

[0095] 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

[0096] The nucleic acids described herein include nucleic acid bases. Primary, standard, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleic acid bases include, but are not limited to, purines, pyrimidines, modified nucleic acid bases, 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.

[0097] 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.

[0098] 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.

[0099] The term “yield” for any given substance, compound, or material as 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 amount, 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. The yield may be affected by the efficiency of the reaction or process, undesirable side reactions, decomposition, the quality of the input substances, compounds, or materials, or the loss of the desired substance, compound, or material at any stage of production.

[0100] 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 contain small amounts of wetting agents, fillers, emulsifiers, or pH buffers, as needed. These compositions may take the form of solutions, suspensions, emulsions, or sustained-release formulations. The formulation should be suitable for the mode of administration.

[0101] 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 about those, or about that less than or about those, or any percentage within the range defined by any two of the aforementioned numbers.

[0102] 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.

[0103] 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.

[0104] The appropriate formulation will vary depending on the chosen route of administration. The formulations and techniques for administering 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, intradermal, aerosol, parenteral delivery (including intramuscular, subcutaneous, intra-arterial, intra-intravenous), intra-portal, intra-articular, intradermal, peritoneal, intrathecal, intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections. Pharmaceutical compositions will generally be formulated to suit a specific intended route of administration.

[0105] 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.

[0106] 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.

[0107] 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 in 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.

[0108] 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 (typically the surface of a cell culture vessel (e.g., a plate, dish, or flask) immersed in culture medium). For example, cells can grow as a monolayer on the surface of a cell culture vessel. Once the growing cells occupy all available space on the surface, they can no longer proliferate 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 seeding this fraction onto a fresh surface in culture medium (e.g., a cell culture vessel). This fraction of cells continues to proliferate and increase until it occupies available space on a new surface, after which this passaging can be repeated continuously.

[0109] The microscopic structure of the liver is composed of polygonal structures called "hepatic lobules." Classically, these lobules have a hexagonal structure, but other geometric shapes are observed depending on the tissue's characteristics. Each lobular unit contains a plate or layer of hepatocytes enclosed by a bundle of blood vessels called the portal vein triplicate, which consists of the portal vein, hepatic artery, and bile duct. Liver activity occurs when blood flows from the peripheral portal vein triplicate across the hepatocytes to the central vein and back into the circulatory system. Due to the asymmetric organization of these lobules, the layer of hepatocytes is divided into three zones. Cells in the "periportal zone" (zone 1) are closest to the portal vein triplicate and receive the most oxygenated blood; cells in the central zone (zone 3) are closest to the central vein and therefore receive the smallest amount of oxygenated blood; and the transitional zone (zone 2) lies between zones 1 and 3. This separation results in each zone of hepatocytes exhibiting different activity. For example, hepatocytes in zone 1 are involved in oxidative liver functions such as gluconeogenesis and oxidative metabolism of fatty acids, while hepatocytes in zone 3 are involved in glycolysis, lipid synthesis, and cytochrome P450-mediated detoxification. In some embodiments, the liver organoids disclosed herein exhibit periportal-like identity, which is similar to the tissue found in the periportal zone of a hepatic lobule, including the functionality and cellular marker features of the periportal zone.

[0110] As used herein, the term “bilirubin” has its general and ordinary meaning as understood herein and refers to a naturally occurring metabolite produced by the normal catabolic breakdown of heme. Bilirubin is produced from the catalytic action of biliverdin by biliverdin reductase. In the liver, bilirubin is conjugated with glucuronic acid by a family of enzymes called UDT-glucuronosyltransferase (UGT). This conjugation makes bilirubin water-soluble, allowing it to be transported in the bile to the small and colonic intestines, where it is further metabolized into waste products. Dysfunction of bilirubin metabolism, particularly due to abnormal function of UGT that interferes with bilirubin conjugation, leads to bilirubin accumulation and is associated with various diseases characterized by hyperbilirubinemia. However, while excess bilirubin is harmful, bilirubin also possesses antioxidant properties and can therefore have beneficial effects in reducing oxidative damage in cells.

[0111] As used herein, the term “hyperbilirubinemia” has its general and ordinary meaning as understood herein and refers to a condition of elevated levels of bilirubin, a natural product of heme catabolism. Bilirubin is filtered from the blood by the liver, converted to water-soluble intermediates, and then released into the intestinal tract in the bile, where it is metabolized by the microbiome and excreted as waste. In newborns, bilirubin levels originally removed by the mother through the placenta may not be adequately removed by the immature liver. Excessive levels of bilirubin may cause severe neurological damage (kernicterus). In adults, hyperbilirubinemia may also result from liver-affecting diseases such as hepatitis and cirrhosis. Neonatal hyperbilirubinemia is treated with phototherapy or, in extreme cases, blood transfusions, but treatment in adults is directed towards the underlying cause.

[0112] As used herein, the terms “L-gulonolactone oxidase” and “GULO” have their general and ordinary meanings as understood herein and refer to an enzyme that catalyzes L-gulonolactone to produce L-xylo-hexa-3-gulonolactone and hydrogen peroxide. L-xylo-hexa-3-gulonolactone then spontaneously converts to ascorbic acid (vitamin C). Thus, this enzyme is involved in the biosynthesis of vitamin C, an essential nutrient involved in many biological functions, including its use as a cofactor for several important enzymes and as an antioxidant. Notably, humans, as well as other rhinorhini primates, certain species of bats and guinea pigs, have evolved to possess non-functional GULO genes. Therefore, these organisms are unable to synthesize ascorbic acid and require vitamin C intake from diet or supplements; vitamin C deficiency can lead to scurvy. Where applicable to the disclosure herein, “functional GULO protein” is a GULO protein that possesses L-gulonolactone catalytic activity resulting in the production of ascorbic acid. Conversely, "inactive" GULO proteins or "non-functional" GULO proteins are those that do not possess catalytic activity for the production of ascorbic acid. Human and human-derived cells contain non-functional GULO proteins and do not have the ability to synthesize ascorbic acid. However, as disclosed herein, human cells may be engineered to express functional GULO proteins to enable ascorbic acid synthesis. These functional GULO proteins may be expressed in human cells (or other cells that are not normally able to synthesize ascorbic acid) by conventional cloning methods, such as genetically engineering cells to have a gene sequence encoding a functional GULO protein.

[0113] 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.

[0114] As used herein, the term “exogenous” has its general and ordinary meaning as understood in light of this specification and refers to external factors originating from outside the biological specimen (e.g., cells, cell populations, organoids, etc.), as opposed to those that are naturally occurring and / or produced by the biological specimen itself. The exogenous components, reagents, and / or conditions as used herein are components, reagents, and / or conditions added to the compositions described herein, but this does not necessarily preclude the possibility that the same components, reagents, and / or conditions may also be present in the biological specimen through endogenous functions.

[0115] The terms “liver organoid” and “hepatocyte organoid” are used interchangeably herein and generally refer to a population of cells differentiated in vitro to form a self-organizing structure that is three-dimensional (3D) and contains one or more functional cell types. Liver organoids differ in several ways from naturally occurring liver tissue. For example, compared to naturally occurring liver tissue, liver organoids have structures with a single lumen, can generally be spherical, and may contain a non-natural basement membrane. The single lumen of liver organoids contains 3D tissue but generally does not form hepatic lobular or cord-like structures like naturally occurring liver tissue. Liver organoids also generally do not contain hematopoietic tissue and adaptive immune cell subsets such as T cell lineages. Furthermore, compared to naturally occurring liver tissue, liver organoids can have different efflux mechanisms because they have three-dimensional structures with tubular structures but without efflux mechanisms. In addition, liver organoids generally cannot receive food intake because they lack intestinal and connected vascular channels.

[0116] Liver organoids may be derived from at least embryonic stem cells (ESCs) or pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs). Liver organoids may also be formed from liver-derived stem cells. Generally, liver organoids can self-assemble via cell sorting and spatially restricted lineage determination through one or more directed steps, which optionally involve the introduction of one or more components, as optionally, but in a manner similar to that which occurs in vivo, but directed in vitro by the careful introduction of exogenous and / or endogenous differentiation factors and / or conditions described herein.

[0117] As used herein, the term “mature liver organoid” refers to a liver organoid that continues to develop from a liver organoid and, in various embodiments, includes luminal projections resembling bile canaliculi, and / or structures having a single lumen and generally being spherical. Mature liver organoids may exhibit lumens having a smaller size and reduced roundness compared to the lumen of a liver organoid. In some embodiments, mature liver organoids may be produced by the addition of exogenous bilirubin and / or amino acid supplementation as described herein. In some embodiments, mature liver organoids may be characterized by expressing reduced levels of AFP, CDX2, and / or NANOG, and / or increased levels of ALB, SLC4A2, and / or HO-1 compared to a liver organoid. In some embodiments, mature liver organoids may be characterized by expressing CYP2E1, CYP7A1, PROXI, MRP3, and / or OATP2. In some embodiments, mature liver organoids may exhibit increased CYP3A4 and / or CYP1A2 protein levels and / or enzymatic activity compared to liver organoids.

[0118] As used herein, the term “hyperbilirubinemia liver organoid” refers to liver organoids exposed to high concentrations of bilirubin, typically provided exogenously by one or more doses, to mimic a hyperbilirubinemia state. In some embodiments, hyperbilirubinemia liver organoids include genetic abnormalities that alter bilirubin metabolism, for example, resulting in increased levels of bilirubin anabolism and / or reduced levels of bilirubin catabolism. Hyperbilirubinemia liver organoids may be characterized by expressing elevated levels of UGT1A1 and / or NRF2 compared to liver organoids not exposed to high concentrations of bilirubin.

[0119] As used herein, the term “tissue culture surface” has its general and ordinary meaning as understood in light of this specification and refers to a substrate surface on which cells can aggregate and / or adhere to facilitate cell growth, differentiation, and / or function.

[0120] As used herein, the term “engineered” refers to entities produced by human hands, including cells, nucleic acids, polypeptides, vectors, and the like. In at least some cases, the engineered entities are synthetic and include elements that do not exist in nature or are not configured in the manner utilized herein. In certain embodiments, constructs and / or vectors are engineered by recombinant nucleic acid technology, and cells are engineered by transfection or transduction of the engineered vector. Because heterologous proteins are either recombinant or synthetic, or cells do not naturally express proteins, cells can be engineered to express heterologous proteins that are not naturally expressed by the cell.

[0121] 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.

[0122] As used herein, the term “embryonic stem cell (ESC)” is also commonly abbreviated as ES cell and, as understood in light herein, refers to pluripotent cells derived from the inner cell mass of the blastocyst, which is an early embryo, having its general and ordinary meaning.

[0123] 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 (stomach wall, 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 may be obtained by induction of 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.

[0124] As used herein, the term “induced pluripotent stem cells (iPSCs)” is commonly abbreviated as iPS cells and, as understood herein, has its general and ordinary meaning, referring to a type of pluripotent stem cell that is artificially obtained, typically from non-pluripotent stem cells, such as adult somatic cells, by inducing the “forced” expression of specific genes. hiPSC refers to human iPSCs. In some methods known in the art, iPSCs can be induced by transfection of non-pluripotent cells, such as adult fibroblasts, with certain stem cell-related genes. 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. In some methods, retroviral systems are used to transform human fibroblasts into pluripotent stem cells using four critical genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In other methods, lentiviral systems are used 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 (POU5FI), certain members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15), certain members of the Klf family (e.g., Klf1l, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, L1N28, Tert, Fbx15, Eras, ECAT15-1, ECAT15-2, Tel1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fthl17, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or any combination thereof.

[0125] As used herein, the term “progenitor cell” has its general and ordinary meaning as understood in light of this specification and encompasses any cell that can be used in the methods described herein, through which one or more progenitor cells acquire the ability to regenerate themselves or to differentiate into one or more specialized cell types. In some embodiments, the progenitor cell is pluripotent or capable of becoming pluripotent. In some embodiments, the progenitor cell is subjected to treatment with an extrinsic factor (e.g., a growth factor) to acquire pluripotency. In some embodiments, the progenitor cell may be totipotent (or omnipotent) stem cell, pluripotent stem cell (artificial or unartificial), multipotent stem cell, oligopotent stem cell, or unipotent stem cell. In some embodiments, the progenitor cell may be derived from an embryo, infant, child, or adult. In some embodiments, the progenitor cell may be a somatic cell subjected to treatment such that pluripotency is conferred through genetic engineering or protein / peptide treatment. Examples of progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (Ec), and epiblast stem cells (EpiSCs).

[0126] 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. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described herein, but it will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cells.

[0127] 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), databases hosted by the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF), the WISC cell bank at 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 at 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), UCOI (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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] PSC differentiation Known methods for producing downstream cell types such as endoderm, foregut endoderm, posterior foregut endoderm, and / or liver lineages from pluripotent cells (e.g., iPSCs or ESCs) are applicable to some embodiments of the methods described herein. In some embodiments, the pluripotent cells are derived from a morula. In some embodiments, the pluripotent stem cells are stem cells. Stem cells used in these methods include, but are not limited to, embryonic stem cells or induced pluripotent stem cells. Embryonic stem cells may be derived from the inner cell mass of an embryo or the gonadal ridge of an embryo. Embryonic stem cells may arise from a variety of animal species, including, but not limited to, various mammalian species, including humans. In some embodiments, human embryonic stem cells are used to produce endoderm, or other downstream cell types such as posterior foregut, posterior foregut endoderm, and / or liver lineages. In some embodiments, iPSCs are used to produce endoderm, or other downstream cell types such as posterior foregut, posterior foregut endoderm, and / or liver lineages. In some embodiments, human iPSCs (hiPSCs) are used to produce embryonic endoderm, or other downstream cell types such as posterior foregut, posterior foregut endoderm, and / or liver lineages.

[0132] 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 stepwise 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 specific signaling pathways in PSCs or any downstream cells.

[0133] 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.

[0134] 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.

[0135] 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 in 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 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.

[0136] 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.

[0137] In some embodiments, stem cells are treated with one or more growth factors to differentiate them into endoderm cells. Examples of such growth factors include those from the TGF-beta superfamily. In some embodiments, one or more growth factors comprise the nodal / activin and / or BMP subgroups of the TGF-beta superfamily of growth factors. In some embodiments, one or more growth factors are selected from the group consisting of nodal, activin A, activin B, BMP4, Wnt3a, or any combination thereof. In some embodiments, stem cells are contacted with activin A. In some embodiments, stem cells are contacted with activin A and BMP4.

[0138] 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 specific types of liver tissue formation. 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.

[0139] Culture and expansion of endoderm, foregut 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 Fiver Injury Screen Using Human Pluripotent Stem Cell-Derived This has been previously discussed in “Organoids” Gastroenterology (2021) 160(3):831-846, PCT Publications WO2018 / 085615, WO2018 / 191673, WO2018 / 226267, WO2019 / 126626, WO2020 / 023245, WO2020 / 069285, and WO2021 / 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.

[0140] In some embodiments, pluripotent stem cells, endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / or downstream hepatocyte types are cultured and expanded as described herein. In some embodiments, pluripotent stem cells, endoderm, posterior foregut, posterior foregut endoderm, and / or foregut endoderm are cultured and expanded as described herein. In some embodiments, pluripotent stem cells, endoderm, posterior foregut, and / or posterior foregut endoderm cells are cultured and expanded as described herein. In some embodiments, foregut endoderm cells are cultured and expanded as described herein.

[0141] In some embodiments, pluripotent stem cells, embryonic endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / 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-β 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.

[0142] In some embodiments, pluripotent stem cells, embryonic endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / or downstream hepatocyte types 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.

[0143] In some embodiments, pluripotent stem cells, embryonic endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / or downstream hepatocyte types 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, BML284, IQ-1, WAY262611, 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, CHIR98014, AZD2858, BIO, AR-A014418, SB216763, SB415286, 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.

[0144] In some embodiments, pluripotent stem cells, embryonic endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / 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.

[0145] In some embodiments, pluripotent stem cells, embryonic endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / or downstream hepatocyte types 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.

[0146] In some embodiments, pluripotent stem cells, embryonic endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / or downstream hepatocyte types 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.

[0147] In some embodiments, pluripotent stem cells, embryonic endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / or downstream hepatocyte types 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.

[0148] In some embodiments, pluripotent stem cells, embryonic endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / 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, BS493, 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.

[0149] In some embodiments, pluripotent stem cells, embryonic endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / or downstream hepatocyte types are converted to hepatocyte types via a “one-step process.” In some embodiments, pluripotent stem cells are converted to hepatocyte types 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.

[0150] In some embodiments, pluripotent stem cells, endoderm, posterior foregut, posterior foregut endoderm, foregut endoderm, and / or downstream hepatocyte types are expanded in cell culture. In some embodiments, pluripotent stem cells (e.g., ESCs and / or iPSCs), endoderm, posterior foregut, posterior foregut endoderm, and / or downstream hepatocyte types are expanded in cell culture. In some embodiments, pluripotent stem cells, endoderm, posterior foregut, posterior foregut endoderm, and / or downstream hepatocyte types are expanded in a basement membrane matrix. In some embodiments, pluripotent stem cells, endoderm, posterior foregut, posterior foregut endoderm, and / or downstream hepatocyte types are expanded in Matrigel®. In some embodiments, pluripotent stem cells, endoderm, posterior foregut, posterior foregut endoderm, and / or downstream hepatocyte types are expanded in a basement membrane matrix that does not contain non-human animal components. In some embodiments, pluripotent stem cells, endoderm, posterior foregut, posterior foregut endoderm, and / or downstream hepatocyte types are expanded in a non-heterogeneous basement membrane matrix. In some embodiments, pluripotent stem cells, endoderm, posterior foregut, posterior foregut endoderm, and / or downstream hepatocyte types are not expanded in Matrigel®. In some embodiments, pluripotent stem cells, endoderm, posterior foregut, posterior foregut endoderm, and / or downstream hepatocyte types are expanded in laminin, collagen IV, entactin, perlecan, fibrin, and / or hydrogel. In some embodiments, pluripotent stem cells, endoderm, posterior foregut, posterior foregut endoderm, and / or downstream hepatocyte types are expanded in a cell culture containing a ROCK inhibitor (e.g., Y-27632).

[0151] In some embodiments, pluripotent stem cells (e.g., ESCs and / or iPSCs) differentiate into endoderm cells. In some embodiments, pluripotent stem cells are differentiated into endoderm cells by contacting the pluripotent stem cells with activin A, BMP4, 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, 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, 10-200 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 BMP4 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, 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, BMP4 at concentrations of 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 contacted with BMP4 at a concentration of 50 ng / mL or about 50 ng / mL.

[0152] This specification provides a method for expanding posterior foregut cells. Exemplary methods are shown in Figure 5H, without being limited by specific features such as timing and growth conditions illustrated in the figure. In some embodiments, the method comprises a) dissociating a posterior foregut cell monolayer into posterior foregut cells and / or posterior foregut endoderm cells; b) seeding the posterior foregut cells and / or posterior foregut endoderm cells onto a tissue culture surface; and c) culturing the posterior foregut cells and / or posterior foregut endoderm cells with a TGF-β pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator. In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells are further cultured with ascorbic acid. In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells are not cultured with ascorbic acid. In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells are further cultured with EGF. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are not cultured with EGF. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are further cultured with ROCK inhibitors. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are not cultured with ROCK inhibitors. In some embodiments, a posterior foregut cell monolayer is dissociated into posterior foregut cells and / or posterior foregut endoderm cells using enzymatic and / or mechanical dissociation. In some embodiments, enzymatic dissociation may involve the use of any conventional enzymatic dissociation solution commonly known in the art, e.g., Accutase, Accumax, trypsin, trypsin / EDTA, collagenase, dispase, TrypEE Express, or TrypLE Select. In some embodiments, mechanical dissociation may involve disrupting cells using a pipette, microchannel, or other device having appropriately sized holes for mechanically shearing cell populations without disrupting individual cells. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are placed on the surface of the tissue container, with a surface area of ​​1 × 10⁻⁶ of the tissue culture surface area. 5 , 2×10 5 , 3 x 10 5 , 4×105 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1 x 10 6 , 2×10 6 , 3 x 10 6 , 4×10 6 , or 5×10 6 cells / cm 2 A cell density that is, approximately, at least, at least about, less than or equal to, or about less than or equal to, or any cell density within the range defined by any two of the aforementioned cell densities, e.g., 1 × 10⁻⁶ of the surface area of ​​the tissue culture surface. 5 ~5×10 6 , 1 x 10 5 ~5×10 5 , 5×10 5 ~5×10 6 , or 3 × 10 5 ~7×10 5 cells / cm 2 They are seeded. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are seeded on the surface of the tissue container, with a surface area of ​​5 × 10⁻⁶ of the tissue culture surface. 5 cells / cm 2 Or approximately 5 x 10 5 cells / cm 2The cells are seeded at a cell density. In some embodiments, the tissue culture surface is coated with a basement membrane matrix or its components. In some embodiments, the basement membrane matrix or its components does not contain non-human animal components so that the basement membrane matrix or its components are non-heterogeneous to humans. In some embodiments, the basement membrane matrix or its components is not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the basement membrane matrix or its components is not Matrigel®, Cultrex®, or Geltrex®. In some embodiments, the basement membrane matrix or its components contains human laminin, collagen IV, entactin, perlecan, fibrin, and / or hydrogel, or other substances that are non-heterogeneous to humans. In some embodiments, the basement membrane matrix or its components is laminin or contains laminin. In some embodiments, the basement membrane matrix or its components is laminin-511 or contains laminin-511. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are cultured until three-dimensional (3D) spheroids spontaneously form. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are cultured for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, about those days, at least those days, at least about those days, less than or equal to those days, or any number of days within the range defined by any two of the aforementioned days, for example, 4–6 days, 2–35 days, 2–15 days, 20–35 days, or 10–20 days.In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are cultured for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, or at least any number of days within the range defined by any two of the aforementioned number of days, for example, 4–6 days, 4–35 days, 4–15 days, 20–35 days, or 10–20 days.

[0153] 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.

[0154] 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.

[0155] 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, alster paulon, kaem paulon, lithium chloride, TDZD8, 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.

[0156] 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.

[0157] In some embodiments of the methods provided herein, the posterior foregut cells and / or posterior 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 posterior foregut cells and / or posterior foregut endoderm cells in step c) are cultured in an EGF-free medium.

[0158] In some embodiments of the methods provided herein, the posterior foregut cells and / or posterior 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 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, 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 posterior foregut cells and / or posterior foregut endoderm cells in step c) are cultured in a medium that does not contain ascorbic acid.

[0159] In some embodiments of the methods provided herein, posterior foregut cells and / or posterior foregut endoderm cells in step c) are cultured in a medium further comprising a ROCK inhibitor. 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 posterior foregut cells and / or posterior foregut endoderm cells in step c) are cultured in a medium that does not contain ROCK inhibitors.

[0160] In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells of the method provided herein may be cultured for multiple passages. In some embodiments, steps a) to c) of the method provided herein are repeated using the cells of step c). In some embodiments, the method further includes passage the cells of step c) one or more times. In some embodiments, the cells of step c) are passaged until the posterior foregut cells and / or posterior foregut endoderm cells cease to spontaneously form spheroids. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are passaged and cultured for 2, 3, 4, 5, 6, 7, 8, 9, 10 days, about those days, at least those days, at least about those days, less than or equal to those days, or any number of days within the range defined by any two of the aforementioned days, for example, 2-10 days, 2-4 days, 2-6 days, 4-10 days, 6-10 days, 4-6 days, or 3-7 days, after which the posterior foregut cells and / or posterior foregut endoderm cells are passaged again. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are passaged and cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or any number of days within the range defined by any two of the aforementioned number of days, e.g., 2–10 days, 2–4 days, 2–6 days, 4–10 days, 6–10 days, 4–6 days, or 3–7 days, after which the posterior foregut cells and / or posterior foregut endoderm cells are passaged again. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are passaged and cultured for 4, 5, or 6 days, after which the posterior foregut cells and / or posterior foregut endoderm cells are passaged again. In some embodiments, the cells are passaged one, two, or three times or less.In some embodiments, the total yield of posterior foregut cells and / or posterior foregut endoderm cells (which may be in the form of spheroids) after the method provided herein, involving multiple passages, is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, or 1000 times the total yield of posterior foregut cells and / or posterior foregut endoderm cells obtained by cell culture without passage, or any multiple within the range defined by any two of the aforementioned multiples, for example, 50 to 1000 times, 50 to 200 times, 200 to 1000 times, or 100 to 300 times.

[0161] In some embodiments, the method further comprises collecting posterior foregut cells and / or posterior foregut endoderm cells and differentiating the posterior foregut cells and / or posterior foregut endoderm cells into liver organoids. In some embodiments, the posterior foregut cells and / or posterior foregut endoderm cells are cultured until three-dimensional (3D) spheroids spontaneously form, and optionally, the spheroids include structures having a single lumen, and the posterior foregut cells and / or posterior foregut endoderm cells are collected from the spheroids. In some embodiments, the method further comprises dissociating the spheroids into individual posterior foregut cells and / or posterior foregut endoderm cells and / or clumps of posterior foregut cells and / or posterior foregut endoderm cells prior to the differentiation step. In some embodiments, prior to the differentiation process, posterior foregut cells and / or posterior foregut endoderm cells are collected from the posterior foregut cell monolayer by dissociating the monolayer into individual posterior foregut cells and / or posterior foregut endoderm cells and / or clumps of posterior foregut cells and / or posterior foregut endoderm cells.

[0162] This specification provides, in several embodiments, methods for expanding posterior foregut cells and / or posterior foregut endoderm and / or foregut endoderm cells. In some embodiments, the method comprises a) dissociating a posterior foregut cell monolayer into posterior foregut cells and / or posterior foregut endoderm cells; b) seeding the posterior foregut cells and / or posterior foregut endoderm cells onto a tissue culture surface; and c) culturing the posterior foregut cells and / or posterior foregut endoderm cells with a TGF-β pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator. In some embodiments, the posterior foregut cell monolayer is dissociated into posterior foregut cells and / or posterior foregut endoderm cells using enzymatic and / or mechanical dissociation. In some embodiments, enzymatic dissociation may involve the use of any conventional enzymatic dissociation solution commonly known in the art, such as Accutase, Accumax, trypsin, trypsin / EDTA, collagenase, dispase, TrypLE Express, or TrypLE Select. In some embodiments, mechanical dissociation may involve disrupting cells using a pipette, microchannel, or other device having appropriately sized holes for mechanically shearing cell populations without disrupting individual cells. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are placed on the surface of a tissue container: 1 × 10⁻⁶ of the surface area of ​​the tissue culture surface. 5 , 2×10 5 , 3 x 10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1 x 10 6 , 2×10 6 , 3 x 10 6 , 4×10 6 , or 5×10 6 cells / cm 2A cell density that is, approximately, at least, at least about, less than or equal to, or approximately less than or equal to, or any cell density within the range defined by any two of the aforementioned cell densities, e.g., 1 × 10⁻⁶ of the surface area of ​​the tissue culture surface. 5 ~5×10 6 , 1 x 10 5 ~5×10 5 , 5×10 5 ~5×10 6 , or 3 × 10 5 ~7×10 5 cells / cm 2 They are seeded. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are seeded on the surface of the tissue container, with a surface area of ​​5 × 10⁻⁶ of the tissue culture surface. 5 cells / cm 2 Or approximately 5 x 10 5 cells / cm 2The cells are seeded at a cell density. In some embodiments, the tissue culture surface is coated with a basement membrane matrix or its components. In some embodiments, the basement membrane matrix or its components does not contain non-human animal components so that the basement membrane matrix or its components are non-heterogeneous to humans. In some embodiments, the basement membrane matrix or its components is not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the basement membrane matrix or its components is not Matrigel®, Cultrex®, or Geltrex®. In some embodiments, the basement membrane matrix or its components contains human laminin, collagen IV, entactin, perlecan, fibrin, and / or hydrogel, or other substances that are non-heterogeneous to humans. In some embodiments, the basement membrane matrix or its components is laminin or contains laminin. In some embodiments, the basement membrane matrix or its components is laminin-511 or contains laminin-511. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are cultured until three-dimensional (3D) spheroids spontaneously form. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are cultured for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, about those days, at least those days, at least about those days, less than or equal to those days, or any number of days within the range defined by any two of the aforementioned days, for example, 2 to 35 days, 2 to 15 days, 20 to 35 days, or 10 to 20 days.In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells are cultured for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, or at least any number of days within the range defined by any two of the aforementioned number of days, for example, 4–35 days, 4–15 days, 20–35 days, or 10–20 days. In some embodiments, steps a)–c) of the method provided herein are repeated using the cells from step c). In some embodiments, the method further includes passage the cells from step c) one or more times. In some embodiments, the cells from step c) are passaged until the posterior foregut cells and / or posterior foregut endoderm cells cease to spontaneously form spheroids. In some embodiments, the cells are passed through one, two, or three times or less. In some embodiments, the total yield of posterior foregut cells and / or posterior foregut endoderm cells (which may be in the form of spheroids) after the method provided herein, involving multiple passages, is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, or 1000 times the total yield of posterior foregut cells and / or posterior foregut endoderm cells obtained by cell culture without passage, or any multiple within the range defined by any two of the aforementioned multiples, for example, 50 to 1000 times, 50 to 200 times, 200 to 1000 times, or 100 to 300 times. In some embodiments, Table 1 provides exemplary concentration ranges for each of the growth factors used in the Method. In embodiments of the Method, any concentration or range for a growth factor under a particular “sub-embodiment” may be used in combination with concentrations or ranges for other growth factors under the same or different “sub-embodiments.” Therefore, the combinations are not limited to those under the same “sub-embodiment.”For example, the presence of TGF-b pathway inhibitors, FGF pathway activators, Wnt pathway activators, and VEGF pathway activators, each defined under any "lower-level embodiment", can be combined with the presence of EGF (any one of EGF "lower-level embodiments" 1 to 3) or the absence of EGF ("lower-level embodiment" 4). The same applies to any combination of the listed growth factors, whether present or absent in the methods embodied herein. Exemplary non-limiting formulations are listed in Table 2. For each of the ranges provided in Table 1 and Table 2, this should be interpreted to include any concentration within the defined range. For example, 100 - 1000 nM TGF-b pathway inhibitor should be interpreted to include 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM TGF-b pathway inhibitor, or any concentration within the range defined by any two of the aforementioned concentrations. Exemplary concentrations within the defined range are provided throughout the present disclosure. In some embodiments, the TGF-b pathway inhibitor is A83-01. In some embodiments, the FGF signaling pathway activator is FGF2. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the VEGF pathway activator is VEGF. In some embodiments, the ROCK inhibitor is Y-27632.

[0163]

Table 1

[0164]

Table 2

[0165] Posterior foregut cells and / or posterior foregut endoderm cells produced by the methods provided herein are also disclosed herein.

[0166] Gene editing In some embodiments, iPSCs, endoderm cells, posterior foregut 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 is described in PCT Publications WO2013 / 176772, WO2014 / 093595, WO2014 / 093622, WO2014 / 093655, WO2014 / 093712, WO2014 / 093661, and WO2014 / 204. This is discussed in Nos. 728, WO2014 / 204729, WO2015 / 071474, WO2016 / 115326, WO2016 / 141224, WO2017 / 023803, and WO2017 / 070633, each of which is expressly incorporated herein in whole by reference.

[0167] Method and composition for producing liver organoids This specification provides methods and compositions for differentiating posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm into liver organoids. In some embodiments, the method comprises i) contacting posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells (optionally, the spheroids include structures having a single lumen), and / or aggregated cells in a microwell or other device as described herein, with a retinoic acid pathway activator; and ii) contacting the cells from step i) with a culture medium (e.g., hepatocyte culture medium (HCM)) containing hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone for a certain period of time, thereby differentiating the posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells into liver organoids. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells may be any of the posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells disclosed herein. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells may be in the form of spheroids, or individual posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells and / or aggregates of posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells derived from dissociating spheroids. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells may be produced by a method that does not involve the use of a heterologous basement membrane matrix. In some embodiments, posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells may be those commonly known in the art.

[0168] In some embodiments of the methods provided herein, the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580. In some embodiments, the retinoic acid pathway activator is retinoic acid. In some embodiments, the retinoic acid pathway activator is provided at a concentration of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 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, 1.0–3.0 μM, 1.0–2.0 μM, 2.0–3.0 μM, or 1.5–2.5 μM. In some embodiments, the retinoic acid pathway activator is provided at a concentration of 2.0 μM or about 2.0 μM.

[0169] In some embodiments, the culture medium (e.g., hepatocyte culture medium) is supplemented with a cMET tyrosine kinase receptor agonist, interleukin 6 (IL-6) family cytokines, and / or corticosteroids. In some embodiments, the cMET tyrosine kinase receptor agonist is selected from the group consisting of HGF, PG-001, phosgonimeton, televalefim, recombinant InlB321 protein, and agonist c-Met antibody, and optionally LMH85. In some embodiments, the IL-6 family cytokines are selected from the group consisting of IL-6, OSM, leukemia suppressor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokines (CLC). In some embodiments, the corticosteroid is selected from the group consisting of dexamethasone, beclomethasone, betamethasone, fluocortolone, halomethasone, and mometasone. In some embodiments, the culture medium (e.g., hepatocyte culture medium) is supplemented with HGF, OSM, and dexamethasone.

[0170] In some embodiments of the methods provided herein, HGF 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 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, HGF is provided at a concentration of 10 ng / mL or about 10 ng / mL.

[0171] In some embodiments of the methods provided herein, OSM is provided at concentrations 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 less than or about those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, concentrations of 10-30 ng / mL, 10-20 ng / mL, 20-30 ng / mL, or 15-25 ng / mL. In some embodiments, OSM is provided at a concentration of 20 ng / mL or about 20 ng / mL.

[0172] In some embodiments of the methods provided herein, dexamethasone is provided at concentrations of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, 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, 50-200 nM, 50-100 nM, 100-200 nM, or 50-150 nM. In some embodiments, dexamethasone is provided at a concentration of 100 nM or about 100 nM.

[0173] In some embodiments of the methods provided herein, the cells of step i) and / or step ii) are brought into contact in a culture medium further containing 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 cells of step i) and / or step ii) are brought into contact in a culture medium that does not contain EGF.

[0174] In some embodiments of the methods provided herein, the cells of step ii) are cultured in a growth medium supplemented with non-essential amino acids, essential amino acids, and glycine. In some embodiments, the supplemented growth medium includes 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of total volume of non-essential amino acids, or a range defined by any two of the aforementioned values, for example, 7-9%, 6-10%, 5-12%, 8-14%, 10-15%, 4-15%, 15-17%, 13-19%, 12-24%, or 10-25%. In some embodiments, the supplemented growth medium consists of approximately 6–10%, 8–14%, 10–15%, 4–15%, 15–17%, 13–19%, 12–24%, or 10–25% of total volume of non-essential amino acids, or approximately 4%, 6%, 8%, 10%, 12%, 14%, or 16%. In some embodiments, the supplemented growth medium contains 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of essential amino acids by total volume, or a range defined by any two of the aforementioned values, for example, 7-9%, 6-10%, 5-12%, 8-14%, 10-15%, 4-15%, 15-17%, 13-19%, 12-24%, or 10-25%. In some embodiments, the supplemented growth medium contains approximately 6-10%, 8-14%, 10-15%, 4-15%, 15-17%, 13-19%, 4%, 6%, 8%, 10%, 12%, 14%, or 16% essential amino acids by total volume.In some embodiments, the supplemented glycine is provided at concentrations of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg / mL, about those concentrations, at least those concentrations, at least about those concentrations, less than or equal to those concentrations, or about less than or equal to those concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 19-21 mg / mL, 18-22 mg / mL, 16-24 mg / mL, 10-30 mg / mL, or 5-34 mg / mL. In some embodiments, the supplemented glycine is provided at concentrations of 18-22 or 20 mg / mL, or about 18-22 or 20 mg / mL. In some embodiments, the growth medium is any standard growth medium commonly used for cell culture, and its compositions. For example, the growth medium may be Eagle's minimal essential medium (MEM), minimal essential medium with alpha modification (a-MEM), Basal Medium Eagle (BME), Dulbecco's modified Eagle's medium (DMEM), or hepatocyte medium (HCM). For illustrative purposes only, Table 3 shows the standard concentrations of non-essential and essential amino acids for MEM, as well as exemplary concentrations of these amino acids after supplementation in some embodiments provided herein. In some embodiments, the supplemented medium concentration is ±10%, ±5%, or ±1% of the values ​​listed in Table 3.

[0175] [Table 3]

[0176] In some embodiments of the method provided herein, the cells of step ii) are further contacted with a low / first concentration of bilirubin, and the liver organoids formed are mature liver organoids. In some embodiments, the low / first concentration of bilirubin is the human fetal physiological concentration of bilirubin. In some embodiments, the low / first concentration of bilirubin is 0.1 to 1 mg / L, 0.5 to 1 mg / L, or 1 mg / L, or about those, less than those, or about less than those. In some embodiments, the low / first concentration of bilirubin is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / L, or about those, less than those, or about less than those, or any concentration within the range defined by any two of the aforementioned concentrations, e.g., 0.1–1 mg / L, 0.1–0.5 mg / L, 0.5–1 mg / L, 0.3–0.7 mg / L, or 0.4–0.6 mg / L. In some embodiments, the low / first concentration of bilirubin is 0.1–3 mg / L, 0.5–3 mg / L, or 3 mg / L, or about those, less than those, or about less than those. In some embodiments, the low / first concentration of bilirubin is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, or 3.0 mg / L, or about those, less than those, or about less than those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, concentrations of 0.1–3 mg / L, 0.5–2.0 mg / L, 0.5–1.5 mg / L, 0.3–2.5 mg / L, or 0.5–1.75 mg / L. In some embodiments, mature liver organoids exhibit luminal projections similar to bile canaliculi, and / or structures having a single lumen and generally being spherical. In some embodiments, mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof, compared to liver organoids not exposed to a low / first dose of bilirubin.In some embodiments, mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, compared to liver organoids not exposed to a low / first dose of bilirubin. In some embodiments, mature liver organoids express CYP2E1, CYP7A1, PROX1, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids not exposed to a low / first dose of bilirubin.

[0177] In some embodiments of the methods provided herein, the cells of step ii) are further contacted with a high / second concentration of bilirubin, and the liver organoids formed are hyperbilirubinemia liver organoids. In some embodiments, the liver organoids, when used in the methods disclosed herein, are 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days old, or about those, at least those, or at least about those, or within a range defined by any two of the aforementioned values, e.g., 18-35, 18-30, 20-25, or 18-25 days old. In some embodiments, the high / second concentration of bilirubin is 2-10 mg / L, 5-10 mg / L, 10 mg / L, or 20 mg / L, or about those, greater than those, or greater than those. In some embodiments, the high / secondary concentration of bilirubin is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / L, or about those, greater than those, or about greater than those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 2-20 mg / L, 2-10 mg / L, 10-20 mg / L, 5-15 mg / L, or 8-12 mg / L. In some embodiments, liver organoids are exposed to the high / secondary concentration of bilirubin for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or within the range defined by any two of the aforementioned values, for example, 1-10, 1-5, 3-8, 5-10, or 7-10 days to form hyperbilirubinemia liver organoids. In some embodiments, hyperbilirubinemia liver organoids express elevated levels of UGT1A1 or NRF2, or both, compared to liver organoids not treated with high / secondary concentrations of bilirubin.

[0178] In some embodiments of the methods provided herein, the liver organoid comprises a gene or mRNA encoding a functional L-gulonolactone oxidase (GULO) protein and / or a functional GULO protein, or both, and the liver organoid is capable of synthesizing ascorbic acid. In some embodiments, the functional GULO protein is mGULO. However, the functional GULO may alternatively be derived from any other animal species containing a functional GULO protein. In some embodiments, the gene encoding the functional GULO protein is conditionally expressed. In some embodiments, the gene is conditionally expressed using a tetracycline induction system or any other system for conditional expression commonly known in the art. In some embodiments, the liver organoid is engineered with the gene encoding the functional GULO protein using CRISPR or any other method of genetic engineering commonly known in the art. In some embodiments, the gene or mRNA encoding the functional GULO protein, or both, is introduced into a mature liver organoid by transfection. In some embodiments, liver organoids containing functional GULO protein express increased levels of NRF2 compared to liver organoids without functional GULO protein. In some embodiments, liver organoids containing functional GULO protein express reduced levels of IL1B, IL6, or TNFa, or any combination thereof, compared to liver organoids without functional GULO protein, when optionally cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid. In some embodiments, liver organoids containing functional GULO protein exhibit reduced caspase-3 activity compared to liver organoids without functional GULO protein, when optionally cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid. In some embodiments, liver organoids containing functional GULO protein express increased levels of ALB compared to liver organoids without functional GULO protein.In some embodiments, liver organoids containing functional GULO protein resemble periportal liver tissue and express periportal liver markers. In some embodiments, the periportal markers include FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, liver organoids containing functional GULO protein exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids without functional GULO protein. In some embodiments, liver organoids containing functional GULO protein exhibit increased bilirubin conjugation activity compared to liver organoids without functional GULO protein. In some embodiments, liver organoids containing functional GULO protein exhibit increased viability in culture compared to liver organoids without functional GULO protein. In some embodiments, the liver organoids are differentiated from pluripotent stem cells that contain functional GULO protein and / or a gene or mRNA encoding functional GULO protein, or both, thereby enabling the pluripotent stem cells to synthesize ascorbic acid.

[0179] In some embodiments of the methods provided herein, the liver organoid contains an inactive UGT1A1 gene, and the liver organoid is a model of Crigler-Nadjar syndrome.

[0180] In some embodiments of the methods provided herein, the method further comprises aggregating posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells in a microwell or other device (e.g., Aggrewell) prior to step i). For example, an exemplary schematic diagram for culturing liver organoids from posterior foregut cells and / or posterior foregut endoderm cells aggregated in a microwell is embodied in FIG. 5I. In some embodiments, each aggregate of posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells comprises from about 250, about 500, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 7500, about 8000, about 8500, about 9000, about 9500, or about 10,000 posterior foregut cells and / or posterior foregut endoderm cells, or any number of posterior foregut cells and / or posterior foregut endoderm cells within a range defined by any two of the foregoing cell numbers. In some embodiments, aggregating posterior foregut cells and / or posterior foregut endoderm cells and / or foregut endoderm cells results in liver organoids of a more uniform size. Further information regarding methods of aggregating posterior foregut cells and / or posterior foregut endoderm cells using other devices such as microwells or Aggrewell to produce liver organoids of a more uniform size can be found in PCT Publication No. WO2021 / 030373, which is hereby expressly incorporated by reference in its entirety.

[0181] In some embodiments of the methods provided herein, the cells of step i) and / or step ii) are not cultured with the basement membrane matrix or its components. In some embodiments, the cells of step i) and / or step ii) are not cultured with the basement membrane matrix or its components which are heterogeneous to humans. In some embodiments, the cells of step i) and / or step ii) are not cultured with the basement membrane matrix or its components isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the cells of step i) and / or step ii) are not brought into contact with Matrigel®, Cultrex®, or Geltrex®.

[0182] In some embodiments of the methods provided herein, the cells in step i) and / or step ii) are cultured in a non-static bioreactor. In some embodiments, the cells in step i) and / or step ii) are cultured in a rotary bioreactor.

[0183] In some embodiments of the methods provided herein, the method further includes the step of cryopreserving liver organoids. In some embodiments, cryopreserving liver organoids includes slow freezing or vitrification cryopreservation. In some embodiments, liver organoids are cryopreserved together with chroman 1, emricasane, polyamine, and trans-ISRIB (CEPT). In some embodiments, chroman 1 is provided at a concentration of 50 nM or about 50 nM. In some embodiments, emricasane is provided at a concentration of 5 μM or about 5 μM. In some embodiments, polyamine is provided at a concentration of 1:1000 or about 1:1000. In some embodiments, trans-ISRIB is provided at a concentration of 7 μM or about 7 μM.

[0184] Liver organoids produced by the methods disclosed herein are disclosed herein.

[0185] When applied to any of the cells disclosed herein, such as pluripotent stem cells, embryonic endoderm, posterior foregut, posterior foregut endoderm, and / or liver organoids, the cells may be derived from a patient. In some embodiments, the patient has a liver disease. In some embodiments, the embryonic endoderm, posterior foregut, posterior foregut endoderm, and / or liver organoids may be derived from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells.

[0186] How to use Methods are also disclosed that include administering one of the liver organoids disclosed herein to a subject in need of such administration. Methods for treating liver-related diseases or disorders in a subject in need of such treatment are also disclosed. In some embodiments, the method includes administering one of the liver organoids disclosed herein to a subject. In some embodiments, the liver organoids are produced from cells derived from the subject. In some embodiments, the cells derived from the subject are induced pluripotent stem cells.

[0187] Furthermore, methods for screening are disclosed herein. In some embodiments, the method includes contacting one of the liver organoids disclosed herein with a candidate compound or composition and evaluating the effect of the candidate compound or composition on the liver organoid. In some embodiments, the liver organoid is a model of liver-related disease or disorder, and evaluating the effect of the candidate compound or composition on the liver organoid includes evaluating the effect of the candidate compound or composition on liver-related disease or disorder. In some embodiments, the liver organoid 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 liver-related disease or disorder.

[0188] Liver-related diseases and disorders The liver organoids of this disclosure can be used for the treatment and / or research or modeling of liver-related diseases and disorders. In some embodiments, the method includes administering either the liver organoids or hepatocytes disclosed herein. Furthermore, liver organoids or hepatocytes disclosed herein are disclosed for use in the manufacture of drugs for the treatment of liver-related diseases or disorders. Furthermore, liver organoids or hepatocytes disclosed herein are disclosed for use in the treatment of subjects requiring treatment of liver-related diseases or disorders.

[0189] The liver-related diseases and disorders related to this disclosure include liver dysfunction and / or liver failure (e.g., hyperammonemia and / or hyperbilirubinemia), hepatitis (e.g., hepatitis A, B, C, D, E, G, TT, and / or autoimmune hepatitis), viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, and metabolic disorders (e.g., metabolic dysfunction-related liver disease (MASLD)). This may include conditions such as MetALD, non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-related steatohepatitis (MASH), autoimmune liver disease, Wilson's disease, metabolic-related fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Nadjar syndrome, urea cycle disorders, Wolmann disease, liver cancer, hepatoblastoma, drug-induced liver injury (DILI), glycogen storage disorders, hemorrhagic disorders, hepatic cysts, and / or alcohol-related liver disease. Those skilled in the art will recognize other liver-related diseases and conditions to which the liver organoids disclosed herein may be relevant.

[0190] For example, liver organoids can be transplanted into subjects with liver dysfunction and / or liver failure, and the transplanted liver organoids will engraft in the subject's liver. After transplantation, the subject may have reduced serum bilirubin and / or ammonia levels, and / or increased serum proteinalbumin, as well as improved symptoms of bile duct stenosis and / or liver regeneration, and may also have an increased survival rate.

[0191] For example, these liver organoids can be used in in vitro human model systems to study hepatocyte function and developmental branching, to study liver-related diseases, to identify and / or screen for therapeutic targets, and / or to identify therapeutic compounds and / or compositions effective in treating liver-related diseases or disorders. Thus, the liver organoids of this disclosure can enable new developments in the treatment and research of liver diseases.

[0192] composition In some embodiments, compositions for carrying out any of the methods disclosed herein are also provided. In some embodiments, compositions produced according to a process provided by any of the methods disclosed herein are also provided. In certain embodiments, any method or composition described herein may be carried out in relation to any other method or composition described herein, and different embodiments may be combined.

[0193] In some embodiments, compositions comprising posterior foregut cells and / or posterior foregut endoderm cells and / or liver organoids produced using one or more of the methods provided herein are provided herein. In some embodiments, compositions comprising at least a portion of posterior foregut cells and / or posterior foregut endoderm cells having spontaneously formed three-dimensional (3D) spheroids are provided herein, and optionally the spheroids include structures having a single lumen.

[0194] In some embodiments, the compositions provided herein are in vitro compositions made outside of a multicellular living organism. In some embodiments, the compositions provided herein can be introduced into a multicellular living organism. In some embodiments, the compositions provided herein include exogenously provided components, reagents, and / or conditions. In some embodiments, the compositions provided herein include exogenously provided components, reagents, and / or conditions that mimic in vivo features desirable for inducing specific cell differentiation and / or organoid organization.

[0195] In some embodiments, provided herein is an in vitro composition comprising posterior foregut cells and / or posterior foregut endoderm cells, at least one exogenous tissue culture surface, at least one exogenous TGF-β pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator. In some embodiments, the composition can also include an endogenous TGF-β pathway inhibitor, FGF pathway activator, Wnt pathway activator, and / or VEGF pathway activator. In some embodiments, the composition includes posterior foregut cells and / or posterior foregut endoderm cells dissociated from a monolayer and / or spheroid. In some embodiments, the composition has posterior foregut cells and / or posterior foregut endoderm cells at a cell density of 1×10 5 、2×10 5 、3×10 5 、4×10 5 、5×10 5 、6×10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、2×10 6 、3×10 6 、4×10 6 、or 5×10 6 cells / cm 2 or greater, precisely or approximately those cell densities, or any cell density within a range defined by any two of the aforementioned cell densities.

[0196] In some embodiments, compositions comprising a tissue culture surface coated with a basement membrane matrix or its components are provided herein. In some embodiments, the basement membrane matrix or its components do not contain non-human animal components. In some embodiments, the basement membrane matrix or its components do not contain non-human animal components so that the basement membrane matrix or its components are heterogeneous to humans. In some embodiments, the basement membrane matrix or its components are not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, are not Matrigel®, are not Cultrex®, and / or Geltrex®. In some embodiments, the basement membrane matrix or its components include human laminin, collagen IV, entactin, perlecan, fibrin, and / or hydrogel.

[0197] In some embodiments, compositions comprising an exogenous TGF-β pathway inhibitor are provided herein. In some embodiments, the exogenous TGF-β pathway inhibitor comprises, essentially derived from, or consists of A83-01, RepSox, LY365947, and / or SB431542. In some embodiments, the exogenous TGF-β pathway inhibitor comprises, essentially derived from, or consists of the TGF-β pathway inhibitor A83-01. In some embodiments, the composition comprises the TGF-β pathway inhibitor at a concentration of 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or about those concentrations, or any concentration within the range defined by any two of the aforementioned concentrations. In some embodiments, the composition comprises the TGF-β pathway inhibitor at a concentration of 500 nM or about 500 nM.

[0198] In some embodiments, compositions comprising exogenous FGF pathway activators are provided herein. In some embodiments, the compositions comprise exogenous FGF pathway activators comprising, essentially comprising, or consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and / or FGF23. In some embodiments, the exogenous FGF pathway activators comprise, essentially comprising, or consisting of FGF2. In some embodiments, the compositions comprise FGF pathway activators at concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or any concentration within the range defined by about those concentrations or any two of the aforementioned concentrations. In some embodiments, the composition contains an FGF pathway activator at a concentration of 5 ng / mL or about 5 ng / mL.

[0199] In some embodiments, compositions comprising exogenous Wnt pathway activators are provided herein. In some embodiments, the compositions comprise exogenous Wnt pathway activators comprising, essentially, or 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, alster paulonne, kaem paulonne, lithium chloride, TDZD8, and TWS119. In some embodiments, the composition comprises an exogenous Wnt pathway activator comprising, essentially, or consisting of CHIR99021. In some embodiments, the composition comprises a Wnt pathway inhibitor at a concentration 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 any concentration within the range defined by about those concentrations or any two of the aforementioned concentrations. In some embodiments, the composition comprises a Wnt pathway activator at a concentration of 3 μM or about 3 μM.

[0200] In some embodiments, compositions comprising an exogenous VEGF pathway activator are provided herein. In some embodiments, the composition comprises an exogenous VEGF pathway activator comprising, essentially derived from, or consisting of VEGF and / or GS4012. In some embodiments, the composition comprises an exogenous VEGF pathway activator comprising, essentially derived from, or consisting of VEGF. In some embodiments, the composition comprises a VEGF pathway activator 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 any concentration within the range defined by about those concentrations, or any two of the aforementioned concentrations. In some embodiments, the composition comprises a VEGF pathway activator at a concentration of 10 ng / mL or about 10 ng / mL.

[0201] In some embodiments, compositions comprising exogenous EGF are provided herein. In some embodiments, compositions not comprising exogenous EGF are provided herein. In some embodiments, compositions comprising EGF at concentrations 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 any concentration within the range defined by about those concentrations or any two of the aforementioned concentrations, are provided herein. In some embodiments, compositions comprising EGF at a concentration of 20 ng / mL or about 20 ng / mL are provided herein.

[0202] In some embodiments, compositions comprising exogenous and / or genetically modified ascorbic acid are provided herein. In some embodiments, compositions not comprising exogenous and / or genetically modified ascorbic acid are provided herein. In some embodiments, compositions comprising ascorbic acid at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL, or any concentration within the range defined by about those concentrations, or any two of the aforementioned concentrations, are provided herein. In some embodiments, compositions comprising ascorbic acid at a concentration of 50 μg / mL or about 50 μg / mL are provided herein.

[0203] In some embodiments, compositions comprising a ROCK inhibitor are provided herein. In some embodiments, compositions not comprising a ROCK inhibitor are provided herein. In some embodiments, the ROCK inhibitor comprises, is essentially derived from, or consists of Y-27632. In some embodiments, compositions comprising a ROCK inhibitor 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 any concentration within the range defined by about those concentrations, or any two of the aforementioned concentrations, are provided herein. In some embodiments, compositions comprising a ROCK inhibitor at a concentration of 10 μM or about 10 μM are provided herein.

[0204] In some embodiments, compositions comprising posterior foregut cells and / or posterior foregut endoderm cells having stem cells and / or differentiated from stem cells are provided herein. In some embodiments, compositions comprising posterior foregut cells and / or posterior foregut endoderm cells having induced pluripotent stem cells and / or differentiated from induced pluripotent stem cells are provided herein. In some embodiments, compositions comprising posterior foregut cells and / or posterior foregut endoderm cells that have been passaged once, twice, or three times are provided herein. In some embodiments, compositions comprising posterior foregut cells and / or posterior foregut endoderm cells that have been passaged less than four times are provided herein.

[0205] In some embodiments, compositions comprising A83-01, FGF2, CHIR99021, VEGF, and / or Y-27632, and optionally further comprising iPSCs, PSCs, and / or posterior foregut cells and / or posterior foregut endoderm cells, are provided herein.

[0206] In some embodiments, compositions are provided herein that comprise a) posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids, and b) a culture medium, optionally comprising hepatocyte culture medium and optionally supplemented with a cMET tyrosine kinase receptor agonist, IL-6 family cytokines, and corticosteroids, the composition optionally further comprising c) a retinoic acid pathway activator. In some embodiments, compositions provided herein comprise a cMET tyrosine kinase receptor agonist. In some embodiments, compositions provided herein comprise a cMET tyrosine kinase receptor agonist comprising, essentially comprising, or consisting of, hepatocyte growth factor (HGF), PG-001, phosgonimeton, telebarefim, recombinant InlB321 protein, and / or the agonist c-Met (e.g., LMH85).

[0207] In some embodiments, compositions comprising IL-6 family cytokines are provided herein. In some embodiments, the IL-6 family cytokines comprise, essentially consist of, or comprise IL-6, oncostatin M (OSM), leukemia suppressor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and / or cardiotrophin-like cytokines (CLCs).

[0208] In some embodiments, compositions comprising a corticosteroid are provided herein. In some embodiments, the corticosteroid comprises, essentially consists of, or comprises dexamethasone, beclomethasone, betamethasone, fluocortone, halomethasone, and / or mometasone.

[0209] In some embodiments, compositions comprising hepatocyte culture medium supplemented with HGF, OSM, and / or dexamethasone are provided herein. In some embodiments, compositions comprising hepatocyte culture medium supplemented with dexamethasone are provided herein. In some embodiments, compositions comprising hepatocyte culture medium supplemented with HGF are provided herein. In some embodiments, compositions comprising hepatocyte culture medium supplemented with OSM are provided herein.

[0210] In some embodiments, compositions comprising a retinoic acid pathway activator are provided herein. In some embodiments, the retinoic acid pathway activator comprises, essentially consists of, or comprises retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and / or AM580. In some embodiments, the retinoic acid pathway activator comprises, essentially consists of, or comprises retinoic acid. In some embodiments, the composition comprises the retinoic acid pathway activator at concentrations of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or any concentration within the range defined by about these concentrations or any two of the aforementioned concentrations. In some embodiments, the composition contains a retinoic acid pathway activator at a concentration of 2.0 μM or about 2.0 μM.

[0211] In some embodiments, the composition contains HGF. In some embodiments, the composition contains HGF 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 any concentration within the range defined by about those concentrations or any two of the aforementioned concentrations. In some embodiments, the composition contains HGF at a concentration of 10 ng / mL or about 10 ng / mL.

[0212] In some embodiments, the composition contains OSM. In some embodiments, the composition contains OSM 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 any concentration within the range defined by about those concentrations or any two of the aforementioned concentrations. In some embodiments, the composition contains OSM at a concentration of 20 ng / mL or about 20 ng / mL.

[0213] In some embodiments, the composition contains dexamethasone. In some embodiments, the composition contains dexamethasone at a concentration of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or any concentration within the range defined by about those concentrations, or any two of the aforementioned concentrations. In some embodiments, the composition contains dexamethasone at a concentration of 100 nM or about 100 nM.

[0214] In some embodiments, the composition contains exogenous bilirubin. In some embodiments, the composition contains both exogenous and endogenous bilirubin. In some embodiments, the composition contains a low concentration of exogenous bilirubin. In some embodiments, the low / first concentration of exogenous bilirubin is the human fetal physiological concentration of bilirubin, or close to it. Human fetal bilirubin levels are generally considered to be about 1 mg / L (0.1 mg / dL), which rapidly rises to 3–10 mg / L (0.3–1.0 mg / dL) within 24 hours postnatally. In some embodiments, the composition is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, or 3.0 mg / L, or about those, less than those, or about less than those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg / L, 0.5 to 2.0 mg / L, 0.5 to 1.5 mg / L, 0.3 to 2.5 mg / L, or This includes exogenous and / or endogenous bilirubin in the following concentrations: 0.5–1.75 mg / L, or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / L, approximately those concentrations, less than those concentrations, approximately less than those concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, e.g., 0.1–1 mg / L, 0.1–0.5 mg / L, 0.5–1 mg / L, 0.3–0.7 mg / L, or 0.4–0.6 mg / L.In some embodiments, the composition is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, or 3.0 mg / L, or about those, less than those, or about less than those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg / L, 0.5 to 2.0 mg / L, 0.5 to 1.5 mg / L, 0.3 to 2.5 mg / L It contains exogenous bilirubin at a concentration of 0.5 to 1.75 mg / L, or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / L, approximately those, less than those, approximately less than those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg / L, 0.1 to 0.5 mg / L, 0.5 to 1 mg / L, 0.3 to 0.7 mg / L, or 0.4 to 0.6 mg / L.

[0215] In some embodiments, compositions comprising mature liver organoids are provided herein. In some embodiments, compositions comprising mature liver organoids exhibiting luminal projections similar to bile canaliculi, and / or structures having a single lumen and generally a spherical shape are provided herein. In some embodiments, compositions comprising mature liver organoids produced via contact with a low dose of exogenous bilirubin are provided herein. In some embodiments, compositions comprising mature liver organoids expressing reduced levels of AFP, CDX2, NANOG, or any combination thereof, compared to liver organoids not contacted with a low dose of bilirubin are provided. In some embodiments, compositions comprising mature liver organoids expressing increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, compared to liver organoids not contacted with a low dose of bilirubin are provided. In some embodiments, compositions comprising mature liver organoids expressing CYP2E1, CYP7A1, PROX1, MRP3, MRP3, or OATP2, or any combination thereof, are provided. In some embodiments, compositions are provided comprising mature liver organoids exhibiting increased CYP3A4 and / or CYP1A2 activity compared to liver organoids not exposed to low doses of bilirubin. In some embodiments, compositions comprising mature liver organoids are provided herein, wherein the cells of the mature liver organoids have been exposed to low doses of exogenous bilirubin, and the mature liver organoids exhibit luminal projections similar to bile canaliculi, and / or structures having a single lumen and generally being spherical. In some embodiments, compositions are provided herein comprising mature liver organoids expressing reduced levels of AFP, CDX2, NANOG, or any combination thereof, compared to liver organoids whose cells have not been exposed to low doses of bilirubin. In some embodiments, compositions are provided herein comprising mature liver organoids expressing increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, compared to liver organoids whose cells have not been exposed to low doses of bilirubin.In some embodiments, compositions comprising mature liver organoids expressing CYP2E1, CYP7A1, PROX1, MRP3, MRP3, or OATP2, or any combination thereof, are provided herein. In some embodiments, compositions comprising mature liver organoids exhibiting increased CYP3A4 and / or CYP1A2 protein levels, and / or enzymatic activity, compared to liver organoids in which the cells were not exposed to low doses of bilirubin, are provided herein.

[0216] In some embodiments, compositions comprising hyperbilirubinemia liver organoids are provided herein, wherein the hyperbilirubinemia liver organoid cells were in contact with a high and / or second concentration of bilirubin. In some embodiments, compositions comprising hyperbilirubinemia liver organoids are provided herein, wherein the high / second concentration of bilirubin was 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / L, about those, greater than those, or about greater than those, or any concentration within the range defined by any two of the aforementioned concentrations, e.g., 2-20 mg / L, 2-10 mg / L, 10-2 The concentrations were 0 mg / L, 5–15 mg / L, or 8–12 mg / L, or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / L, approximately those, greater than those, or approximately greater than those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 4–20 mg / L, 2–10 mg / L, 10–20 mg / L, 5–15 mg / L, or 8–12 mg / L. In some embodiments, compositions comprising hyperbilirubinemia liver organoids are provided herein, which express elevated levels of UGT1A1 or NRF2, or both, compared to liver organoids not treated with a high / second concentration of bilirubin.

[0217] Furthermore, in some embodiments, compositions are provided herein comprising posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids, which have been engineered to contain a gene or mRNA encoding a functional L-gulonolactone oxidase (GULO) protein and / or a functional GULO protein, or both, the posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids, which can synthesize ascorbic acid. In some embodiments, compositions are provided herein comprising posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids, which have been engineered to express a functional GULO protein, where the functional GULO protein is mouse GULO (mGULO). In some embodiments, the gene encoding the functional GULO protein is conditionally expressed. In some embodiments, the gene encoding the functional GULO protein is constitutively expressed. In some embodiments, the gene encoding the functional GULO protein is conditionally expressed using a tetracycline induction system.

[0218] In some embodiments, compositions comprising posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids are provided herein, which are engineered to contain a gene encoding a functional GULO protein using CRISPR-mediated knock-in. In some embodiments, compositions comprising posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids are provided herein, which contain a functional GULO coding gene or mRNA, or both, the functional gene being introduced into the posterior foregut cells and / or posterior foregut endoderm cells, liver organoids, mature liver organoids and / or progenitor cells by transfection. In some embodiments, compositions comprising posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids are provided herein, which are engineered to contain a gene encoding a functional GULO protein using adenovirus-mediated gene transfection. In some embodiments, compositions comprising posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids are provided herein, which are engineered to contain genes encoding functional GULO proteins using adeno-associated virus-mediated gene transfection.

[0219] In some embodiments, the compositions provided herein comprise liver organoids and / or mature liver organoids containing functional GULO protein, wherein the liver organoids and / or mature liver organoids express increased levels of NRF2 compared to liver organoids and / or mature liver organoids that do not contain functional GULO protein. In some embodiments, the compositions provided herein comprise liver organoids and / or mature liver organoids containing functional GULO protein, wherein the liver organoids and / or mature liver organoids express reduced levels of IL1B, IL6, or TNFa, or any combination thereof, compared to liver organoids and / or mature liver organoids that do not contain functional GULO protein. In some embodiments, liver organoids and / or mature liver organoids containing functional GULO protein exhibit reduced caspase-3 activity compared to liver organoids and / or mature liver organoids that do not contain functional GULO protein. In some embodiments, liver organoids and / or mature liver organoids containing functional GULO protein express increased levels of ALB compared to liver organoids and / or mature liver organoids not containing functional GULO protein. In some embodiments, liver organoids and / or mature liver organoids resemble periportal liver tissue and / or express periportal liver markers. In some embodiments, the periportal liver markers include or consist of FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, liver organoids and / or mature liver organoids containing functional GULO protein exhibit increased CYP3A4 and / or CYP1A2 protein levels and / or enzyme activity compared to liver organoids and / or mature liver organoids not containing functional GULO protein. In some embodiments, liver organoids and / or mature liver organoids containing functional GULO protein exhibit increased bilirubin conjugation activity compared to liver organoids and / or mature liver organoids not containing functional GULO protein.In some embodiments, liver organoids and / or mature liver organoids containing functional GULO protein exhibit increased viability in culture compared to liver organoids and / or mature liver organoids not containing functional GULO protein. In some embodiments, the liver organoids and / or mature liver organoids are differentiated from pluripotent stem cells that contain functional GULO protein and / or a gene or mRNA encoding functional GULO protein, or both, thereby enabling the pluripotent stem cells to synthesize ascorbic acid.

[0220] Furthermore, in some embodiments, compositions described in Table 1, Table 2, or Table 3 are also provided herein.

[0221] In some embodiments, a solution of non-essential amino acids is used in an amount of exactly or about 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 11 vol%, 12 vol%, 13 vol%, 14 vol%, or 15 vol% (specifically or about 890 mg / L alanine, 1320 mg / L asparagine, 1330 mg / L aspartic acid, 750 mg / L glycine, 105 mg / L serine, 1150 mg / L proline, and 1470 mg / L glutamic acid). (Containing), exactly or about 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 11 vol%, 12 vol%, 13 vol%, 14 vol%, or 15 vol% of essential amino acid solution (exactly or about arginine 6320 mg / L, cysteine ​​1200 mg / L, histidine 2100 mg / L, isoleucine 2620 mg / L, leucine 2620 mg / L, lysine 3625 mg / L, methionine 755 mg / L, phenylalanine) (Containing 1650 mg / L, threonine 2380 mg / L, tryptophan 510 mg / L, tyrosine 1800 mg / L, and valine 2340 mg / L), and exactly or about 65% by volume, 66% by volume, 67% by volume, 68% by volume, 69% by volume, 70% by volume, 71% by volume, 72% by volume, 73% by volume, 74% by volume, 75% by volume, 76% by volume, 77% by volume, 78% by volume, 79% by volume, 80% by volume, 81% by volume, 82% by volume, 83% by volume, 84% by volume Compositions are provided herein that comprise an amino acid supplement liquid component comprising 85% by volume, 86% by volume, 87% by volume, 88% by volume, 89% by volume, or 90% by volume of hepatocyte culture medium (HCM), and further supplemented with exactly or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg / mL of glycine.

[0222] In some embodiments, exactly or about 14% non-essential amino acid solution (containing exactly or about 890 mg / L alanine, 1320 mg / L asparagine, 1330 mg / L aspartic acid, 750 mg / L glycine, 105 mg / L serine, 1150 mg / L proline, and 1470 mg / L glutamic acid), exactly or about 6% by volume essential amino acid solution (containing exactly or about 6320 mg / L arginine, 1200 mg / L cysteine, 2100 mg / L histidine, isoleucine, etc.) A composition is provided herein that comprises an amino acid supplement liquid component containing exactly or about 80 volume% hepatocyte culture medium (HCM), further supplemented with exactly or about 20 g / L of glycine. In some embodiments, the composition provided herein may have a pH of about 6 to 8, or pH 6.5 to 7.5, or exactly or about pH 7.0. In some embodiments, the composition provided herein may contain hepatocyte growth factor (HGF), oncostatin M, dexamethasone, and / or ascorbic acid.

[0223] In some embodiments, the compositions provided herein include hepatic lineage-determined cells differentiated from endoderm cells of an embryonic body using retinoic acid. In some embodiments, the compositions provided herein include hepatic lineage-determined cells characterized as hepatic organoids. In some embodiments, the compositions provided herein include hepatic organoids characterized as secreting increased levels of albumin and / or urea compared to hepatic organoids contained in HCM without amino acid supplementation. In some embodiments, the compositions provided herein include hepatic organoids characterized as expressing increased levels of liver maturation-related gene expression compared to hepatic organoids contained in HCM without amino acid supplementation. In some embodiments, the compositions provided herein include hepatic organoids characterized as expressing reduced levels of vimentin compared to hepatic organoids contained in HCM without amino acid supplementation. In some embodiments, the compositions provided herein do not expressly include non-human animal basement membrane matrix or its components. In some embodiments, the compositions provided herein do not expressly contain mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, Matrigel®, Cultrex®, and / or Geltrex®.

[0224] In some embodiments, hyperbilirubinemia liver organoids containing naturally occurring and / or engineered mutations in the UDP-glucuronosyltransferase family 1 member A1 (UGT1A1) gene are also provided herein. In some embodiments, hyperbilirubinemia liver organoids are provided herein, which were produced by contacting progenitor cells, progenitor liver organoids, and / or progenitor mature liver organoids with exogenous bilirubin for at least two rounds. In some embodiments, hyperbilirubinemia liver organoids produced from clone-derived cells and / or iPSCs are also provided herein.

[0225] In some embodiments, cryopreserved compositions comprising liver organoids, chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) are provided herein. In some embodiments, cryopreserved compositions comprising mature liver organoids, chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) are provided herein. In some embodiments, cryopreserved compositions comprising hyperbilirubinergic liver organoids, chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) are provided herein.

[0226] Pharmaceutical compositions according to various embodiments of this disclosure may include one or more additional pharmaceutically acceptable components, which may include carriers, excipients, and / or stabilizers that are nontoxic to cells or mammals to which they are exposed at the doses and concentrations used, or that have acceptable toxicity levels. As used herein, “pharmaceutically acceptable,” “diluent,” “excipient,” and / or “carrier” have their general and ordinary meanings as understood herein and are intended to include any solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent and absorption retardant, etc., that are 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 contain small amounts of wetting agents, fillers, emulsifiers, or pH buffers, as needed. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, etc. The formulation should be suitable for the mode of administration.

[0227] 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.

[0228] A pharmaceutical composition may contain one or more "pharmaceutically acceptable salts," which may include, but are not limited to, relatively non-toxic inorganic and organic acids or base addition salts of the composition or excipients, including 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 from 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.

[0229] The appropriate formulation will vary depending on the chosen route of administration. The formulations and techniques for administering 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, intradermal, aerosol, parenteral delivery (including intramuscular, subcutaneous, intra-arterial, intra-intravenous), intra-portal, intra-articular, intradermal, peritoneal, intrathecal, intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections. Pharmaceutical compositions will generally be formulated to suit a specific intended route of administration.

[0230] As used herein, “carrier” has its general and ordinary meaning as understood herein and may refer 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.

[0231] As used herein, “diluent” may refer to a component in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable, having its general and ordinary meaning as understood in light of this specification. 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 include, but are not limited to, buffered aqueous solutions such as phosphate-buffered saline that mimics the composition of human blood.

[0232] Dosage and route of administration Embodiments of the present disclosure may include methods of administering or treating an animal, which may include administering at least one treatment in an amount effective to treat a disease, condition, or disorder that an organism has, is suspected to have, or is susceptible to, or to produce a desired physiological effect. In some embodiments, the disease, condition, or disorder may be a liver-related disease or disorder.

[0233] In some embodiments, at least one treatment may include a composition or pharmaceutical composition that can be administered to an animal (e.g., a mammal, primate, monkey, or human) in amounts of about 0.005 to about 50 mg / kg body weight, about 0.01 to about 15 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, about 0.5 to about 7 mg / kg body weight, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 10 mg / kg, about 12 mg / kg, or about 15 mg / kg. With respect to some conditions, the dose may be about 0.5 mg / kg human body weight or about 6.5 mg / kg human body weight. In some cases, certain subjects (e.g., mammals, mice, rabbits, cats, pigs, or dogs) can be administered doses of approximately 0.005 to approximately 50 mg / kg body weight, approximately 0.01 to approximately 15 mg / kg body weight, approximately 0.1 to approximately 10 mg / kg body weight, approximately 0.5 to approximately 7 mg / kg body weight, approximately 0.005 mg / kg, approximately 0.01 mg / kg, approximately 0.05 mg / kg, approximately 0.1 mg / kg, approximately 1 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 20 mg / kg, approximately 30 mg / kg, approximately 40 mg / kg, approximately 50 mg / kg, approximately 80 mg / kg, approximately 100 mg / kg, or approximately 150 mg / kg. Naturally, those skilled in the art will understand that many concentrations can be used in the methods disclosed herein, and that any number of concentrations can be adjusted and tested to find one that achieves the desired result in a given situation, using the guidelines provided herein in part. In some embodiments, the dose or therapeutically effective dose of the compounds disclosed herein is sufficient to achieve plasma concentrations of the compound or its active metabolite within the range described herein, for example, about 1 to 10 nM, 10 to 100 nM, 0.1 to 1 μM, 1 to 10 μM, 10 to 100 μM, 100 to 200 μM, 200 to 500 μM, or more preferably about 500 to 1000 μM, 10 to 1000 nM, or 0.1 to 1 μM.

[0234] In other embodiments, the treatment may be administered in combination with one or more other therapeutic agents for a given disease, condition, or disorder.

[0235] Compounds and pharmaceutical compositions are preferably prepared and administered in dose units. Solid dose units are tablets, capsules, and suppositories. Different daily doses may be used for the treatment of a subject, depending on the activity of the compound, the mode of administration, the nature and severity of the disease or disorder, and the age and weight of the subject.

[0236] However, under certain circumstances, a higher or lower daily dose may be appropriate. The daily dose can be administered both as a single dose in the form of individual dose units or several smaller dose units, and as multiple doses of subdivided doses at specific intervals.

[0237] Treatment can be administered topically or systemically at a therapeutically effective dose. The effective dose for this use, of course, depends on the severity of the disease or disorder, as well as the subject's body weight and overall condition. Typically, the dose used in vitro can provide useful guidance for the amount useful for in situ administration of the pharmaceutical composition, and animal models can be used to determine the effective dose for treating a particular disorder.

[0238] Various considerations are described, for example, in Langer, 1990, Science, 249:1527 and Goodman and Gilman's (eds.), 1990 (same literature), each of which is incorporated herein by reference and for all purposes. Doses for parenteral administration of active pharmaceutical products can be converted to corresponding oral doses by multiplying the parenteral dose by an appropriate conversion factor. For general applications, parenteral dose in mg / mL × 1.8 = corresponding oral dose in milligrams ("mg"). For oncological applications, parenteral dose in mg / mL × 1.6 = corresponding oral dose in mg. The average adult weight is approximately 70 kg. See, for example, Miller-Keane, 1992, Encyclopedia & Dictionary of Medicine, Nursing & Allied Health, 5th Ed., (WBSaunders Co.), pp. 1708 and 1651.

[0239] However, it is also understood that a specific dose level for any given patient depends on various factors, including the activity of the particular compound used, age, weight, general health status, sex, diet, administration time, route of administration, elimination rate, drug combination, and the severity of the particular disease being treated.

[0240] In some embodiments, administration may include one or more therapeutic unit doses combined with a pharmaceutically acceptable carrier, and may also include other drugs, pharmaceuticals, carriers, adjuvants, diluents, and excipients. In certain embodiments, the carrier, vehicle, or excipient can facilitate administration, delivery, and / or improve the preservation of the composition. In other embodiments, one or more carriers may include, but are not limited to, salines, e.g., ordinary saline, Ringer's solution, PBS (phosphate-buffered saline), and mixtures of various salts, including potassium salts and phosphates, with or without sugar additives such as glucose. Carriers may include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, bactericidal antibiotics, and solutes that make the formulation isotonic with the body fluids of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. In other embodiments, one or more excipients may include, but are not limited to, water, saline, dextrose, glycerol, ethanol, and combinations thereof. Non-toxic adjuncts such as wetting agents, buffers, or emulsifiers may also be added to the composition. Oral formulations may contain commonly used excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.

[0275] The amount of the active component in a unit dose preparation may vary or be adjusted from 0.1 mg to 10,000 mg, more typically from 1.0 mg to 1,000 mg, and most typically from 10 mg to 500 mg, depending on the specific application and the potency of the active component. The composition may also contain other suitable therapeutic agents as needed.

[0241] Therapies can be administered to the subject by any number of preferred routes of administration or formulations. Therapies such as immunotherapy can also be used to treat subjects for a variety of diseases. Subjects include, but are not limited to, mammals, primates, monkeys (e.g., macaques, rhesus monkeys, or pig-tailed macaques), humans, dogs, cattle, cattle, pigs, birds (e.g., chickens), mice, rabbits, and rats. In the specific embodiments described herein, the subject is human.

[0242] The route of administration of the therapeutic compounds described herein may be any preferred route. The route of administration may be, but is not limited to, oral, parenteral, cutaneous, nasal, rectal, vaginal, and ocular. In other embodiments, the route of administration may be parenteral, mucosal, intravenous, subcutaneous, topical, intradermal, oral, sublingual, intranasal, or intramuscular. The choice of route of administration may depend on the identity of the compound (e.g., the physical and chemical properties of the compound) as well as the age and weight of the animal, the specific disease (e.g., type of cancer), and the severity of the disease (e.g., stage or severity of cancer). Of course, a combination of routes of administration may be administered as desired.

[0243] Some embodiments of the present disclosure include methods for providing a treatment comprising one or more doses of one or more compositions, the compositions may be the same or different if there are more than one dose.

[0244] toxicity The ratio between toxicity and therapeutic effect for a particular treatment is the therapeutic index, which can be expressed as the ratio between LD50 (the amount of compound that is lethal in 50% of the population) and ED50 (the amount of compound that is effective in 50% of the population). Compounds exhibiting a high therapeutic index are preferred. Therapeutic index data obtained from in vitro assays, cell culture assays, and / or animal studies may be used when formulating a range of doses for use in humans. Doses of such compounds are preferably within the range of plasma concentrations containing an ED50 that is little to no toxicity. Doses may vary within this range depending on the dosage form used and the route of administration utilized. See, for example, Fingl et al., THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch.1, p.1, 1975. The exact formula, route of administration, and dosage may be selected by individual practitioners, taking into account the patient's condition and the specific method by which the compound is used. For in vitro formulations, the exact formula and dosage may be selected by individual practitioners, taking into account the patient's condition and the specific method by which the compound is used.

[0245] kit In some embodiments, kits providing means for carrying out any of the methods described herein are also disclosed herein. In some embodiments, kits comprising any of the compositions described herein or means for producing the compositions are also disclosed herein.

[0246] In some embodiments, the kit can be prepared from readily available components and reagents. For example, such a kit may include any one or more of the following components and / or reagents: enzymes, reaction tubes, buffers, surfactants, primers, probes, antibodies, cell culture media, differentiation-inducing reagents, amino acid mixtures / supplements, engineered constructs and / or polynucleotides, transcription inducers, bilirubin, ascorbic acid, retinoic acid pathway activators, corticosteroids, cMET tyrosine kinase receptor agonists, IL-6 family cytokines, TGF-β pathway inhibitors, FGF pathway activators, Wnt pathway activators, VEGF pathway activators, ROCK inhibitors, and / or cells. In some embodiments, the components and reagents may be packaged together in any combination and / or individually. In some embodiments, the kit may contain concentrated components and reagents in concentrations exceeding or at the working concentrations provided herein. In some embodiments, individual components may be provided in concentrated amounts in the kit, and in some embodiments, components are provided individually at the same concentrations as they would be present in solution with other components. In some embodiments, the concentrations of the components may be 1x, 2x, 5x, 10x, or 20x or more. In some embodiments, the kit may include components that can be individually packaged or placed in containers such as tubes, bottles, vials, syringes, or other suitable container means.

[0247] In some embodiments, the kit is housed in a container. The kit may further include instructions for using the kit to evaluate cell expression and / or differentiation. The agents in the kit for measuring expression and / or determining differentiation may include multiple PCR probes and / or primers for qRT-PCR, and / or multiple antibodies or fragments thereof for evaluating the expression of appropriate biomarkers to classify the cellular state.

[0248] In some embodiments, the kit is manufactured using and in compliance with Good Manufacturing Practices (GMP).

[0249] Although embodiments have been described in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the embodiments defined in the appended claims. Furthermore, it should be understood that all examples in this disclosure are provided as non-limiting examples. [Examples]

[0250] The following non-limiting examples are provided to further illustrate the embodiments disclosed herein. Those skilled in the art will understand that the techniques disclosed in the following examples represent approaches that have been shown to function well in the implementation of the embodiments and can therefore be considered to constitute exemplary forms for such implementation. However, those skilled in the art will understand that many modifications can be made in light of this disclosure to the particular embodiments disclosed, and that similar or comparable results can be obtained without departing from the spirit and scope of the embodiments disclosed herein.

[0251] Example 1. Protocol for freezing liver organoids Cryopreservation protocol using CELLBANKER® 1 Cellbanker® 1 (AMSBIO), containing 50 nM chroman 1, 5 μM emricasane, 1:1000 polyamine, and 7 μM trans-ISRIB (CEPT), was maintained on ice or at 4°C.

[0252] Human liver organoids (HLOs) from days 20–24 were collected from 6-well plates and filtered through a 150 μM mesh into 50 mL conical tubes (total volume should not exceed approximately 25 mL). Approximately 20 mL of cold PBS was added to reach a final volume of approximately 45 mL. The suspension was pipetted up and down five times to wash the organoids. The tubes were then centrifuged at 300 × g for 5 minutes at 4°C to pelletize the HLOs. The supernatant was aspirated and further washed with 10–20 mL of fresh PBS, followed by repeated centrifugation and aspiration. The washed HLOs were resuspended in CELLBANKER® 1+CEPT frozen medium in two cryovials per well of the 6-well plate, with 0.75 mL of frozen medium suspension per cryovial. The cryovials were then transferred to a -80°C freezer container for 24 hours, and then to liquid nitrogen for longer storage.

[0253] Cryopreservation protocol using Cell Reservoir One (for vitrification) Cell Reservoir One (for vitrification) (Nacalai) containing CEPT was maintained on ice or at 4°C.

[0254] The same HLO collection and washing process as described above was performed. The washed HLO was resuspended in Cell Reservoir One (for vitrification) + CEPT freezing medium and rapidly transferred to cryopreservation tubes. The tubes were immersed in liquid nitrogen to a height of 2 / 3 for 10 seconds using tweezers, and then fully immersed. This process should be performed within 60 seconds or faster. The tubes were then transferred to a liquid nitrogen storage tank for long-term storage.

[0255] CELLBANKER(registered trademark) 1 decompression protocol A standard thawing procedure can be performed. Briefly, the cryovial was immersed in 37°C water for up to 2 minutes until a small amount of solid medium remained in the vial. Preheated medium was added to the vial, and the contents were transferred to a conical tube along with fresh medium. The suspension was pipetteed to wash the thawed HLO, and then centrifuged at 300×g for 5 minutes at room temperature. The medium was aspirated, and the HLO was resuspended in fresh medium containing CEPT (and optionally Matrige® 1).

[0256] Defrosting protocol for Cell Reservoir One (for vitrification) 10 mL of cell culture medium was preheated to 37°C in a centrifuge tube. The sterile plate was also preheated in a 37°C incubator. The cryopreservation tube containing the frozen cells was removed from the liquid nitrogen storage tank, the cap was removed, and all liquid nitrogen in the tube was discarded. More than 800 μL of preheated cell culture medium was added to the tube, and the HLO was rapidly thawed by several pipetting operations. The larger the volume used, the faster the sample thaws. The appropriate volume of medium should be added depending on the size of the tube. The thawed cell suspension was then transferred to a sterile centrifuge tube. The cryopreservation tube was washed with fresh cell culture medium, and the thawed cell suspension was added. The HLO was centrifuged at 300xg for 5 minutes at room temperature. The medium was aspirated, and the HLO was resuspended in fresh medium containing CEPT (and optionally Matrigel®).

[0257] Example 2. Freezing and thawing of liver organoid cultures Human liver organoids (22 days into culture) were frozen in CELLBANKER® 1 or vitrified (-80°C for 24 hours, then transferred to liquid nitrogen storage). An exemplary protocol is provided in Example 1.

[0258] For the experiment, organoids were thawed and immediately placed in hepatocyte culture medium. Live / dead staining was performed on the same day as thawing. Culture medium was also collected on the day of thawing and 48 hours later, and albumin secretion was detected. A schematic diagram of this is shown in Figure 1A.

[0259] Figure 1B shows the results of viability / death staining of thawed liver organoids. Organoids were stained with calcein AM (to label viable cells), ethidium homodimer-1 (to label dead cells), and NucBlue (to label nuclei). Human liver organoids showed minimal damage after short-term freezing, suggesting that freezing is a viable approach for organoid transport. The viability of organoids subjected to vitrification (e.g., using Cell Reservoir One (for vitrification)) was slightly higher than that of organoids cryopreserved by slow freezing (e.g., using CELLBANKER® 1).

[0260] Figure 1C shows the results of measuring albumin (ALB) secretion in liver organoids thawed according to the protocol provided in Example 1, compared to an unfrozen liver organoid control. The liver organoids did not show albumin secretion immediately after thawing following either slow freezing or vitrification cryopreservation. However, liver organoids cryopreserved by vitrification recovered up to 70% of their ALB secretion function after 3 days of recovery culture compared to the unfrozen control.

[0261] Enhanced maturation of liver organoids in Examples 3.2D and 3D Approaches to promote the maturation of liver organoids in culture were investigated. Previous methods for liver organoid growth have used hepatocyte culture medium, and supplementation of this medium with additional amino acids was tested. Amino acid (AA) supplementation medium was prepared as a mixture of 14% non-essential amino acid solution (100×, containing alanine, asparagine, aspartic acid, glycine, serine, proline, and glutamic acid; ThermoFisher, Grand Island, NY), 6% essential amino acid solution (50×, containing arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine; ThermoFisher, Grand Island, NY), and 80% hepatocyte medium (HCM) (Hepatocyte Culture Medium Bullet Kit; Lonza, Walkersville, MD), neutralized to pH 7.0 with NaOH, and further supplemented with 20 g / L glycine. Optionally, the hepatocyte culture medium may be supplemented with hepatocyte growth factor (HGF), oncostatin M, and / or dexamethasone, among other hepatocyte growth factors.

[0262] Liver organoids were prepared by induction of differentiation from endoderm and liver lineage determination using retinoic acid according to a conventional approach, and were cultured in AA-supplemented medium for 8, 10, or 12 days after retinoic acid induction.

[0263] The expression of liver-specific genes—albumin (ALB), cytochrome P450 family 3 subfamily A member 4 (CYP3A4), phosphoenolpyruvate carboxykinase 1 (PCK1), and glucose-6-phosphate catalytic subunit (G6PC)—in these liver organoids was quantified by RT-qPCR using liver organoids cultured in HCM without AA supplementation as a control (Figure 2A). Liver organoids cultured in AA-supplemented medium for 8, 10, and 12 days showed enhanced expression of all of these genes compared to control liver organoids.

[0264] Liver organoids cultured in AA supplementation medium for 8, 10, and 12 days after retinoic acid induction (and 14 days for urea quantification) were used to quantify albumin secretion and urea production compared to control liver organoids cultured in HCM without AA supplementation (Figure 2B). Longer exposure to AA supplementation medium (12 or 14 days) resulted in enhanced albumin secretion and urea production compared to control liver organoids.

[0265] Pluripotent stem cells were engineered with a luciferase reporter driven by a PCK1 promoter, enabling the measurement of gluconeogenesis, and differentiated into liver organoids. An exemplary PCK1 luciferase reporter can be found, for example, in PCT Publication WO2021 / 262676, which is expressly incorporated herein by reference in its entirety. PCK1 expression in these liver organoids having this construct can be quantified by luciferase activity, either in living cells or after lysis. A schematic diagram of this process is shown in Figure 2C.

[0266] Liver organoids were grown in AA-supplemented medium or, as a control, in HCM without AA supplementation. Bright-field images of these organoids are shown in Figure 2D. Liver organoids grown in AA-supplemented medium showed increased PCK1 expression compared to the control, as measured by luciferase activity, both in living cells and after lysis (Figure 2E).

[0267] The use of AA supplementation medium can also be extended beyond 3D organoids to two-dimensional (2D) hepatocyte cultures from pluripotent stem cell differentiation. To test the effect of the medium on 2D hepatocyte cultures, AA supplementation medium was used to replace HCM without AA supplementation after liver induction at various time points in culture (counting from early pluripotent stem cell differentiation, at days 12, 14, 16, 18, 21, or 34 of culture). A schematic diagram of this process is shown in Figure 3A, where A is HCM only, B is HCM + AA starting at day 12, and C-G are HCM replaced with HCM + AA at days 14, 16, 18, 21, and 34, respectively.

[0268] Figure 3B shows bright-field images of 2D hepatocyte cultures grown in AA-supplemented medium at various time points. Under all conditions, the cells exhibit normal hepatocyte morphology.

[0269] Lactate production was compared between 2D hepatocyte cultures grown in AA-supplemented medium and HCM without AA supplementation (Figure 3C). At each time point, fresh medium was replaced the day before, and test samples were collected 24 hours later. Hepatocytes grown in AA-supplemented medium showed reduced lactate production, suggesting a shift in metabolism from glucose-dependent to pyruvate-dependent compared to cells grown in conventional hepatocyte growth medium.

[0270] Albumin secretion from 2D hepatocyte cultures after replacing HCM without AA supplementation with AA supplementation medium was quantified at various time points (Figure 3D). Hepatocytes to which AA supplementation medium was added after 12 days of culture showed the highest albumin secretion. Increased albumin secretion compared to control hepatocytes was observed even for longer culture periods (Figure 3E).

[0271] Gene expression of ALB, E-cadherin (E-cad), CYP3A4, G6PC, pyruvate kinase M1 / 2 (PKM), and PCK1 was quantified in 58-day 2D hepatocytes grown in either HCM without AA supplementation or in AA supplementation medium supplemented at various time points in culture (Figure 3F). Overall, AA supplementation enhances liver maturation and gene expression. Interestingly, PCK1 and G6PC expression (which are normally suppressed by insulin added to the culture medium) decreased with increasing duration of AA supplementation, suggesting that AA supplementation normalizes the metabolism of liver organoids.

[0272] Pluripotent stem cells were engineered to express mScarlet under the PCK1 promoter, which acts as a reporter for gluconeogenesis, and differentiated into hepatocytes in 2D culture. Hepatocytes were grown in either HCM without AA supplementation or AA-supplemented medium. Prior to fluorescence quantification, cells were also optionally cultured for 24 hours without insulin (in DMEM / F12 supplemented with 0.2% BSA). Figure 3G shows mScarlet fluorescence images of these hepatocytes. Cells grown in AA-supplemented medium showed increased mScarlet expression driven by the PCK1 promoter 24 hours after insulin starvation, suggesting that these cells exhibited increased metabolism compared to controls.

[0273] 57-day 2D hepatocyte cultures grown in either HCM without AA supplementation or HCM with AA supplementation medium replaced on day 12 were probed for either EpCAM (epithelial marker), vimentin (mesenchymal marker), and either DAPI or hepatocyte nuclear factor 4 alpha (HNF4α) (nucleus) (Figure 3H). Hepatocytes differentiated from pluripotent stem cells cultured in HCM without AA supplementation showed vimentin-positive mesenchymal cells, but this vimentin signal was not present in cells cultured in AA supplementation medium, suggesting that culturing cells in AA supplementation medium potentially reduces mesenchymal cells and enriches hepatocytes.

[0274] Example 4. Generation of human organoids without Matrigel® embedding The culture of 3D human liver organoids without the use of Matrigel® containing heterogeneous components was briefly examined in PCT Publication WO2018 / 085615 (which is expressly incorporated herein by reference in its entirety). A schematic diagram of this protocol is shown in Figure 4A. Posterior foregut cells and / or posterior foregut endoderm cells differentiated from pluripotent stem cells spontaneously form three-dimensional structures after retinoic acid induction and subsequent liver culture conditions (e.g., contact with hepatocyte growth factor, oncostatin M, and dexamethasone). These three-dimensional structures can be pipetteed from the original cell layer and further cultured to grow into liver organoids (Figure 4B). These liver organoids grown under conditions without Matrigel® exhibit normal organoid morphology (Figure 4C) and predicted liver functions, including albumin secretion (Figure 4D) and expression of ALB, alpha-fetoprotein (AFP), HNF4α, retinol-binding protein 4 (RBP4), and alpha-1 antitrypsin (AAT) (Figure 4E).

[0275] Organoids grown without Matrigel® have advantages because they are not grown with heterologous animal components and can therefore be used for human purposes. Furthermore, they can grow to relatively large sizes that are easy to handle. However, these organoids may have greater size variability than organoids grown in Matrigel®, may exhibit abnormal morphology (as Matrigel® helps maintain 3D morphology), and may be less suitable for cryopreservation (which may reduce scaling for manufacturing purposes). In addition, conventional protocols also involve the use of Matrigel® during culture steps other than organoid maturation, such as pluripotent stem cell culture.

[0276] To overcome one or more of these potential drawbacks, we explored alternative approaches for culturing organoids with heterogeneous components without using Matrigel® or other basement membrane matrices.

[0277] A method for culturing foregut cells after initial induction using a combination of FGF2, EGF, VEGF, CHIR99201, and A83-01 was briefly discussed in PCT Publication WO2018 / 191673. Modified versions of this combination, with or without EGF, can be used to expand foregut cells arising from the differentiation of endoderm cells using FGF pathway activators (e.g., FGF4) and Wnt pathway activators (e.g., CHIR99021).

[0278] A schematic diagram for expanding foregut cells before subsequent differentiation into hepatic lineage is shown in Figure 5A. After induction of the foregut into the endoderm of the embryo, the cells are dissociated into single cells using a standard enzymatic dissociation solution such as Accutase, and can then be passaged in EP medium (Advanced DMEM / F12, B27 / N2 / HEPES / Glutamax, 5 ng / mL FGF2, 10 ng / mL VEGF, 3 μM CHIR99021, 500 nM A83-01, and 50 μg / mL ascorbic acid) supplemented with 10 μM Y-27632 (ROCK inhibitor) of choice.

[0279] The ability of these dissociated foregut cells to grow on substrates other than Matrigel® for expansion purposes was investigated. Culture plates were coated with either Matrigel® or laminin, basement membrane matrix components that can be manufactured and produced without animal products. Basement membrane matrix-like coatings are preferable for culture because foregut cells would otherwise not adhere properly to the plate for growth. Foregut cells expanded on both Matrigel® and laminin-coated plates grew well over a 7-day culture period and began to form 3D spheroids (Figure 5B). After further culture (days 8-10), fully formed spheroids that could be further matured into liver organoids were observed (Figure 5C). There was no significant difference in the rate of initial cell adhesion, but the efficiency of spheroid formation was better with laminin coating than with Matrigel® coating.

[0280] The number of dissociated foregut cells used to seed onto plates for enlargement was also tested: 1.0 × 10⁶ 6 or 5.0 × 10 6 Foregut cells were seeded onto laminin-coated 6-well plates (9.6 cm per well). 2 The cells were grown for 5 days (Figure 5D), with a surface area of ​​5.0 × 10⁶ cells. Spheroid formation began earlier as the number of seeded cells increased (5.0 × 10⁶ cells at each time point). 6 (As seen in larger spheroids on cell seeding plates). Therefore, spheroid formation depends on cell density.

[0281] Because enlarged foregut cells spontaneously form spheroids, they can be used as a source of spheroids to produce scaling. Spontaneously formed 3D spheroids were collected from foregut cell cultures at day 12 and used for liver organoid maturation. By continuing the culture on the plates after spheroid collection, additional spheroids were formed over an additional 23 days (up to day 35 of culture) (Figure 5E). These newly formed spheroids showed the same ability to mature into liver organoids.

[0282] Following the initial dissociation, plating, and collection of foregut cells, additional passages of proliferative foregut cells may be performed. Four passages (passages every 10 days) from the first 8-day foregut culture differentiated from pluripotent stem cells were tested. Spheroids were formed up to the third passage but not in the fourth passage (Figure 5F). In the fourth passage, mesenchymal cells were predominantly proliferated. Three passages from the initial foregut culture can result in an approximately 200-fold increase in cell number, providing a significant advantage for scaling. Gene expression for foregut cells from each passage was quantified for caudal type homeobox 2 (CDX2), forkhead box A2 (FOXA2), AFP, vimentin (VIM), SRY-box transcription factor 17 (SOX17), HNF4α, and ALB (Figure 5G). CDX2 and SOX17 expression was maintained in passage 4. FOXA2 and HNF4α expression peaked in passage 2.

[0283] Overall, to obtain the continuous formation of human liver organoids from a single cell passage of foregut cells, the dissociated foregut cells were plated on a laminin (e.g., laminin-511) coating in EP medium (containing FGF4) with a high density of plating (e.g., 5 × 10⁶). 6 Cells / 6-well plate [9.6cm] 2They can be grown in ]). Foregut cells should be passaged for up to 3 passages, as mesenchymal cells grow primarily in subsequent passages and spheroids cease to form. A schematic diagram of liver organoid formation, starting from pluripotent stem cells and including foregut cell passages for scaling, is illustrated in Figure 5H, where “scalable foregut organoids” refers to spontaneously formed foregut spheroids that are collected for maturation into liver organoids. The “organoid reformation” process represents the successive passages of foregut cells and the spontaneous formation of further foregut spheroids, which are collected and mature into liver organoids.

[0284] This process of expanding foregut cells to increase liver organoid production can be further modified using other devices such as microwell plates (e.g., Aggrewell plates (StemCell Technologies)) or formation plates designed to aggregate foregut cells for more uniform organoid formation. These approaches using devices to produce uniform organoids are discussed in PCT Publication WO2021 / 030373 (which is expressly incorporated herein by reference in its entirety). A schematic diagram of liver organoid formation, starting from pluripotent stem cells and including foregut cell passage for scaling and the use of devices to aggregate foregut cells, is illustrated in Figure 5I.

[0285] Example 5. Scalable 3D bioreactor liver organoid formation Liver organoids without Matrigel® are difficult to maintain in static culture. The use of 3D rotational culture to maintain suspended liver organoids was investigated. A schematic diagram of this process is shown in Figure 6A. Spheroids formed from foregut induction (e.g., formed on a basement membrane matrix or its components that do not contain non-human animal components as described herein) and cultures using the method described in the previous examples were collected and grown in hepatocyte culture medium in rotational culture. The spheroids were able to mature into liver organoids over 15 days under these rotational culture conditions (Figure 6B). Gene expression of AFP, HNF4α, FOXA2, ALB, CDX2, and VIM was quantified in liver organoids grown in rotational culture (3D) and compared to passage 1 (P1) foregut cells (Figure 6C). The liver organoids showed high expression of ALB and AFP, indicating mature hepatocytes. The expression of CDX2 and HNF4α did not differ significantly from that of foregut cells. Therefore, this demonstrates that liver organoids can be cultured without the need for Matrigel® or other basement membrane matrices when grown in rotational culture.

[0286] Example 6. Additional materials and methods animal All animal experiments were conducted with the approval of the Institutional Review Board and the Animal Experimentation Committee. Adult Gunn (Gunn-Ugt1a1j / BluHsdRrrc) rats (breeding pairs, 9-12 weeks old) were obtained from the Rat Resource & Research Center (RRRC, Columbia, MO). The rats were housed in standard rat cages with wood chip bedding, maintained at a temperature of 20-24°C and a relative humidity of 45-55% under a 12-hour:12-hour light-dark cycle. All animals were free-range with standard solid feed (Cincinnati Lab Supply, Cincinnati, OH) prior to the experiment. All animals were handled in accordance with the facility's guidelines and regulations.

[0287] Maintaining PSC Human iPSC strain 72.3 (RRID:CVCL_A1BW) was obtained from the Pluripotent Stem Cell Facility at Cincinnati Children's Hospital Medical Center (CCHMC), jointly managed by CN. Mayhew and JM. Wells. Undifferentiated hiPSCs were cultured on dishes coated with laminin-511 E8 fragment (Nippi) in StemFit medium (Ajinomoto Company) containing 100 ng / mL of basic fibroblast growth factor (FGF; R&D Systems) at 37°C, 5% CO2, and 95% air.

[0288] Human liver organoid (HLO) production Pluripotent stem cells were placed in 24-well plates coated with laminin iMatrix-511Silk, in a 2x10⁶ arrangement. 5Cells were seeded at a cell / well density and maintained in StemFit medium containing Y-27632. On day 2, the medium was replaced with fresh StemFit medium. The following day, cells were treated with RPMI medium mixed with activin A and BMP4 to generate endoderm. On day 4, the medium was replaced with RPMI, activin A, and 0.2% dFBS, and on day 5, it was changed to 2% dFBS. From days 6 to 8, cells were supplied with FGF4 and CHIR99021 in Advanced DMEM (supplemented with B27, N2, 10 mM HEPES, 2 mM L-glutamine, and gentamicin-amphotericin) to induce posterior foregut. On day 9, cells were dissociated into single-cell suspension using Accutase treatment. Next, this single-cell suspension was mixed with 50% Matrigel® and 50% EP medium (Advanced DMEM / F12, B27 / N2 / HEPES / Glutamax, 5 ng / mL FGF2, 10 ng / mL VEGF, 3 μM CHIR99021, 500 nM A83-01, and 50 μg / mL ascorbic acid), and seeded as 50 μl droplets in 6-well plates. These cells were supplied with EP medium every 48 hours for 4 days to generate organoids. These organoids were then treated with Advanced DMEM and retinoic acid (RA) every 48 hours for 4 days to identify liver lineages. Subsequently, the organoids were supplied with hepatocyte culture medium (HCM), hepatocyte growth factor (HGF), oncostatin M, and dexamethasone every 3-4 days to generate HLOs.

[0289] HLO maturation by low-dose bilirubin To induce HLO maturation, organoids at day 15 were supplied with complete HCM containing a low dose of bilirubin (1 mg / L) plus supplements. This treatment was continued until day 30, when the organoids were harvested. Bright-field images were captured using a KEYENCE BZ-X710 fluorescence microscope (Keyence), and the images were analyzed using the ImageJ suite. RNA was isolated using the Rneasy mini-kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. qPCR was performed using the QuantStudio 5 real-time PCR system (Thermo Fisher Scientific Inc.) with the TaqMan gene expression master mix (Applied Biosystems). All primer and probe information for each target gene was obtained from the Universal Probe Library Assay Design Center website (available from lifescience.roche.com / en_us / brands / universal-probe-library.html on the World Wide Web). For whole-mount immunostaining, organoids were fixed in 4% PFA, permeabilized with 0.1% PBST, blocked with 5% normal donkey serum in 0.1% PBST, and stained with appropriate primary and secondary antibodies. Images were captured using a Nikon A1 inverted confocal microscope.

[0290] Bilirubin and drug processing On day 27, mature organoids were treated for 5 days with bilirubin (1–10 mg / L), doxycycline (100 ng / mL; added 3 days prior to activating gene expression), and / or further with drugs such as hydrocortisone, dexamethasone, ketoconazole, and mifepristone (1–2 μM each), and then collected for downstream assays. Bilirubin assays were performed using a colorimetric kit (Abcam ab235627). RNA was isolated using the Rneasy mini-kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. qPCR was performed using the QuantStudio 5 real-time PCR system (Thermo Fisher Scientific Inc.) with the TaqMan gene expression master mix (Applied Biosystems). All primer and probe information for each target gene was obtained from the Universal Probe Library Assay Design Center (available on the World Wide Web at lifescience.roche.com / en_us / brands / universal-probe-library.html). Images were captured using a KEYENCE BZ-X710 fluorescence microscope (Keyence).

[0291] Organoid transplantation into the portal vein HLO was collected on day 27, dissociated into organoid fragments by repeated pipetting, washed with PBS, and resuspended in HCM containing 2% FBS and a CEPT cocktail (50 nM Chroman 1, 5 μM Emricasan, 1:1000 polyamine, and 7 μM trans-ISRIB) to increase viability. Recipient rats were treated with a single dose of retrolucin (5 mg / kg) and tacrolimus (0.8 mg / kg) four days prior to transplantation. A midline incision was made, the intestine was pushed to one side, and 200 μL was injected into the portal vein in 3 × 10⁴ oz infusions by clamping the portal vein posteriorly using a 32 g 1 inch needle to control bleeding.3 Organoids (approximately 5 x 10) 5 Cells were injected. Excessive blood loss was prevented by applying SURGICEL SnoW Absorbable Hemostat (Ethicon). The animals were then closed with 5-0 bicryl-coated surgical sutures (Ethicon) and GLUture (Zoetis Inc.), and buprenorphine (0.1 mg / kg) was administered as an analgesic. The animals were then maintained with doxycycline (2 mg / kg) and tacrolimus injections every 3-4 days until the day of collection. Blood was collected periodically using the retroorbital method as needed.

[0292] Live cell imaging and functional assays For live imaging of organoids, Celldiscoverer 7 (Zeiss) was used, and images were acquired every 30 minutes for 7 days. Bilirubin conjugation was visualized using 5 μM fluorescent UnaG, which was incubated with HLO medium and imaged for 2 days.

[0293] RNA sequencing and analysis RNA was isolated using the Rneasy Mini Kit (Qiagen). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit for RT-PCR (Applied Biosystems) according to the manufacturer's protocol. qPCR was performed using the TaqMan gene expression master mix (Applied Biosystems) on the QuantStudio 5 real-time PCR system (Applied Biosystems). All samples were amplified in the TaqMan gene expression assay and normalized with an 18S rRNA endogenous control. For RNA sequencing, the quality of the extracted RNA was assessed using the Agilent 2100 Bioanalyzer (Agilent). A sequence library was prepared using the TruSeq Stranded mRNA Kit (Illumina) and sequenced using the NovaSeq 6000 (Illumina). Reads were aligned to the human genome assembly hg38 and quantified using the pseudo-mapper Salmon (v1.8.0). Gene expression analysis was performed using the R Bioconductor package DESeq2 (v1.36.0). The read count matrix was normalized by a size factor, and variance stabilizing transformation (VST) was applied to the normalized expression data. Data was visualized using the clusterProfiler (v.4.4.1) and pheatmap (v1.0.12) packages.

[0294] Alternatively, all-transcriptome RNA sequencing of HLO (including bilirubin-treated and mifepristone-treated) was performed on isolated total RNA using the Illumina NovaSeq platform with services from Novogene Co., Ltd. (Beijing, China). The RNA sequencing parameter was 150 bp paired-end sequencing at a depth of 20 M reads per sample. Fastq read files were obtained for each sample and then aligned using Salmon, a pseudo-mapping tool that aligns and quantifies transcripts using RNA-seq data. Raw transcript counts and normalized transcripts per million (TPM) values ​​were obtained and analyzed for differential expression using DESeq2. For differential expression, statistical and biological significance was set to P<0.05, FDR<0.05, log change multiplier>1, and the lowest three transcript counts from three of the six samples were used. For heatmap visualization and hierarchical clustering analysis, we used hclust and pheatmap, respectively. Finally, we performed path analysis using biomaRt and org.Hs.eg.db from R version 4.0.3.

[0295] mGULO Editing The mouse GULO (L-gulonolactone oxidase) (mGULO) cDNA sequence was searched from NCBI. A 5' linker and Kozak sequence were added to the beginning of the sequence, and an HA tag was added to the end of the sequence. Furthermore, P2A-mCherry was added after the HA tag, and a 3' linker was added to the end. Then, a custom gene was synthesized and cloned into the pAAVS1-Ndi-CRISPRi(Gen1)PCSF#117 vector using restriction sites AflII and AgeI. The vector has a Teton system and was then Neo using Gateway technology. r A selection marker was inserted.

[0296] mGULO iPSC generation and maintenance Next, the PCSF#117 vector containing the modified GULO sequence was inserted into the AAVS1 locus of 72.3 iPSC cell lines using lentivirus-mediated CRISPR / Cas9. The correct clones were then selected using G418. The viable clones were then validated for accurate insertion, random insertion, and copy number using PCR, and verified by DNA sequencing. The edited iPSCs were then seeded on laminin iMatrix-511 Silk-coated cell culture plates and maintained in StemFit Basic04 complete medium containing Y-27632. Cells were passaged every 4–7 days using Accutase up to passage 40 (p40). mGULO HLO was generated according to the HLO generation protocol described herein. mGULO protein expression was validated using the GLUO ELISA kit (MBS2890737, MyBioSource, San Diego, CA).

[0297] mGULO HLO treatment and bilirubin visualization using bilirubin On day 24, mGULO expression was induced by treating mGULO HLO with doxycycline (Dox) (100 ng / mL). Subsequently, on day 27, mature organoids were treated with bilirubin and Dox for 5 days and then collected for downstream assays. Bilirubin assays were performed using a colorimetric kit (ab235627) and UnaG, a green-to-dark light-switching fluorescent protein that fluoresces only upon bilirubin binding, to measure and visualize unconjugated and conjugated bilirubin. Images were captured using a KEYENCE BZ-X710 fluorescence microscope (Keyence).

[0298] ChIP-PCR and ChIP-qPCR ChIP experiments were performed using the High Sensitivity ChIP Kit (Abcam). Briefly, organoids were fixed with PFA, then total chromatin was prepared, sonicated to an optimal size of 300 bp, and confirmed by gel electrophoresis. The chromatin was used for immunoprecipitation with either EP300 antibody or IgG1 isotype control. DNA fragments were amplified using custom primers for PCR and qPCR, and enrichment ratio data were normalized to immunoprecipitation from the IgG control.

[0299] Protein expression assay Albumin secretion was measured by collecting 200 μL of supernatant from HLOs cultured in HCM and stored at -80°C until use. The supernatant was assayed using the Human Albumin ELISA Quantitation Set kit (Bethyl Laboratories) according to the manufacturer's instructions.

[0300] For the mouse GULO expression assay, organoids were dissociated and washed with PBS. Then, cells were lysed with RIPA lysis and extraction buffer and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) to extract total protein, and the assay was performed using the mouse GULO / L-gulonolactone oxidase ELISA kit (MyBioSource.com) according to the manufacturer's instructions.

[0301] Metabolite assay Bilirubin levels were measured by collecting the supernatant of bilirubin-treated HLO and rat serum. The supernatant and serum were assayed using the Bilirubin Assay Kit (Total and Direct, Colorimetric) (abcam) and the Bilirubin Assay Kit (Sigma-Aldrich) according to the manufacturer's instructions.

[0302] Cellular antioxidant levels were measured by recovering HLO, washing it in PBS, and plating it onto a 96-well assay plate. The levels were then quantified using the Cellular Antioxidant Assay Kit (abcam) according to the manufacturer's instructions.

[0303] Activity assay The CYP3A4 and CYP1A2 assays were performed by recovering HLO, washing it in PBS, plating it onto 96-well assay plates, and treating it with rifampicin and omeprazole, respectively, for 24 hours. The assays were then carried out using the P450-Glo CYP3A4 and CYP1A2 assay (Promega) and normalized using the CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer's instructions.

[0304] The apoptosis assay was performed by lysing HLO and assaying the lysate with the Caspase-3 Assay Kit (Colorimetric) (abcam) according to the manufacturer's instructions.

[0305] Rat serum was assayed using an aspartate aminotransferase (AST) activity assay kit and an alanine transaminase (ALT) activity assay kit (Sigma-Aldrich).

[0306] Quantification and statistical analysis Statistical analysis was performed using R software v4.2.0 with unpaired two-tailed Student's t-test, Dunn-Holland-Wolfe test, or Welch's test. Statistical analysis of stiffness measurements was performed using the nonparametric Kruskal-Wallis and post-hoc Dunn-Holland-Wolfe tests. For comparisons between two independent groups, if the groups were independent and had unequal variances, the nonparametric Brunner-Manzel test was used unless otherwise specified. A p-value < 0.05 was considered statistically significant. The N-value refers to biologically independent copies. Image analysis was open-label.

[0307] Example 7. Low doses of bilirubin promote maturation of fetal-like liver organoids. The role of bilirubin in liver development was investigated using a human liver organoid model. Figure 7A shows an exemplary schematic diagram for preparing liver organoids treated with a low concentration of bilirubin (e.g., 1 mg / L) similar to the physiological concentration in human fetuses (approximately 10 times lower than the physiological concentration in adults). Bilirubin was added to early organoids that had differentiated into the liver lineage. Exemplary methods for producing liver organoids have bee...

Claims

1. A method for enlarging posterior foregut cells and / or posterior foregut endoderm cells, a) Dissociating the posterior foregut and / or posterior foregut endoderm cell monolayer into posterior foregut cells and / or posterior foregut endoderm cells, b) Seeding the posterior foregut cells and / or posterior foregut endoderm cells onto the tissue culture surface, c) A method comprising culturing the posterior foregut cells and / or posterior foregut endoderm cells together with a TGF-β pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator.

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

3. The posterior foregut cells and / or posterior foregut endoderm cells are 1×10 ５ , 2×10 ５ , 3×10 ５ , 4×10 ５ , 5×10 ５ , 6×10 ５ , 7×10 ５ , 8×10 ５ , 9×10 ５ , 1×10 ６ , 2×10 ６ , 3×10 ６ , 4×10 ６ , or 5×10 ６ cells / cm ２ , or at about those cell densities, or at any cell density within the range defined by any two of the aforementioned cell densities, seeded onto the surface of the tissue container, the method according to claim 1 or 2.

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

5. The method according to claim 4, wherein the basement membrane matrix or its components does not contain non-human animal components such that the basement membrane matrix or its components are heterogeneous to humans, optionally the basement membrane matrix or its components are not isolated from mouse Angelbreth-Holm-Swarm (EHS) sarcoma cells, optionally the basement membrane matrix or its components are not Matrigel®, Cultrex®, or Geltrex®.

6. The method according to claim 4 or 5, wherein the basement membrane matrix or its components include human laminin, collagen IV, entactin, perlecan, fibrin, and / or hydrogel.

7. The method according to any one of claims 1 to 6, wherein the posterior foregut cells and / or posterior foregut endoderm cells are cultured until three-dimensional (3D) spheroids spontaneously form, and optionally the 3D spheroids include a structure having a single lumen and / or the spheroids do not contain hematopoietic tissue and acquired immune cells.

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

9. The method according to any one of claims 1 to 8, wherein the TGF-β pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542, with the selection being optional from A83-01.

10. The method according to any one of claims 1 to 9, wherein 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the TGF-β pathway inhibitor is provided at a concentration of 500 nM or about 500 nM.

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

12. The method according to any one of claims 1 to 11, wherein 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the FGF pathway activator is provided at a concentration of 5 ng / mL or about 5 ng / mL.

13. The Wnt pathway activators are Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML284, IQ-1, WAY262611, CHIR990 The method according to any one of claims 1 to 12, comprising the group consisting of 21, CHIR98014, AZD2858, BIO, AR-A014418, SB216763, SB415286, aloysin, indirubin, alsterpolone, kaempaulone, lithium chloride, TDZD8, and TWS119, optionally selected from CHIR99021.

14. The method according to any one of claims 1 to 13, wherein the Wnt pathway activator is provided at a concentration 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the Wnt pathway activator is provided at a concentration of 3 μM or about 3 μM.

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

16. The method according to any one of claims 1 to 15, wherein 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the VEGF pathway activator is provided at a concentration of 10 ng / mL or about 10 ng / mL.

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

18. The method according to claim 17, wherein the 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the EGF is provided at a concentration of 20 ng / mL or about 20 ng / mL.

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

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

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

22. The method according to claim 21, wherein 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the ROCK inhibitor is provided at a concentration of 10 μM or about 10 μM.

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

24. The method according to claim 23, wherein the cells in step c) are passaged until the posterior foregut cells and / or posterior foregut endoderm cells cease to spontaneously form spheroids.

25. The method according to claim 23 or 24, wherein the cells in step c) are passaged three or fewer times.

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

27. The method according to claim 26, further comprising culturing the posterior foregut cells and / or posterior foregut endoderm cells until a three-dimensional (3D) spheroid is spontaneously formed, collecting the posterior foregut cells and / or posterior foregut endoderm cells from the spheroid, and optionally dissociating the spheroid into individual posterior foregut cells and / or posterior foregut endoderm cells and / or clumps of posterior foregut cells and / or posterior foregut endoderm cells before the differentiation step, wherein optionally the spheroid comprises a structure having a single lumen and / or the spheroid does not contain hematopoietic tissue and acquired immune cells.

28. The method according to claim 26, wherein, prior to the differentiation step, the posterior foregut cell monolayer is dissociated into individual posterior foregut cells and / or posterior foregut endoderm cells and / or clumps of posterior foregut cells and / or posterior foregut endoderm cells to collect the posterior foregut cells and / or posterior foregut endoderm cells from the posterior foregut cell monolayer.

29. A method for differentiating posterior foregut cells and / or posterior foregut endoderm cells into liver organoids, i) optionally, posterior foregut cells and / or posterior foregut endoderm cells, which are in the form of spheroids, optionally, in the form of individual cells or cell clusters dissociated from spheroids, optionally, the spheroids comprising structures having a single lumen and / or the spheroids not containing hematopoietic tissue and acquired immune cells, and / or optionally, cells aggregated in a microwell or other device as described herein, being brought into contact with a retinoic acid pathway activator. ii) A method comprising bringing the cells from step i) into contact with a culture medium, optionally wherein the culture medium is a hepatocyte culture medium, for a certain period of time, thereby differentiating the posterior foregut cells and / or posterior foregut endoderm cells into liver organoids.

30. The method according to claim 29, wherein the culture medium is supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid.

31. The method according to claim 30, wherein the cMET tyrosine kinase receptor agonist is selected from the group consisting of hepatocyte growth factor (HGF), PG-001, phosgonimeton, telebarefim, recombinant InlB321 protein, and agonist c-Met antibody, and optionally from LMH85.

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

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

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

35. The method according to claim 29, wherein the culture medium is supplemented with dexamethasone.

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

37. The method according to any one of claims 29 to 36, wherein the posterior foregut cells and / or posterior foregut endoderm cells are in the form of a spheroid, or individual posterior foregut cells and / or posterior foregut endoderm cells and / or aggregates of posterior foregut cells and / or posterior foregut endoderm cells derived from dissociating the spheroid, and optionally the spheroid includes a structure having a single lumen and / or the spheroid does not contain hematopoietic tissue and acquired immune cells.

38. The method according to any one of claims 29 to 37, wherein the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580, and optionally selected from retinoic acid.

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

40. The method according to claim 34, wherein the HGF 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the HGF is provided at a concentration of 10 ng / mL or about 10 ng / mL.

41. The method according to claim 34, wherein the OSM 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 concentrations, or at any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the OSM is provided at a concentration of 20 ng / mL or about 20 ng / mL.

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

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

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

45. The method according to claim 44, wherein the supplemented growth medium contains 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of non-essential amino acids by total volume, or a range defined by any two of the above values, and optionally, the supplemented growth medium contains approximately 4–10%, 6–12%, 10–16%, 12–15%, 13–19%, or approximately 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% of non-essential amino acids by total volume.

46. The method according to claim 44 or 45, wherein the supplemented growth medium contains, by total volume, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of essential amino acids, or a range defined by any two of the above values, and optionally, the supplemented growth medium contains, by total volume, approximately 4–10%, 6–12%, 10–16%, 12–15%, 13–19%, or approximately 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% of essential amino acids.

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

48. The method according to any one of claims 29 to 47, wherein the cells of step ii) are further brought into contact with a low / first concentration of bilirubin, and the liver organoids formed are mature liver organoids.

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

50. The low / first concentration of bilirubin is a) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, or 3.0 mg / L, or approximately these, less than these, or approximately less than these, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg / L, 0.5 to 2.0 mg / L, 0.5 to 1.5 mg / L, 0.3 to 2.5 mg / L, The method according to claim 48 or 49, wherein the concentration is either 0.5 to 1.75 mg / L, or b) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / L, about those, less than those, about less than those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg / L, 0.1 to 0.5 mg / L, 0.5 to 1 mg / L, 0.3 to 0.7 mg / L, or 0.4 to 0.6 mg / L.

51. The method according to any one of claims 48 to 50, wherein the mature liver organoid has luminal projections similar to bile canaliculi and / or a structure having a single lumen and generally a spherical shape, and / or the mature liver organoid does not contain hematopoietic tissue and acquired immune cells.

52. The method according to any one of claims 48 to 51, wherein the mature liver organoid expresses reduced levels of AFP, CDX2, NANOG, or any combination thereof, compared to liver organoids that have not been exposed to the low / first dose of bilirubin.

53. The method according to any one of claims 48 to 52, wherein the mature liver organoid expresses increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, compared to liver organoids that have not been exposed to the low / first dose of bilirubin.

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

55. The method according to any one of claims 48 to 54, wherein the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids that have not been contacted with the low / first dose of bilirubin.

56. The method according to any one of claims 29 to 55, wherein the cells of step ii) are further brought into contact with a high / second concentration of bilirubin, and the liver organoids formed are hyperbilirubinemia liver organoids.

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

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

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

60. The method according to claim 59, wherein the functional GULO protein is mouse GULO (mGULO).

61. The method according to claim 59 or 60, wherein the gene encoding the functional GULO protein is conditionally expressed using a tetracycline induction system of optional choice.

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

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

64. The method according to any one of claims 59 to 63, wherein the liver organoid containing the functional GULO protein expresses an increased level of NRF2 compared to a liver organoid that does not contain the functional GULO protein.

65. The method according to any one of claims 59 to 64, wherein the liver organoid containing the functional GULO protein expresses reduced levels of IL1B, IL6, or TNFa, or any combination thereof, compared to liver organoids that do not contain the functional GULO protein, when optionally cultured in an ascorbic acid-depleted medium or in the absence of ascorbic acid.

66. The method according to any one of claims 59 to 65, wherein the liver organoid containing the functional GULO protein exhibits reduced caspase-3 activity when optionally cultured in an ascorbic acid-depleted medium or in the absence of ascorbic acid, compared to liver organoids that do not contain the functional GULO protein.

67. The method according to any one of claims 59 to 66, wherein the liver organoid containing the functional GULO protein expresses an increased level of ALB compared to a liver organoid that does not contain the functional GULO protein.

68. The method according to any one of claims 59 to 67, wherein the liver organoid containing the functional GULO protein resembles periportal liver tissue and expresses a periportal liver marker.

69. The method according to claim 68, wherein the periportal liver marker includes FAH, ALB, PAH, CPS1, HGD, or any combination thereof.

70. The method according to any one of claims 59 to 69, wherein the liver organoid containing the functional GULO protein exhibits increased CYP3A4 and CYP1A2 activity compared to liver organoids not containing the functional GULO protein.

71. The method according to any one of claims 59 to 70, wherein the liver organoid containing the functional GULO protein exhibits increased bilirubin conjugation activity compared to liver organoids not containing the functional GULO protein.

72. The method according to any one of claims 59 to 71, wherein the liver organoid containing the functional GULO protein exhibits an increased viability in culture compared to liver organoids not containing the functional GULO protein.

73. The method according to any one of claims 59 to 72, wherein the liver organoid is differentiated from the pluripotent stem cell, comprising a functional GULO protein and / or a gene or mRNA encoding the functional GULO protein, or both, thereby enabling the pluripotent stem cell to synthesize ascorbic acid.

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

75. The method according to any one of claims 29 to 74, further comprising agglutinating the posterior foregut cells and / or posterior foregut endoderm cells in a microwell or other apparatus (e.g., Aggrewell) prior to step i), wherein agglutinating the posterior foregut cells and / or posterior foregut endoderm cells results in liver organoids of a more uniform size.

76. The method according to any one of claims 29 to 75, wherein the cells of step i) and / or step ii) are not cultured with a basement membrane matrix or its components, optionally, the cells of step i) and / or step ii) are not cultured with a basement membrane matrix or its components that are heterologous to humans, optionally, the cells of step i) and / or step ii) are not cultured with a basement membrane matrix or its components isolated from mouse Angelbreth-Holm-Swarm (EHS) sarcoma cells, and optionally, the cells of step i) and / or step ii) are not brought into contact with Matrigel®, Cultrex®, or Geltrex®.

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

78. The method according to claim 77, wherein the liver organoids are cultured in a static or non-static bioreactor, then dissociated into single cells, and subsequently reconstructed and / or expanded through a further culture step in a static or non-static bioreactor, optionally a 3D bioreactor, or optionally a 3D rotating bioreactor.

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

80. The method according to claim 79, wherein the cryopreservation of the liver organoids includes slow freezing or vitrification cryopreservation, and optionally the liver organoids are cryopreserved together with chroman 1, emricasane, polyamine, and trans-ISRIB (CEPT).

81. The method according to any one of claims 1 to 80, wherein the posterior foregut cells and / or posterior foregut endoderm cells are derived from pluripotent stem cells, optionally embryonic stem cells, or induced pluripotent stem cells.

82. The method according to any one of claims 1 to 81, wherein the posterior foregut cells and / or posterior foregut endoderm cells are derived from a subject, optionally selected, a subject having a liver-related disease or disorder.

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

84. Posterior foregut cells and / or posterior foregut endoderm cells, or liver organoids, produced by the method described in any one of claims 1 to 83.

85. An in vitro composition comprising pluripotent stem cells, endoderm of an embryo, posterior foregut, posterior foregut endoderm, and / or downstream hepatocyte type, and at least one exogenous tissue culture surface, at least one exogenous TGF-β pathway inhibitor, at least one exogenous FGF pathway activator, at least one exogenous Wnt pathway activator, and at least one exogenous VEGF pathway activator.

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

87. The posterior foregut cells and / or posterior foregut endoderm cells comprise a surface area of ​​1 × 10⁻⁶ of the tissue culture surface. ５ , 2 x 10 ５ , 3 x 10 ５ , 4 x 10 ５ , 5 x 10 ５ , 6 x 10 ５ , 7 x 10 ５ , 8 x 10 ５ , 9 x 10 ５ , 1 x 10 ６ , 2 x 10 ６ , 3 x 10 ６ , 4 x 10 ６ , or 5 x 10 ６ cells / cm ２ The in vitro composition according to claim 85 or 86, wherein the cell density is exactly or approximately those cell densities, or any cell density within the range defined by either of the aforementioned cell densities.

88. The in vitro composition according to any one of claims 85 to 87, wherein the tissue culture surface is coated with a basement membrane matrix or its components.

89. The in vitro composition according to claim 88, wherein the basement membrane matrix or its components does not contain non-human animal components such that the basement membrane matrix or its components are heterogeneous to humans, optionally the basement membrane matrix or its components are not isolated from mouse Angelbreth-Holm-Swarm (EHS) sarcoma cells, optionally the basement membrane matrix or its components are not Matrigel®, Cultrex®, or Geltrex®.

90. The in vitro composition according to claim 88 or 89, wherein the basement membrane matrix or its components comprises human laminin, collagen IV, entactin, perlecan, fibrin, and / or hydrogel.

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

92. The in vitro composition according to any one of claims 85 to 91, wherein the TGF-β pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542, and optionally the TGF-β pathway inhibitor includes A83-01 or is A83-01.

93. The in vitro composition according to any one of claims 85 to 92, wherein the TGF-β pathway inhibitor is at a concentration of 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within the range defined by about those concentrations, or any two of the aforementioned concentrations, and optionally, the TGF-β pathway inhibitor is at a concentration of 500 nM or about 500 nM.

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

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

96. The Wnt pathway activators are 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 An in vitro composition according to any one of claims 85 to 95, wherein the Wnt pathway activator is selected from the group consisting of BIO, AR-A014418, SB216763, SB415286, aloysin, indirubin, alsterpolone, kaempaulone, lithium chloride, TDZD8, and TWS119, and optionally comprises CHIR99021 or is CHIR99021.

97. The in vitro composition according to any one of claims 85 to 96, wherein the Wnt pathway activator is 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 any concentration within the range defined by about those concentrations, or any two of the aforementioned concentrations, and optionally, the Wnt pathway activator is at a concentration of 3 μM or about 3 μM.

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

99. The in vitro composition according to any one of claims 85 to 98, wherein the VEGF pathway activator is 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 any concentration within the range defined by about those concentrations, or any two of the aforementioned concentrations, and optionally, the VEGF pathway activator is at a concentration of 10 ng / mL or about 10 ng / mL.

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

101. The in vitro composition according to any one of claims 85 to 100, wherein the EGF is 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 concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, and optionally, the EGF is at a concentration of 20 ng / mL or about 20 ng / mL.

102. The in vitro composition according to any one of claims 85 to 101, wherein the composition further comprises exogenous and / or genetically modified ascorbic acid, or the composition does not contain exogenous and / or genetically modified ascorbic acid.

103. The in vitro composition according to any one of claims 85 to 102, wherein the ascorbic acid is at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL, or any concentration within the range defined by about those concentrations, or any two of the aforementioned concentrations, and optionally, the ascorbic acid is at a concentration of 50 μg / mL or about 5 μg / mL.

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

105. The in vitro composition according to any one of claims 85 to 104, wherein the ROCK inhibitor is 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 any concentration within the range defined by about those concentrations, or any two of the aforementioned concentrations, and optionally, the ROCK inhibitor is at a concentration of 10 μM or about 10 μM.

106. The in vitro composition according to any one of claims 85 to 105, wherein the posterior foregut cells and / or posterior foregut endoderm cells, embryonic endoderm, posterior foregut endoderm, and / or downstream hepatocytes differentiate from stem cells, and optionally, the posterior foregut cells and / or posterior foregut endoderm cells, embryonic endoderm, posterior foregut endoderm, and / or downstream hepatocytes differentiate from induced pluripotent stem cells.

107. The in vitro composition according to any one of claims 85 to 107, wherein the posterior foregut cells and / or posterior foregut endoderm cells, embryonic endoderm, posterior foregut endoderm, and / or downstream hepatocytes have been passaged less than four times.

108. The in vitro composition according to claim 108, wherein the cells include or are essentially composed of posterior foregut cells and / or posterior foregut endoderm cells.

109. The in vitro composition according to any one of claims 85 to 108, wherein the TGF-β pathway inhibitor is A83-01, the FGF pathway activator is FGF2, the Wnt pathway activator is CHIR99021, the VEGF pathway activator is VEGF, and the ROCK inhibitor is Y-27632.

110. A liver organoid produced by the method described in any one of claims 29 to 83.

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

112. The in vitro composition according to claim 111, wherein the cMET tyrosine kinase receptor agonist is selected from the group consisting of hepatocyte growth factor (HGF), PG-001, phosgonimeton, telebarefim, recombinant InlB321 protein, and agonist c-Met antibody, and optionally from LMH85.

113. The in vitro composition according to claim 111 or 112, wherein the IL-6 family cytokine is selected from the group consisting of IL-6, oncostatin M (OSM), leukemia suppressor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokine (CLC).

114. The in vitro composition according to any one of claims 111 to 113, wherein the corticosteroid is selected from the group consisting of dexamethasone, beclomethasone, betamethasone, fluocortone, halomethasone, and mometasone.

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

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

117. The in vitro composition according to any one of claims 111 to 116, wherein the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580, and optionally, the retinoic acid pathway activator contains retinoic acid or is retinoic acid.

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

119. The in vitro composition according to any one of claims 115 to 118, wherein the HGF is 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 any concentration within the range defined by about those concentrations, or any two of the aforementioned concentrations, and optionally the HGL is at a concentration of 10 ng / mL or about 10 ng / mL.

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

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

122. The in vitro composition according to any one of claims 111 to 121, wherein the composition does not contain exogenous EGF.

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

124. The biliruby is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, or 3.0 mg / L, or approximately these, less than these, or approximately less than these, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg / L, 0.5 to 2.0 mg / L, 0.5 to 1.5 mg / L, 0.3 to 2.5 mg / L, or 0.5 to The in vitro composition according to claim 123, wherein the concentration is 1.75 mg / L, or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / L, approximately those, less than those, approximately less than those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg / L, 0.1 to 0.5 mg / L, 0.5 to 1 mg / L, 0.3 to 0.7 mg / L, or 0.4 to 0.6 mg / L.

125. The in vitro composition according to claim 124, wherein the composition comprises a mature liver organoid, the mature liver organoid having luminal projections similar to bile canaliculi and / or a structure having a single lumen and generally a spherical shape, and / or the mature liver organoid does not contain hematopoietic tissue and acquired immune cells.

126. The in vitro composition according to claim 125, wherein the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof, compared to liver organoids that have not been exposed to a low dose of bilirubin.

127. The in vitro composition according to claim 125 or 126, wherein the mature liver organoid expresses increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, compared to liver organoids that have not been contacted with the low dose of bilirubin.

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

129. The in vitro composition according to any one of claims 125 to 128, wherein the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids that have not been contacted with a low dose of bilirubin.

130. An in vitro composition comprising a mature liver organoid, wherein the cells of the mature liver organoid are in contact with a low dose of bilirubin, optionally the low dose of bilirubin is provided exogenously, the mature liver organoid exhibits luminal projections similar to bile canaliculi, and / or a structure having a single lumen and generally spherical shape, and / or the mature liver organoid does not contain hematopoietic tissue and adaptive immune cells.

131. The in vitro composition according to claim 130, wherein the mature liver organoid expresses reduced levels of AFP, CDX2, NANOG, or any combination thereof, compared to liver organoids in which the cells were not exposed to a low dose of bilirubin.

132. The in vitro composition according to claim 130 or 131, wherein the mature liver organoid expresses increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, compared to liver organoids in which the cells were not exposed to a low dose of bilirubin.

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

134. The in vitro composition according to any one of claims 130 to 133, wherein the mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids in which the cells were not exposed to a low dose of bilirubin.

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

136. The aforementioned high / second concentration of bilirubin was 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / L, approximately those, greater than those, or approximately greater than those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 2–20 mg / L, 2–10 mg / L, 10–20 mg / L, 5–15 mg / L, or 8–12 mg / L. The in vitro composition according to claim 135, or the concentration being 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / L, about those, greater than those, or about greater than those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 4–20 mg / L, 2–10 mg / L, 10–20 mg / L, 5–15 mg / L, or 8–12 mg / L.

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

138. The in vitro composition according to any one of claims 111 to 137, wherein the posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids contain a functional L-gulonolactone oxidase (GULO) protein and / or a gene or mRNA encoding the functional GULO protein, or both, and the posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids are capable of synthesizing ascorbic acid.

139. The in vitro composition according to claim 138, wherein the functional GULO protein is mouse GULO (mGULO).

140. The in vitro composition according to claim 138 or 139, wherein the gene encoding the functional GULO protein is conditionally expressed using a tetracycline induction system of optional choice.

141. The in vitro composition according to claim 138 or 139, wherein the posterior foregut cells and / or posterior foregut endoderm cells, liver organoids and / or mature liver organoids are manipulated using CRISPR to contain the gene encoding the functional GULO protein.

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

143. The in vitro composition according to any one of claims 138 to 142, wherein the liver organoid and / or mature liver organoid containing the functional GULO protein expresses an increased level of NRF2 compared to the liver organoid and / or mature liver organoid that does not contain the functional GULO protein.

144. The in vitro composition according to any one of claims 138 to 143, wherein the liver organoid and / or mature liver organoid containing the functional GULO protein express reduced levels of IL1B, IL6, or TNFa, or any combination thereof, compared to liver organoids and / or mature liver organoids that do not contain the functional GULO protein.

145. The in vitro composition according to any one of claims 138 to 144, wherein the liver organoid and / or mature liver organoid containing the functional GULO protein exhibit reduced caspase-3 activity compared to the liver organoid and / or mature liver organoid not containing the functional GULO protein.

146. The in vitro composition according to any one of claims 138 to 145, wherein the liver organoid and / or mature liver organoid containing the functional GULO protein expresses an increased level of ALB compared to the liver organoid and / or mature liver organoid that does not contain the functional GULO protein.

147. The in vitro composition according to any one of claims 138 to 146, wherein the liver organoid and / or mature liver organoid containing the functional GULO protein resembles periportal liver tissue and expresses a periportal liver marker.

148. The in vitro composition according to claim 147, wherein the periportal liver marker comprises FAH, ALB, PAH, CPS1, HGD, or any combination thereof.

149. The in vitro composition according to any one of claims 138 to 148, wherein the liver organoid and / or mature liver organoid containing the functional GULO protein exhibit increased CYP3A4 and CYP1A2 activity compared to the liver organoid and / or mature liver organoid not containing the functional GULO protein.

150. The in vitro composition according to any one of claims 138 to 149, wherein the liver organoid and / or mature liver organoid containing the functional GULO protein exhibits increased bilirubin conjugation activity compared to the liver organoid and / or mature liver organoid not containing the functional GULO protein.

151. The in vitro composition according to any one of claims 138 to 150, wherein the liver organoid and / or mature liver organoid containing the functional GULO protein exhibits an increased viability in culture compared to the liver organoid and / or mature liver organoid not containing the functional GULO protein.

152. The in vitro composition according to any one of claims 138 to 151, wherein the liver organoid and / or mature liver organoid comprises a functional GULO protein and / or a gene or mRNA encoding the functional GULO protein, or both, and is differentiated from the pluripotent stem cell, thereby enabling the pluripotent stem cell to synthesize ascorbic acid.

153. A method comprising administering a liver organoid or composition according to any one of claims 110 to 152 to a subject in need thereof.

154. A method for treating a subject in need of treatment for a liver-related disease or disorder, comprising administering one or more liver organoids or compositions described in claims 110 to 152 to the subject.

155. The method according to claim 153 or 154, wherein the liver organoid is produced from cells derived from the subject, and optionally, the cells derived from the subject are induced pluripotent stem cells.

156. The method according to claim 154 or 155, wherein administration includes transplanting the liver organoid or composition into the subject.

157. The method according to any one of claims 154 to 156, wherein the liver-related disease or disorder includes one or more of the following: liver dysfunction and / or liver failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic disease, autoimmune liver disease, Wilson's disease, metabolic-related fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Nadjar syndrome, urea cycle disorders, Wolmann disease, liver cancer, hepatoblastoma, metabolic dysfunction-related liver disease (MASLD), MetALD, metabolic dysfunction-related fatty liver disease (MASH), drug-induced liver injury (DILI), glycogen storage disorder, hemorrhagic disease, hepatic cyst, acetaminophen-induced acute liver injury, and / or alcohol-related liver disease.

158. The method according to any one of claims 154 to 157, wherein the liver dysfunction and / or liver failure includes hyperammonemia and / or hyperbilirubinemia, or the metabolic disorder includes non-alcoholic fatty liver disease (NAFLD), or the non-alcoholic fatty liver disease (NAFLD) includes metabolic dysfunction-associated steatohepatitis (MASH), or the hepatitis includes hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, hepatitis TT, and / or autoimmune hepatitis.

159. The method according to any one of claims 154 to 158, wherein the subject has reduced serum bilirubin and / or ammonia levels and / or increased serum proteinalbumin after transplantation.

160. The method according to any one of claims 154 to 159, wherein the subject has symptoms of improved bile duct stenosis and / or liver regeneration after transplantation.

161. The method according to any one of claims 154 to 160, wherein the subject has an increased survival rate after transplantation.

162. The method according to any one of claims 154 to 161, wherein the liver organoid engrafts in the target liver.

163. The method according to any one of claims 154 to 162, wherein the liver organoid is treated with amino acid (AA) supplementation.

164. The method according to any one of claims 154 to 163, wherein the liver organoid is treated with amino acid (AA) supplementation for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days or more prior to transplantation.

165. The method according to any one of claims 154 to 164, wherein the liver-related disease or disorder includes acetaminophen acute liver injury.

166. The method according to any one of claims 154 to 165, wherein the method can be used in a process compliant with Good Manufacturing Practices (GMP).

167. A method for screening, comprising contacting a liver organoid described in any one of claims 84 to 152 with a candidate compound or composition, and evaluating the effect of the candidate compound or composition on the liver organoid.

168. The method according to claim 167, wherein the liver organoid is a model of liver-related disease or disorder, and evaluating the effect of the candidate compound or composition on the liver organoid is equivalent to evaluating the effect of the candidate compound or composition on the liver-related disease or disorder.

169. The method according to claim 167 or 168, wherein the liver organoid is produced from cells derived from the subject, and optionally, the cells derived from the subject are induced pluripotent stem cells.

170. The method according to claim 169, wherein the subject has a liver-related disease or disorder.

171. The method according to any one of claims 167 to 170, wherein the method can be used in a process compliant with Good Manufacturing Practices (GMP).

172. A composition comprising the amino acid supplementation liquid components listed in Table 3.

173. A composition comprising a cocktail of growth factors according to the embodiments in Table 1 or Table 2.

174. A solution of non-essential amino acids in exactly or about 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 11 vol%, 12 vol%, 13 vol%, 14 vol%, or 15 vol% (containing exactly or about 890 mg / L alanine, 1320 mg / L asparagine, 1330 mg / L aspartic acid, 750 mg / L glycine, 105 mg / L serine, 1150 mg / L proline, and 1470 mg / L glutamic acid), A solution of essential amino acids in exactly or approximately 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 11 vol%, 12 vol%, 13 vol%, 14 vol%, or 15 vol% (approximately arginine 6320 mg / L, cysteine ​​1200 mg / L, histidine 2100 mg / L, isoleucine 2620 mg / L, leucine 2620 mg / L, lysine 3625 mg / L, methionine 755 mg / L, phenylalanine 1 mg / L) (containing 650 mg / L, threonine 2380 mg / L, tryptophan 510 mg / L, tyrosine 1800 mg / L, and valine 2340 mg / L), and exactly or about 65% by volume, 66% by volume, 67% by volume, 68% by volume, 69% by volume, 70% by volume, 71% by volume, 72% by volume, 73% by volume, 74% by volume, 75% by volume, 76% by volume, 77% by volume, 78% by volume, 79% by volume, 80% by volume, 81% by volume, 82% by volume, 83% by volume, A composition comprising 84% by volume, 85% by volume, 86% by volume, 87% by volume, 88% by volume, 89% by volume, or 90% by volume of hepatocyte culture medium (HCM), and further comprising an amino acid supplement liquid component supplemented with exactly or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg / mL of glycine.

175. Precisely or approximately 14% non-essential amino acid solution (containing precisely or approximately 890 mg / L alanine, 1320 mg / L asparagine, 1330 mg / L aspartic acid, 750 mg / L glycine, 105 mg / L serine, 1150 mg / L proline, and 1470 mg / L glutamic acid), precisely or approximately 6% by volume essential amino acid solution (precisely or approximately 6320 mg / L arginine, 1200 mg / L cysteine, 2100 mg / L histidine, 2620 mg / L isoleucine, The composition according to any one of claims 172 to 174, comprising an amino acid supplement liquid component comprising exactly or about 80 volume% of hepatocyte culture medium (HCM), and an amino acid supplement liquid component further supplemented with exactly or about 20 g / L of glycine.

176. The composition according to any one of claims 172 to 175, wherein the pH is approximately pH 6 to 8, or pH 6.5 to 7.5, or exactly or approximately pH 7.

0.

177. The composition according to any one of claims 172 to 176, further comprising hepatocyte growth factor (HGF), oncostatin M, dexamethasone, and / or ascorbic acid.

178. The composition according to any one of claims 172 to 177, further comprising liver lineage-determined cells differentiated from endoderm cells of an embryo using retinoic acid.

179. The composition according to claim 178, wherein the liver lineage-determined cells are characterized as liver organoids.

180. The composition according to claim 179, characterized in that the liver organoids secrete increased levels of albumin and urea compared to liver organoids contained in HCM without amino acid supplementation.

181. The composition according to claim 179 or 180, characterized in that the liver organoids express increased levels of liver maturation-related gene expression compared to liver organoids contained in HCM without amino acid supplementation.

182. The composition according to any one of claims 179 to 181, characterized in that the liver organoid expresses a reduced level of vimentin compared to liver organoids contained in HCM without amino acid supplementation.

183. The composition according to any one of claims 172 to 182, wherein the composition does not contain non-human animal components such that the basement membrane matrix or its components are heterogeneous to humans.

184. The composition according to claim 182, wherein the composition does not contain mouse Angelbreth-Holm-Swarm (EHS) sarcoma cells, Matrigel®, Cultrex®, and / or Geltrex®.

185. In vitro hyperbilirubinemia liver organoids containing naturally occurring and / or engineered mutations in the UDP-glucuronosyltransferase family 1 member A1 (UGT1A1) gene.

186. An in vitro hyperbilirubinemia liver organoid, wherein the hyperbilirubinemia liver organoid is produced by exposing progenitor cells, progenitor liver organoids, and / or progenitor mature liver organoids to exogenous bilirubin for at least two rounds.

187. The in vitro hyperbilirubinemia liver organoid according to claim 185 or 186, wherein the hyperbilirubinemia liver organoid is clonal and / or iPSC-derived.

188. A cryopreserved composition comprising liver organoids, chroman 1, emricasane, polyamine, and trans-ISRIB (CEPT).

189. A cryopreserved composition comprising mature liver organoids, chroman 1, emricasane, polyamine, and trans-ISRIB (CEPT).

190. A cryopreserved composition comprising hyperbilirubinemia liver organoids, chroman 1, emricasane, polyamine, and trans-ISRIB (CEPT).

191. A kit comprising means for carrying out the method described in any one of claims 1 to 83 or 153 to 171.

192. A kit comprising a composition, or means for producing the composition according to any one of claims 84-152, 172-184, or 188-190, or means for producing the liver organoid according to any one of claims 185-187.

193. Use of the method, composition, or kit according to any one of claims 1 to 192 as a medicine, a means for the treatment and / or prevention of disease, a means for diagnosis, and / or medical research.

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