Clinical grade organoids

Abstract

Disclosed herein are improved methods of expanding cell populations and methods of producing organoids from pluripotent stem cells, as well as compositions and uses thereof, compositions comprising liquid components that can be used to carry out these methods for supplementing amino acids, and kits comprising components for carrying out these methods and / or products of using these methods. These methods can be carried out without the need for xenogeneic and ambiguous basement membrane matrices for cell culture, which enables the achievement of good production specification (GMP) compliant methods for producing organoids for various uses such as clinical screening and treatment, methods for treating liver-related diseases or conditions and methods for screening candidate compounds or compositions for treating liver-related diseases or conditions are included.

Description

[0001] Statement on Federally Funded Research and Development This invention was completed with government support under licenses UH3DK119982-02 and DP2 DK128799-01 granted by the National Institutes of Health (NIH). The government holds certain rights to this invention. Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 493,269 entitled “CLINICAL-GRADE ORGANOIDS”, filed March 30, 2023, pursuant to 35 USC § 119(e), which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates in general to organoid compositions and methods for preparing organoid compositions. These methods can be performed without the use of xenogeneic basement membrane matrix 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, typically involves the use of a basement membrane matrix (also known as an extracellular matrix), which provides a biological niche including extracellular proteins and growth factors that support cell growth. A popular basement membrane matrix used for biological research is the extracellular matrix produced from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. However, this basement membrane matrix is ​​incompatible with human clinical purposes because it is composed of xenogeneic mouse components, is undefined (i.e., varies between batches), and may carry pathogens.

[0005] Therefore, there is a need for organoid compositions and other compounds that are not of xenogeneic origin and are involved in organoid production without using the basement membrane matrix. Summary of the Invention

[0006] Embodiments of this disclosure include methods for expanding hindbrain cells and / or hindbrain endoderm cells, the methods comprising: a) dissociating a hindbrain endoderm cell monolayer into hindbrain cells and / or hindbrain endoderm cells; b) seeding the hindbrain cells and / or hindbrain endoderm cells onto a tissue culture surface; and c) culturing the hindbrain cells and / or hindbrain 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, enzymatic dissociation and / or mechanical dissociation can be used to dissociate the foregut endoderm cell monolayer into hinforegut cells and / or hinforegut endoderm cells. In some embodiments, hinforegut cells and / or hinforegut endoderm cells can be separated at a ratio equal to or about 1 × 10⁻⁶. 5 1, 2×10 5 1, 3×10 5 1, 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 5 1×10 6 1, 2×10 6 1, 3×10 6 1, 4×10 6 One or 5×10 6 cells / cm 2 The cell density of the surface area of ​​the tissue culture surface, or any cell density having a range defined by any two of the aforementioned cell densities, is seeded onto the surface of the tissue container.

[0008] In some embodiments, the tissue culture surface is coated with a basement membrane matrix or a component thereof. In some embodiments, the basement membrane matrix or its component does not contain non-human animal components that would make the basement membrane matrix or its component xenogeneic to humans, optionally wherein the basement membrane matrix or its component is not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the basement membrane matrix or its component is not Matrigel ® Cultrex ® Or Geltrex ® In some embodiments, the basement membrane matrix or its components comprise human adhesion proteins, collagen IV, nestin, basement membrane glycans, fibrin, and / or hydrogel.

[0009] In some embodiments, hindgut cells and / or hindgut endoderm cells can be cultured until they can spontaneously form three-dimensional (3D) spheroids, optionally wherein the spheroids contain structures with a single lumen, and / or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells. In some embodiments, hindgut cells and / or hindgut endoderm cells can 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 selected from A83-01, RepSox, LY365947, and SB431542, optionally A83-01. In some embodiments, the TGF-β pathway inhibitor may be provided at a concentration equal to or about 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the TGF-β pathway inhibitor is provided at a concentration equal to or about 500 nM. In some embodiments, the FGF pathway activator may be selected from FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23, optionally FGF2. In some embodiments, the FGF pathway activator may be provided at a concentration equal to or about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, or 10 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the FGF pathway activator is provided at a concentration equal to or about 5 ng / mL. In some implementations, the Wnt pathway activator may be selected from Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8 and TWS119, optionally CHIR99021.In some embodiments, the Wnt pathway activator may be provided at a concentration equal to or about 1 µM, 1.5 µM, 2 µM, 2.5 µM, 3 µM, 3.5 µM, 4 µM, 4.5 µM, 5 µM, 5.5 µM, 6 µM, 6.5 µM, 7 µM, 7.5 µM, or 8 µM, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the Wnt pathway activator is provided at a concentration equal to or about 3 µM. In some embodiments, the VEGF pathway activator may be selected from the group consisting of VEGF or GS4012, optionally VEGF. In some embodiments, the VEGF pathway activator may be provided at a concentration equal to or about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the VEGF pathway activator is provided at a concentration equal to or about 10 ng / mL.

[0011] In some embodiments, the hindbrain cells and / or hindbrain endoderm cells of step c) may be cultured in a medium that also includes EGF, or in a medium that does not include EGF. In some embodiments, EGF may be provided at a concentration equal to or about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein EGF is provided at a concentration equal to or about 20 ng / mL. In some embodiments, the hindgut cells and / or hindgut endoderm cells of step c) may be cultured in a medium that also includes ascorbic acid, or in a medium that does not include ascorbic acid. In some embodiments, ascorbic acid may be provided at a concentration equal to or about 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL, or at any concentration within the range defined by any two of the foregoing concentrations, optionally wherein ascorbic acid is provided at a concentration equal to or about 50 µg / mL.

[0012] In some embodiments, the hindbrain cells and / or hindbrain endoderm cells of step c) may be cultured in a medium that also includes a ROCK inhibitor, or in a medium that does not contain the ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632. In some embodiments, the ROCK inhibitor may be provided at a concentration equal to or about 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, 9 µM, 10 µM, 11 µM, 12 µM, 13 µM, 14 µM, 15 µM, 16 µM, 17 µM, 18 µM, 19 µM, or 20 µM, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the ROCK inhibitor is provided at a concentration equal to or about 10 µM.

[0013] In some embodiments, the cells in step c) may be passaged once or multiple times. In some embodiments, the cells in step c) may be passaged until the hindgut cells and / or hindgut endoderm cells no longer spontaneously form spheroids. In some embodiments, the cells in step c) may be passaged no more than three times.

[0014] In some embodiments, hindbrain cells and / or hindbrain endoderm cells can be cultured, and the hindbrain cells and / or hindbrain endoderm cells can differentiate into liver organoids. In some embodiments, hindbrain cells and / or hindbrain endoderm cells can be cultured until they spontaneously form three-dimensional (3D) spheroids, optionally wherein the spheroids contain structures with a single lumen, and / or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells, and the hindbrain cells and / or hindbrain endoderm cells can be collected from the spheroids, optionally further including dissociating the spheroids into individual clumps of hindbrain cells and / or hindbrain endoderm cells and / or hindbrain cells and / or hindbrain endoderm cells prior to the differentiation step. In some embodiments, the foregut endoderm cells and / or the foregut endoderm cells are collected from the foregut endoderm cell monolayer prior to the differentiation step by dissociating the foregut endoderm cell monolayer into individual clumps of foregut and / or foregut endoderm cells and / or foregut and / or foregut endoderm cells.

[0015] Further embodiments of this disclosure include methods for differentiating hindbrain cells and / or hindbrain endoderm cells into liver organoids, the methods comprising: i) contacting hindbrain cells and / or hindbrain endoderm cells with a retinoic acid pathway activator, said hindbrain cells and / or hindbrain endoderm cells optionally in the form of spheroids, optionally in the form of individual cells or cell clusters detached from spheroids, and / or optionally as cells aggregated in micropores or other devices as described herein, optionally wherein the spheroids contain structures having a single lumen, and / or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells; and ii) contacting the cells of step i) with a culture medium for a period of time, thereby differentiating the hindbrain cells and / or hindbrain endoderm cells into liver organoids, optionally wherein the culture medium is a hepatocyte culture medium.

[0016] In some embodiments, the culture medium is supplemented with a cMET tyrosine kinase receptor agonist, IL-6 family cytokines, and corticosteroids. In some embodiments, the cMET tyrosine kinase receptor agonist may be selected from hepatocyte growth factor (HGF), PG-001, fosgonimeton, terevalefim, recombinant InlB321 protein, and agonistic c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokines may be selected from IL-6, oncogene 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 the group consisting of dexamethasone, beclomethasone, betamethasone, fluclomethasone, halometasone, 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, the foregut cells and / or foregut endoderm cells may comprise foregut cells and / or foregut endoderm cells produced by the methods described above. In some embodiments, the foregut cells and / or foregut endoderm cells may be in the form of globular bodies or individual foregut cells and / or foregut endoderm cells, and / or in the form of clumps of foregut cells and / or foregut endoderm cells derived from dissociated globular bodies, optionally wherein the globular bodies contain structures having a single lumen, and / or wherein the globular bodies 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, BS 493, TTNPB, and AM580, optionally retinoic acid. In some embodiments, the retinoic acid pathway activator may be provided at concentrations of 1.0 µM, 1.1 µM, 1.2 µM, 1.3 µM, 1.4 µM, 1.5 µM, 1.6 µM, 1.7 µM, 1.8 µM, 1.9 µM, 2.0 µM, 2.1 µM, 2.2 µM, 2.3 µM, 2.4 µM, 2.5 µM, 2.6 µM, 2.7 µM, 2.8 µM, 2.9 µM, or 3.0 µM, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the retinoic acid pathway activator may be provided at a concentration equal to or about 2.0 µM. In some embodiments, HGF may be provided at a concentration equal to or about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein HGF is provided at a concentration equal to or about 10 ng / mL. In some embodiments, OSM is provided at a concentration equal to or about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein OSM is provided at a concentration equal to or about 20 ng / mL. In some embodiments, dexamethasone may be provided at concentrations equal to or about 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein dexamethasone is provided at a concentration equal to or about 100 nM. In some embodiments, the cells of step i) and / or step ii) are not contacted with EGF.

[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 comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of non-essential amino acids by total volume, or non-essential amino acids within the range defined by any two of the foregoing values, optionally wherein the supplemented growth medium comprises about 4%-10%, 6%-12%, 10%-16%, 12%-15%, 13%-19% by total volume, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% of non-essential amino acids. In some embodiments, the supplemented growth medium comprises 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of essential amino acids by total volume, or essential amino acids within the range defined by any two of the foregoing values, optionally wherein the supplemented growth medium comprises about 4%-10%, 6%-12%, 10%-16%, 12%-15%, 13%-19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, or 16% of essential amino acids by total volume. In some embodiments, the supplemented glycine may be provided at a concentration equal to or about 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, or 35 mg / mL, or at any concentration within the range defined by any two of the foregoing concentrations, optionally wherein the supplemented glycine is provided at a concentration equal to or about 18 mg / mL to 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, wherein the resulting liver organoids are mature liver organoids. In some embodiments, the low / first concentration of bilirubin may be bilirubin at physiological concentrations in human fetuses. In some embodiments, the low / first concentration of bilirubin may be, may be about, may be less than, or may be less than about: a) 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, 1.25 mg / L, 1.5 mg / L, 1.75 mg / L, 2.0 mg / L, 2.25 mg / L, 2.5 mg / L, 2.75 mg / L, or 3.0 mg / L, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 0.1 mg / L to 3 mg / L, 0.5 mg / L to 2.0 mg / L, 0.5 mg / L to 1.5 mg / L, 0.3 mg / L to 2.5 mg / L, or 0.5 mg / L to 1.75 mg / L); or b) Any concentration within the range defined by any two of the foregoing concentrations (e.g., 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, or 1 mg / L), or any concentration within the range defined by any two of the foregoing concentrations (e.g., 0.1 mg / L to 1 mg / L, 0.1 mg / L to 0.5 mg / L, 0.5 mg / L to 1 mg / L, 0.3 mg / L to 0.7 mg / L, or 0.4 mg / L to 0.6 mg / L).

[0021] In some embodiments, mature liver organoids may exhibit luminal projections similar to bile canaliculi, and / or have a single lumen and generally a spherical shape, and / or contain no hematopoietic tissue or acquired immune cells. In some embodiments, compared to liver organoids not exposed to a low / first dose of bilirubin, mature liver organoids may express reduced levels of AFP, CDX2, NANOG, or any combination thereof. In some embodiments, compared to liver organoids not exposed to a low / first dose of bilirubin, mature liver organoids may express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof. In some embodiments, mature liver organoids may express CYP2E1, CYP7A1, PROX1, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, compared to liver organoids not exposed to a low / first dose of bilirubin, mature liver organoids may exhibit increased CYP3A4 and CYP1A2 activity.

[0022] In some implementations, the cells in step ii) may be further exposed to a high / second concentration of bilirubin, wherein the resulting liver organoids are hyperbilirubinemic liver organoids. In some embodiments, the high / second concentration of bilirubin can be, can be about, can be greater than, or can be greater than about: a) 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 2 mg / L to 20 mg / L, 2 mg / L to 10 mg / L, 10 mg / L to 20 mg / L, 5 mg / L to 15 mg / L, or 8 mg / L to 12 mg / L); or b) Concentrations of 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L, or any range defined by any two of the foregoing concentrations (e.g., 4 mg / L to 20 mg / L, 2 mg / L to 10 mg / L, 10 mg / L to 20 mg / L, 5 mg / L to 15 mg / L, or 8 mg / L to 12 mg / L). In some embodiments, relative to liver organoids not treated with a high / second concentration of bilirubin, hyperbilirubinemic liver organoids may express elevated levels of UGT1A1 or NRF2 or both.

[0023] In some embodiments, liver organoids may include a functional L-gulonolactone oxidase (GULO) protein and / or a gene or mRNA encoding the functional GULO protein, or both, wherein the liver organoids are capable of synthesizing ascorbic acid. In some embodiments, the functional GULO protein may be mouse GULO (mGULO). In some embodiments, a tetracycline-inducible system may be optionally used to conditionally express the gene encoding the functional GULO protein. In some embodiments, liver organoids may be genetically engineered using CRISPR to encode the functional GULO protein. In some embodiments, the gene or mRNA encoding the functional GULO protein, or both, may be introduced into the liver organoids via transfection. In some embodiments, liver organoids including the functional GULO protein may express increased levels of NRF2 compared to liver organoids that do not include the functional GULO protein. In some embodiments, liver organoids containing functional GULO proteins may optionally express reduced levels of IL1β, IL6, or TNFα, or any combination thereof, when cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid, compared to liver organoids that do not contain functional GULO proteins. In some embodiments, liver organoids containing functional GULO proteins may optionally exhibit reduced caspase-3 activity when cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid, compared to liver organoids that do not contain functional GULO proteins. In some embodiments, liver organoids containing functional GULO proteins may express increased levels of ALB, compared to liver organoids that do not contain functional GULO proteins. In some embodiments, liver organoids containing functional GULO proteins may resemble periportal liver tissue and express periportal liver markers. In some embodiments, periportal liver markers may include FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, liver organoids including functional GULO proteins may exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids that do not include functional GULO proteins. In some embodiments, liver organoids including functional GULO proteins may exhibit increased bilirubin conjugation activity compared to liver organoids that do not include functional GULO proteins. In some embodiments, liver organoids including functional GULO proteins may exhibit increased viability in culture compared to liver organoids that do not include functional GULO proteins. In some embodiments, the liver organoids have been differentiated from pluripotent stem cells that include functional GULO proteins and / or genes or mRNA encoding functional GULO proteins, or both, thereby enabling the pluripotent stem cells to synthesize ascorbic acid.In some implementations, liver organoids include an inactive UGT1A1 gene, wherein the liver organoids can be used as a model of Crigler-Najjar syndrome.

[0024] In some embodiments, prior to step i), hindforegut cells and / or hindforegut endoderm cells may be aggregated in micropores or other devices (e.g., Aggrewell), wherein aggregation of hindforegut cells and / or hindforegut endoderm cells may produce liver organoids of more uniform size. In some embodiments, the cells of steps i) and / or ii) are not cultured with basement membrane matrix or components thereof, optionally wherein the cells of steps i) and / or ii) are not cultured with human xenogeneic basement membrane matrix or components thereof, optionally wherein the cells of steps i) and / or ii) are not cultured with basement membrane matrix or components thereof isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the cells of steps i) and / or ii) are not cultured with Matrigel ® Cultrex ® Or Geltrex ® Contact. In some embodiments, the cells of step i) and / or step ii) and / or the liver organoids formed therefrom can be cultured in a static or non-static bioreactor, optionally a rotating bioreactor, optionally a 3D bioreactor, optionally a 3D rotating bioreactor. In some embodiments, after culturing in a static or non-static bioreactor, the liver organoids can be dissociated into single cells and can subsequently be reconstituted and / or expanded via additional culture steps in a static or non-static bioreactor, optionally a 3D bioreactor, optionally a 3D rotating bioreactor. In some embodiments, these methods also include cryopreservation of liver organoids. In some embodiments, cryopreservation of liver organoids includes slow freezing or vitrification, optionally wherein the liver organoids can be cryopreserved with chroman I, emricasan, polyamines, and trans-ISRIB (CEPT).

[0025] In some embodiments, the foregut cells and / or foregut endoderm cells are derived from pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells. In some embodiments, the foregut cells and / or foregut endoderm cells are derived from a subject, optionally a subject suffering from liver-related disease or condition. In some embodiments, the method can be used in a Good Manufacturing Practice (GMP) compliant process. Embodiments of this disclosure also include foregut cells and / or foregut endoderm cells or liver organoids generated by any of the foregoing methods.

[0026] Further embodiments of this disclosure include in vitro compositions comprising: pluripotent stem cells, shaped endoderm, foregut endoderm, ventral foregut endoderm and / or downstream hepatocyte types, 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.

[0027] In some embodiments, the composition comprises hindbrain cells and / or hindbrain endoderm cells, and the hindbrain cells and / or hindbrain endoderm cells are dissociated hindbrain cells and / or hindbrain endoderm cells. In some embodiments, the hindbrain cells and / or hindbrain endoderm cells may be greater than or equal to, exactly or about 1 × 10⁻⁶ cells. 5 1, 2×10 5 1, 3×10 5 1, 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 5 1×10 6 1, 2×10 6 1, 3×10 6 1, 4×10 6 One or 5×10 6 cells / cm 2 The cell density of the surface area of ​​the tissue culture surface, or any cell density within a range defined by any two of the aforementioned cell densities. In some embodiments, the tissue culture surface may be coated with a basement membrane matrix or a component thereof. In some embodiments, the basement membrane matrix or its component does not contain non-human animal components that would make the basement membrane matrix or its component xenogeneic to humans, optionally wherein the basement membrane matrix or its component is not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the basement membrane matrix or its component is not Matrigel ® Cultrex ® Or Geltrex ® In some embodiments, the basement membrane matrix or its components comprise human adhesion proteins, collagen IV, nestin, basement membrane glycans, fibrin, and / or hydrogel.

[0028] In some embodiments, at least a portion of the hindbrain cells and / or hindbrain endoderm cells may be spontaneously formed three-dimensional (3D) spheroids, optionally wherein the spheroids contain structures with a single lumen, and / or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells. In some embodiments, the TGF-β pathway inhibitor may be selected from A83-01, RepSox, LY365947, and SB431542; optionally wherein the TGF-β pathway inhibitor includes or is A83-01. In some embodiments, the TGF-β pathway inhibitor may have a concentration equal to or about 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the TGF-β pathway inhibitor has a concentration equal to or about 500 nM. In some embodiments, the FGF pathway activator may be selected from FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23, optionally wherein the FGF pathway activator includes or is FGF2. In some embodiments, the FGF pathway activator may have a concentration equal to or about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, or 10 ng / mL, or any concentration within the range defined by any two of the foregoing concentrations, optionally wherein the FGF pathway activator has a concentration equal to or about 5 ng / mL. In some embodiments, the Wnt pathway activator may be selected from Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, alloxan, indirubin, atporon, tamparone, lithium chloride, TDZD 8, and TWS119, optionally including or being CHIR99021 as the Wnt pathway activator.In some embodiments, the Wnt pathway activator may have a concentration equal to or about 1 µM, 1.5 µM, 2 µM, 2.5 µM, 3 µM, 3.5 µM, 4 µM, 4.5 µM, 5 µM, 5.5 µM, 6 µM, 6.5 µM, 7 µM, 7.5 µM, or 8 µM, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the Wnt pathway activator has a concentration equal to or about 3 µM. In some embodiments, the VEGF pathway activator may be selected from VEGF or GS4012, optionally wherein the VEGF pathway activator includes or is VEGF. In some embodiments, the VEGF pathway activator may have a concentration equal to or about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the VEGF pathway activator has a concentration equal to or about 10 ng / mL. In some embodiments, the composition further includes exogenous EGF, or the composition may not include exogenous EGF. In some embodiments, EGF may have a concentration equal to or about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the EGF has a concentration equal to or about 20 ng / mL. In some embodiments, the composition further includes exogenous ascorbic acid and / or genetically modified ascorbic acid, or the composition does not include exogenous ascorbic acid and / or genetically modified ascorbic acid. In some embodiments, ascorbic acid may have a concentration equal to or about 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL or 100 μg / mL, or any concentration within the range defined by any two of the foregoing concentrations, optionally wherein ascorbic acid has a concentration equal to or about 50 μg / mL.In some embodiments, the composition may further include a ROCK inhibitor, or may be cultured in a medium that does not contain a ROCK inhibitor, optionally wherein the ROCK inhibitor includes or is Y-27632. In some embodiments, the ROCK inhibitor may have a concentration equal to or about 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, 9 µM, 10 µM, 11 µM, 12 µM, 13 µM, 14 µM, 15 µM, 16 µM, 17 µM, 18 µM, 19 µM, or 20 µM, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the ROCK inhibitor has a concentration equal to or about 10 µM.

[0029] In some embodiments, hindbrain cells and / or hindbrain endoderm cells, defined endoderm, ventral foregut endoderm, and / or downstream hepatocytes can differentiate from stem cells. In some embodiments, hindbrain cells and / or hindbrain endoderm cells, defined endoderm, ventral foregut endoderm, and / or downstream hepatocytes can differentiate from induced pluripotent stem cells. In some embodiments, hindbrain cells and / or hindbrain endoderm cells, defined endoderm, ventral foregut endoderm, and / or downstream hepatocytes have been passaged less than four times. In some embodiments, the cells comprise hindbrain cells and / or hindbrain endoderm cells or are substantially composed of hindbrain cells and / or hindbrain 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 this disclosure include liver organoids produced by any of the foregoing methods.

[0031] Further embodiments of this disclosure include in vitro compositions comprising: a) hindgut cells and / or hindgut 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; and wherein the composition optionally additionally 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, forgonidone, trevallefime, recombinant InlB321 protein, and agonistic c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokines may be selected from IL-6, oncogene 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, fluclomethasone, halometasone, 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, BS 493, TTNPB, and AM580, optionally wherein the retinoic acid pathway activator includes or is retinoic acid. In some embodiments, the retinoic acid pathway activator may be at a concentration of 1.0 µM, 1.1 µM, 1.2 µM, 1.3 µM, 1.4 µM, 1.5 µM, 1.6 µM, 1.7 µM, 1.8 µM, 1.9 µM, 2.0 µM, 2.1 µM, 2.2 µM, 2.3 µM, 2.4 µM, 2.5 µM, 2.6 µM, 2.7 µM, 2.8 µM, 2.9 µM, or 3.0 µM, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the retinoic acid pathway activator has a concentration equal to or about 2.0 µM. In some embodiments, HGF may have a concentration equal to or about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein HGF has a concentration equal to or about 10 ng / mL.In some embodiments, OSM may have a concentration equal to or about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein OSM has a concentration equal to or about 20 ng / mL. In some embodiments, dexamethasone may have a concentration equal to or about 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein dexamethasone has a concentration equal to or about 100 nM. In some embodiments, the composition does not include exogenous EGF.

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

[0033] Further embodiments of this disclosure include in vitro compositions comprising mature liver organoids, wherein the cells of the mature liver organoids are contacted with a low dose of bilirubin, optionally wherein the low dose of bilirubin is exogenously provided, and the mature liver organoids exhibit luminal projections similar to bile ducts, and / or have a single lumen and generally a spherical shape, and / or wherein the mature liver organoids do not contain hematopoietic tissue and acquired immune cells. In some embodiments, compared to liver organoids in which the cells are not contacted with a low dose of bilirubin, the mature liver organoids may express reduced levels of AFP, CDX2, NANOG, or any combination thereof. In some embodiments, compared to liver organoids in which the cells are not contacted with a low dose of bilirubin, the mature liver organoids may express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof. In some embodiments, the mature liver organoids may express CYP2E1, CYP7A1, PROX1, MRP3, MRP3, or OATP2, or any combination thereof. In some implementations, mature liver organoids may exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids in which cells have not been exposed to low doses of bilirubin.

[0034] In some embodiments, the composition further includes hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoid cells are contacted with a high concentration and / or a second concentration of bilirubin. In some embodiments, the high / second concentration of bilirubin is, is about, greater than, or greater than about: 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L, or a range defined by any two of the aforementioned concentrations (e.g., 2 mg / L to 20 mg / L, 2 mg / L to 10 mg / L, 10 mg / L to 20 mg / L, 5 mg / L to 15 mg / L). Any concentration within the range of 4 mg / L or 8 mg / L to 12 mg / L; or any concentration within the range of 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L, or any range defined by any two of the aforementioned concentrations (e.g., 4 mg / L to 20 mg / L, 2 mg / L to 10 mg / L, 10 mg / L to 20 mg / L, 5 mg / L to 15 mg / L, or 8 mg / L to 12 mg / L). In some embodiments, relative to liver organoids not treated with a high / second concentration of bilirubin, hyperbilirubinemic liver organoids may express elevated levels of UGT1A1 or NRF2 or both. In some embodiments, the foregut cells and / or foregut endoderm cells, liver organoids and / or mature liver organoids include a functional L-gulonolactone oxidase (GULO) protein and / or a gene or mRNA encoding the functional GULO protein, or both, wherein the foregut cells and / or 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, a tetracycline-inducible system is optionally used to conditionally express the gene encoding the functional GULO protein. In some embodiments, foregut cells and / or foregut endoderm cells, liver organoids and / or mature liver organoids can be engineered using CRISPR to include a gene encoding the functional GULO protein. In some embodiments, the gene or mRNA encoding the functional GULO protein, or both, is introduced into the liver organoids via transfection.In some embodiments, liver organoids including the functional GULO protein and / or mature liver organoids may express increased levels of NRF2 compared to liver organoids and / or mature liver organoids that do not contain the functional GULO protein. In some embodiments, liver organoids including the functional GULO protein and / or mature liver organoids may express decreased levels of IL1β, IL6, or TNFα, or any combination thereof, compared to liver organoids and / or mature liver organoids that do not contain the functional GULO protein. In some embodiments, liver organoids including the functional GULO protein and / or mature liver organoids may exhibit decreased caspase-3 activity compared to liver organoids and / or mature liver organoids that do not contain the functional GULO protein. In some embodiments, liver organoids including the functional GULO protein and / or mature liver organoids may express increased levels of ALB compared to liver organoids and / or mature liver organoids that do not contain the functional GULO protein. In some embodiments, liver organoids and / or mature liver organoids including functional GULO proteins may resemble periportal liver tissue and express periportal liver markers. In some embodiments, periportal liver markers may include FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, liver organoids including functional GULO proteins and / or mature liver organoids may exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids and / or mature liver organoids that do not contain functional GULO proteins. In some embodiments, liver organoids including functional GULO proteins and / or mature liver organoids may exhibit increased bilirubin conjugation activity compared to liver organoids and / or mature liver organoids that do not contain functional GULO proteins. In some embodiments, liver organoids including functional GULO proteins and / or mature liver organoids may exhibit increased viability in culture compared to liver organoids and / or mature liver organoids that do not contain functional GULO proteins. In some implementations, liver organoids and / or mature liver organoids have been differentiated from pluripotent stem cells, which include functional GULO proteins and / or genes or mRNAs encoding functional GULO proteins or both, thereby enabling the pluripotent stem cells to synthesize ascorbic acid.

[0035] Further embodiments of this disclosure include methods for administering the aforementioned liver organoids or compositions to a subject in need, and methods for treating liver-related diseases or conditions in a subject in need, the methods including administering one or more of the aforementioned liver organoids or compositions to the subject.

[0036] In some embodiments, the liver organoids have been generated from cells derived from the subject, optionally wherein 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 conditions include one or more types of liver dysfunction and / or failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic diseases, autoimmune liver diseases, Wilson's disease, metabolic-associated fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Najjar syndrome, urea cycle disorder, Wolman's disease, hepatocellular carcinoma, hepatoblastoma, metabolic dysfunction-associated liver disease (MASLD), MetALD, metabolic dysfunction-associated steatohepatitis (MASH), drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic diseases, liver cysts, acetaminophen-induced acute liver injury, and / or alcohol-related liver disease. In some embodiments, liver dysfunction and / or failure includes hyperammonemia and / or hyperbilirubinemia; or metabolic diseases including non-alcoholic fatty liver disease (NAFLD); or NAFLD including metabolic dysfunction-associated steatohepatitis (MASH); or hepatitis including hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, TT hepatitis, and / or autoimmune hepatitis. In some embodiments, the subject may have decreased serum bilirubin and / or ammonia levels and / or increased serum protein levels after transplantation. In some embodiments, the subject may have improved bile duct stricture and / or liver regeneration symptoms after transplantation. In some embodiments, the subject may have increased survival rates after transplantation. In some embodiments, liver organoids are implanted into the subject's liver. In some embodiments, the liver organoids have been treated with amino acid (AA) supplements. In some embodiments, the liver organoids are treated with amino acid (AA) supplements for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to transplantation. In some embodiments, liver-related diseases or conditions include acetaminophen-induced acute liver injury. In some embodiments, this method can be used in processes conforming to Good Manufacturing Practices (GMP).

[0037] Further embodiments of this disclosure include methods for screening, which include contacting the aforementioned liver organoids with candidate compounds or compositions and evaluating the effects of the candidate compounds or compositions on the liver organoids. In some embodiments, the liver organoids may be models of liver-related diseases or conditions, and determining the effects of candidate compounds or compositions on the liver organoids includes evaluating the effects of candidate compounds or compositions on liver-related diseases or conditions. In some embodiments, the liver organoids have been generated from cells derived from a subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells. In some embodiments, the subject suffers from a liver-related disease or condition. In some embodiments, the method may be used in a Good Manufacturing Practice (GMP) compliant process.

[0038] Further embodiments of this disclosure include compositions comprising a liquid component of supplementary amino acids according to Table 3. Additional embodiments include compositions comprising a mixture of growth factors according to the embodiments of Table 1 or Table 2.

[0039] Further embodiments of this disclosure include compositions comprising a liquid component containing supplemental amino acids, the liquid component comprising exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by volume a solution of non-essential amino acids (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), by volume A solution containing exactly or approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of essential amino acids (containing exactly or approximately 6320 mg / L arginine, 1200 mg / L cysteine, 2100 mg / L histidine, 2620 mg / L isoleucine, 2620 mg / L leucine, 3625 mg / L lysine, 755 mg / L methionine, 1650 mg / L phenylalanine, 2380 mg / L threonine, 510 mg / L tryptophan, 1800 mg / L tyrosine, and 2... 340 mg / L valine) and hepatocyte culture medium (HCM) at exactly or approximately 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% by volume, supplemented with exactly or approximately 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, or 11 mg / mL. , 12mg / mL, 13mg / mL, 14mg / mL, 15mg / mL, 16mg / mL, 17mg / mL, 18mg / mL, 19mg / mL, 20mg / mL, 21mg / mL, 22mg / mL, 23mg / mL, 2 4mg / mL, 25mg / mL, 26mg / mL, 27mg / mL, 28mg / mL, 29mg / mL, 30mg / mL, 31mg / mL, 32mg / mL, 33mg / mL, 34mg / mL or 35mg / mL glycine.In some embodiments, the composition comprises a liquid component for supplementing amino acids, which includes exactly or about 14% of a solution of non-essential amino acids (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% by volume of a solution of essential amino acids (containing exactly or about 6320 mg / L arginine). The composition contains (acid, 1200 mg / L cysteine, 2100 mg / L histidine, 2620 mg / L isoleucine, 2620 mg / L leucine, 3625 mg / L lysine, 755 mg / L methionine, 1650 mg / L phenylalanine, 2380 mg / L threonine, 510 mg / L tryptophan, 1800 mg / L tyrosine, and 2340 mg / L valine) and hepatocyte culture medium (HCM) at exactly or about 80% by volume, supplemented with exactly or about 20 g / L glycine. In some embodiments, the pH is between about pH 6 and 8, or between pH 6.5 and 7.5, or exactly or about pH 7.0. In some embodiments, the composition further comprises hepatocyte growth factor (HGF), oncogene M, dexamethasone, and / or ascorbic acid. In some embodiments, the composition further comprises hepatic lineage-typed cells differentiated from morphologically defined endoderm cells using retinoic acid. In some implementations, liver lineage-typed cells are characterized as liver organoids.

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

[0041] In some embodiments, the composition does not contain non-human animal components that make the basement membrane matrix or its components xenogeneic 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 this disclosure include in vitro hyperbilirubinemia liver organoids, which include UDP-glucuronyltransferase family 1 member A1 (…UGT1A1 The liver organoids are naturally occurring and / or engineered mutations in the iPSC. In some embodiments, hyperbilirubinemia liver organoids are produced by exogenous bilirubin through at least two rounds of contacting precursor cells, precursor liver organoids, and / or precursor mature liver organoids. In some embodiments, the hyperbilirubinemia liver organoids are clonal and / or derived from iPSCs.

[0043] Further embodiments of this disclosure include: compositions comprising cryopreserved liver organoids, chroman I, enledgeridine, polyamines, and trans-ISRIB (CEPT), and / or compositions comprising cryopreserved mature liver organoids, chroman I, enledgeridine, polyamines, and trans-ISRIB (CEPT), and / or compositions comprising cryopreserved hyperbilirubinemia liver organoids, chroman I, enledgeridine, polyamines, and trans-ISRIB (CEPT).

[0044] Further embodiments of this disclosure include: kits comprising tools for performing any of the foregoing methods; and / or kits comprising compositions or tools for producing any of the foregoing compositions; and / or kits for producing any of the foregoing liver organoids. Further embodiments of this disclosure include the use of these methods, compositions, or kits as pharmaceuticals, tools for treating and / or preventing diseases, diagnostic tools, and / or for medical research. Attached Figure Description

[0045] In addition to the features described herein, other features and variations will become apparent from the following description of the accompanying drawings and exemplary embodiments. It should be understood that these drawings depict embodiments and are not intended to limit the scope.

[0046] Figure 1A An implementation scheme for cryopreserving liver organoids and optional subsequent steps is illustrated.

[0047] Figure 1B An implementation scheme depicting fluorescence microscopy images of liver organoids, which have been frozen and thawed (compared to non-frozen organoids) using slow freezing and vitrification cryopreservation methods to observe the abundance of live cells (labeled with calcein AM) and dead cells (labeled with ethidium homodimer-1).

[0048] Figure 1C A quantitative implementation scheme for albumin secretion in liver organoids that have been frozen and thawed using slow freezing and vitrification cryopreservation methods is described (compared to non-frozen organoids).

[0049] Figure 2A depicts liver organoids cultured with amino acid (AA) supplemented medium at different culture times. ALB , CYP3A4 , PCK1 and G6PC Implementation scheme for RT-qPCR quantification of expression.

[0050] Figure 2B illustrates the implementation scheme for quantifying albumin secretion and urea production in liver organoids cultured with AA-supplemented medium at different culture times.

[0051] Figure 2C A schematic implementation of an embodiment depicting the detection of activities, such as PCK1 activity, using live cells or cell lysates in liver organoids grown in AA-supplemented medium using a luciferase reporter factor method.

[0052] Figure 2D An implementation scheme depicting bright-field microscopy images of organoids grown in AA-supplemented medium and standard hepatocyte culture medium (HCM) is described.

[0053] Figure 2E illustrates the implementation scheme for quantifying PCK1 expression in liver organoids grown in AA-supplemented medium, as measured by luciferase reporter factor, in live cell and cell lysate assays, compared to liver organoids grown in standard hepatocyte culture medium.

[0054] Figure 3A illustrates a schematic implementation scheme for two-dimensional (2D) hepatocyte differentiation from pluripotent stem cells and cultures using AA-supplemented culture medium added at different times during culture.

[0055] Figure 3B An embodiment depicting bright-field images of 2D hepatocyte cultures grown with AA-supplemented medium at different culture times is described.

[0056] Figure 3C An implementation scheme for quantifying lactate production in 2D hepatocytes grown in AA-supplemented medium, compared to those grown in standard hepatocyte culture medium, is described.

[0057] Figure 3D An implementation scheme for quantifying albumin secretion in 2D hepatocytes grown in AA-supplemented medium at different culture times, compared to those grown in standard hepatocyte culture medium, is described.

[0058] Figure 3E An implementation scheme for quantifying albumin secretion in 2D hepatocytes grown long-term in AA-supplemented medium is described, wherein the albumin secretion rate is normalized relative to the rate of hepatocytes grown in standard hepatocyte medium.

[0059] Figure 3F depicts 2D hepatocytes grown in AA-supplemented medium at different culture times compared to those grown in standard hepatocyte culture medium. ALB , E- cadherin, CYP3A4 , G6PC , PKM and PCK1 An implementation plan for RT-qPCR quantification of gene expression.

[0060] Figure 3G Fluorescence and bright-field microscopy images of engineered 2D hepatocytes expressing mScarlet under PCK1 reporter factor were depicted, compared with those grown in standard hepatocyte culture medium with or without insulin starvation, and with AA-supplemented culture medium with or without insulin starvation.

[0061] Figure 3H An embodiment is described that describes a fluorescence microscopy image of the expression of EpCAM (epithelial cells), vimentin (mesenchymal cells), and DAPI or HNF4α (nucleus) in 2D hepatocytes grown in AA-supplemented medium compared with those grown in standard hepatocyte culture medium.

[0062] Figure 4A Depicting the use of Matrigel ® A schematic diagram of an implementation scheme for culturing liver organoids in the presence of other basement membrane matrices with heterogeneous components.

[0063] Figure 4B An embodiment depicting a bright-field image of spontaneously forming 3D spheroids from a foregut cell monolayer is described, wherein the spheroids can be transferred to mature into liver organoids without the use of Matrigel. ® Or other basement membrane matrices with heterogeneous components.

[0064] Figure 4C Depicting the display without Matrigel ® An implementation scheme for bright-field and fluorescence microscopy images of organoids grown under specific conditions, exhibiting normal organoid morphology.

[0065] Figure 4D Depicting in the absence of Matrigel ® Organoids grown under specific conditions, according to previous protocols in Matrigel ® A quantitative implementation scheme for comparing albumin secretion between organoids and 2D hepatocyte cultures grown under certain conditions.

[0066] Figure 4E Depicting in the absence of Matrigel® Organoids grown under specific conditions, according to previous protocols in Matrigel ® Between organoids grown under certain conditions and 2D hepatocyte cultures ALB , AFP , HNF4α , RBP4 and AAT Implementation scheme for RT-qPCR quantification of expression.

[0067] Figure 5A An implementation scheme is illustrated, showing the passage and expansion of foregut cells after pluripotent stem cell differentiation for large-scale organoid production.

[0068] Figure 5B The image depicts plate inoculation on laminin or Matrigel. ® Implementation scheme of bright-field microscopy images of foregut cell growth (days 1-7) and spontaneous formation of spheroids on coated plates.

[0069] Figure 5C An embodiment depicting bright-field microscopic images showing further growth of foregut cells (days 8–10) and complete formation of globules, wherein the cells are seeded onto Matrigel plates. ® Cells seeded on a plate coated with laminin appeared to induce more efficient spheroid formation compared to cells on a plate coated with laminin.

[0070] Figure 5D A bright-field microscopy image depicting the growth (days 1-5) of foregut cells and the spontaneous formation of spheroids when plates are seeded at different seeding densities on laminin-coated plates is described.

[0071] Figure 5E An embodiment is described that shows bright-field microscopy images of additional spheroids produced from additional cultures as spheroids are collected from the initial spontaneous formation of foregut cells.

[0072] Figure 5F depicts a bright-field microscopy image and quantitative implementation of the total potential cell number obtainable through multiple passages of foregut cells initially differentiated from pluripotent stem cells. Globules form during passages 1–3, not from passage 4 foregut cells.

[0073] Figure 5G depicts foregut cells from generations 1-4. CDX2 , FOXA2 , AFP , VIM , SOX17 , HNF4α and ALB Implementation scheme for RT-qPCR quantification of expression.

[0074] Figure 5H An implementation scheme depicting liver organoid formation starting from pluripotent stem cells and including schematic diagrams for large-scale foregut cell passage is described.

[0075] Figure 5I The illustration depicts an embodiment of liver organoid formation starting from pluripotent stem cells and including foreguinal cell passage for large-scale processing, as well as a schematic representation of using a device to aggregate foreguinal cells to improve the uniformity of organoid size and shape.

[0076] Figure 6A Depicting in the absence of Matrigel ® A schematic diagram of the implementation scheme for 3D rotational culture of liver organoids grown under certain conditions.

[0077] Figure 6B The text describes how liver organoids can be cultured using 3D rotation in Matrigel-free environments. ® An implementation scheme for growing bright-field images under certain conditions.

[0078] Figure 6C depicts liver organoids grown in 3D rotation culture compared to first-generation foregut cells. AFP , HNF4α , FOXA2 , ALB , CDX2 and VIM The implementation plan for RT-qPCR quantification.

[0079] Figure 7A An implementation scheme for generating HLO and ripening with low doses of bilirubin is illustrated.

[0080] Figure 7B An embodiment of HLO treated with a low dose of bilirubin (1 mg / L) compared to a control is depicted in bright-field image, and the lumen outline is shown using ImageJ, with arrows indicating luminal protrusions similar to bile canaliculi found in human livers.

[0081] Figure 7C depicts an embodiment comparing the lumen size and roundness of HLOs in control and 1 mg / L bilirubin-treated HLOs.

[0082] Figure 7D depicts the immature marker genes and maturation marker genes of control organoids and organoids treated with 1 mg / L bilirubin compared to human liver samples. ALB , NANOG , SLC4A2 , HO-1 , AFP and CDX2 ) RT-qPCR implementation plan.

[0083] Figure 7E illustrates the implementation scheme for the assay of CYP3A4 and CYP1A2 activities in response to rifampicin and omeprazole in control and 1 mg / L bilirubin-treated HLOs (RLU: relative optical units; CTG: CellTiter-Glo assay).

[0084] Figures 7F-7H An implementation scheme for immunofluorescence of mature liver enzymes and transport proteins in liver organoids treated with 1 mg / L bilirubin was described. Figure 7F The detection of CYP2E1 and MRP3 was described. Figure 7G The detection of CYP7A1 and MRP1 was described. Figure 7H The detection of PROX1 and OATP2 was described.

[0085] Figure 8A An embodiment depicting a bright-field image of HLO with ascorbic acid depletion on day 15, compared to a control.

[0086] Figure 8B An implementation of an exemplary workflow for generating mGULO iPSCs is described.

[0087] Figure 8C depicts a linear diagram of the synthetic mGULO-mCherry gene designed for expression in hiPSCs under a doxycycline-activated TetOn system, and a vector diagram of the pAAVS1-Ndi-CRISPRi (Gen1) plasmid used to clone the mGULO gene into hiPSCs.

[0088] Figure 8D An implementation scheme for generating an HLO using an iPSC modified to express a TetOn mGULO is depicted.

[0089] Figure 8E A scheme depicting bright-field and fluorescence images of mCherry expression in doxycycline (Dox)-treated mGULO HLOs compared to control HLOs is described.

[0090] Figure 8F Bright-field and fluorescence images of mCherry expression in ascorbic acid-depleted mGULO HLOs were depicted on day 18 with and without Dox treatment.

[0091] Figure 8G illustrates the ELISA implementation scheme for mGULO protein expression and cellular antioxidant concentration in mGULO HLO treated with Dox (10 ng / mL or 100 ng / mL) compared to the control HLO.

[0092] Figure 8HThe inflammatory and detoxification marker genes in ascorbic acid-depleted Dox-treated mGULO HLO were depicted compared to ascorbic acid-depleted control or mGULO HLO. NRF2 , 1L1B , IL6 and TNFα ) RT-qPCR implementation plan.

[0093] Figure 8I An implementation scheme for the assay of caspase 3 activity in ascorbic acid-depleted Dox-treated mGULO HLO compared to an ascorbic acid-depleted control or mGULO HLO is described.

[0094] Figure 8J An implementation scheme of a heatmap derived from RNA-seq was depicted, showing that Dox-treated mGULO HLO expressed portal vein perivascular markers compared to control HLO.

[0095] Figure 8K An implementation scheme for gene upregulation categorized by function was described, showing that the periportal pathway was overrepresented in Dox-treated mGULO HLO.

[0096] Figure 8L Bright-field images of Dox-treated mGULO and control HLO with or without 1 mg / L bilirubin treatment are depicted, along with luminal profiles using ImageJ, where arrows indicate luminal protrusions similar to bile canaliculi found in human livers.

[0097] Figure 8M depicts an embodiment comparing the lumen size and roundness of Dox-treated mGULO HLO or control HLO with or without 1 mg / L bilirubin treatment.

[0098] Figure 8N An implementation scheme for quantifying albumin expression in Dox-treated mGULO HLO or control HLO with or without 1 mg / L bilirubin treatment is described.

[0099] Figure 8O An embodiment depicting bright-field images of mGULO HLO treated with bilirubin and different concentrations of Dox (0 ng / mL, 10 ng / mL, 100 ng / mL or 1000 ng / mL Dox) is described.

[0100] Figure 8P illustrates the implementation scheme for the assay of CYP3A4 and CYP1A2 activities in response to rifampin and omeprazole in mGULO HLO in control or Dox-treated mGULO at 1 mg / L bilirubin.

[0101] Figure 8QAn implementation scheme for the UnaG assay was described, showing the loss of fluorescence, indicating bilirubin conjugation even in the presence of deep yellow bilirubin.

[0102] Figure 8R An implementation scheme for UnaG assay of mGULO organoids treated with Dox, compared with a control, is described.

[0103] Figure 8S A quantitative implementation scheme was described, showing the total percentage of active organoids and organoids carrying conjugated bilirubin in Dox-treated mGULO organoids compared to the control.

[0104] Figure 9A An implementation scheme for generating HLO and treating with different concentrations of bilirubin is illustrated.

[0105] Figure 9B An implementation scheme for treating HLO with bilirubin (0 mg / L-10 mg / L) and bright-field images after 1 day and 4 days is described.

[0106] Figure 9C The study depicted the effects of treating organoids with different concentrations of bilirubin compared to untreated organoids. UGT1A1 and NRF2 Implementation plan for RT-qPCR of genes.

[0107] Figure 9D An implementation scheme was described for a patient with Crigler-Najjar syndrome (CNS) from which CNS iPSCs arise. DNA sequencing of the patient revealed a nonsense mutation c.858C>A (p.Cys280X) in the UGT1A1 gene.

[0108] Figure 9E An implementation scheme depicts fluorescence images showing the expression of the classic pluripotency markers Sox2 and Oct4 in CNS iPSCs derived from patients with Crigler-Najjar syndrome.

[0109] Figure 9F A bright-field image depicting a scheme showing that, according to a standard scheme, CNS iPSCs can differentiate into defined endoderm (DE) and liver organoids (hLO) is described.

[0110] Figure 9G An embodiment is described that displays fluorescence images of liver organoids generated by CNS iPSCs expressing the proliferation marker Ki67, the liver-specific marker AFP, and the epithelial marker ECAD.

[0111] Figure 9HAn embodiment depicting bright-field images of CNS HLOs treated with bilirubin (10 mg / L) and a control (0 mg / L bilirubin) after 1 and 4 days shows that these HLOs suffered from bilirubin toxicity.

[0112] Figure 9I The CNS HLO was described 10 days after treatment with bilirubin (10 mg / L) and the levels of bilirubin-treated HLO. UGT1A1 Implementation scheme for bright-field imaging of CNS HLO transfected with mRNA.

[0113] Figure 9J Describes the measurement Figure 9I An implementation scheme for the determination of unconjugated bilirubin (UCB) and conjugated bilirubin (CB) in HLO.

[0114] Figure 9K An implementation scheme for bilirubin determination of unconjugated bilirubin (UCB) and conjugated bilirubin (CB) in mGULO HLO treated with 10 mg / L bilirubin and Dox (0 ng / mL, 10 ng / mL, 100 ng / mL or 1000 ng / mL) is described.

[0115] Figure 10A An embodiment depicting bright-field images of liver organoids treated with 10 mg / L bilirubin and the glucocorticoid agonist hydrocortisone (HC; 1 μM or 5 μM) or dexamethasone (Dex; 1 μM or 5 μM) is described.

[0116] Figure 10B Describes the measurement Figure 10A An implementation plan for the determination of unconjugated and conjugated bilirubin in liver organoids.

[0117] Figure 10C An embodiment depicting bright-field images of liver organoids treated with 10 mg / L bilirubin and the glucocorticoid antagonist ketoconazole (KCZ; 1 μM or 5 μM) or mifepristone (Mif; 1 μM or 5 μM) was described.

[0118] Figure 10D Describes the measurement Figure 10C An implementation plan for the determination of unconjugated and conjugated bilirubin in liver organoids.

[0119] Figure 10E The study described the effects of organoids treated with 10 mg / L bilirubin compared to those treated with 10 mg / L bilirubin and hydrocortisone, dexamethasone, ketoconazole, or mifepristone. UGT1A1 and NRF2 Implementation plan for RT-qPCR of genes.

[0120] Figure 10F depicts an implementation scheme comparing enriched pathways obtained from RNA sequencing between organoids treated with 10 mg / L bilirubin and 1 μM mifepristone compared to the control, as well as a GSEA plot comparing enriched ROS and xenobiotic metabolism.

[0121] Figure 10G An implementation scheme for a Venn diagram depicting differentially expressed genes in ROS and xenobiotic metabolism was described.

[0122] Figure 10H illustrates the implementation schemes for ChIP-PCR and CHIP-qPCR of organoids treated with 10 mg / L bilirubin and 1 μM mifepristone (Mife) or 1 μM dexamethasone (Dex).

[0123] Figure 11A illustrates the implementation scheme of the workflow for orthotopic transplantation of HLO in rodents.

[0124] Figure 11B The implementation scheme for albumin ELISA in serum collected from Gunn rats transplanted with mGULO HLO or sham surgery at different time points after transplantation is described.

[0125] Figure 11C Describing the effects of transplantation Figure 11B Implementation plan for bilirubin determination in Gunn rats.

[0126] Figure 11D depicts the effects of transplantation on... Figure 11B Implementation plan for AST and ALT measurement in Gunn rats.

[0127] Figure 12A The text describes the cultivation of organisms in a 3D bioreactor with or without Matrigel. ® An implementation scheme for an exemplary process of subjecting HLO induced by conventional methods to amplification culture under certain conditions.

[0128] Figure 12B An implementation scheme for HLO growth after 15 days of culture in a 3D bioreactor is described, compared with conventional static culture.

[0129] Figure 12C The text describes the cultivation process in a 3D bioreactor, even without the addition of Matrigel. ® The group obtained the implementation plan for the large HLO.

[0130] Figure 13 An implementation scheme was described in which similar expression levels of E-cadherin, vimentin, and Prox1 were observed, indicating that HLOs obtained from 3D bioreactor cultures have properties comparable to those obtained from static cultures.

[0131] Figure 14A An implementation scheme is described for dissociating HLOs induced by conventional methods into single cells and for passage and reconstitution of HLOs using a 3D bioreactor.

[0132] Figure 14B Depicting the presence and absence of Matrigel ® In this case, an implementation scheme was adopted to induce uniform HLO by reconstructing HLO in a 3D bioreactor after 6 days.

[0133] Figure 15A A schematic diagram of an implementation scheme for a model of salvage from acute liver injury caused by acetaminophen via HLO transplantation is depicted.

[0134] Figure 15B An implementation scheme for HLO imagery used for porting is described.

[0135] Figure 15C The implementation scheme of Kaplan-Meier survival curves for salvaging acute liver injury via HLO transplantation was described. Detailed Implementation

[0136] In the following detailed description, reference is made to the accompanying drawings, which form part of this specification. In the drawings, similar symbols generally identify similar components unless the context otherwise requires. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter set forth herein. It will be readily understood that, as generally described herein and shown in the accompanying drawings, aspects of this disclosure can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0137] The following descriptions of various embodiments are exemplary and illustrative only and should not be construed as limiting or restricting in any way. Other embodiments, features, objects, and advantages of this teaching will be apparent from the description and drawings, as well as from the claims.

[0138] The disclosure herein uses affirmative language to describe multiple embodiments. This disclosure also includes embodiments that exclude, in whole or in part, the subject matter, such as substances or materials, methods, steps and conditions, schemes or procedures.

[0139] It should be understood that any use of subheadings in this document is for organizational purposes and should not be construed as limiting the application of those subheading features to the various embodiments described herein. Each feature described herein is applicable to and can be used in all the various embodiments discussed herein, and all features described herein can be used in any intended combination, regardless of the specific example embodiment described herein. It should also be noted that the exemplary descriptions using specific features are primarily for informational purposes and not to limit the design, sub-features, and functionality of the explicitly described features in any way.

[0140] Overview The development of methods for generating human liver organoids from pluripotent stem cells has enabled the study of liver function that closely resembles that of the natural liver. Compared to methods involving the combination of primary hepatocytes or differentiation from adult pluripotent cells, the differentiation of pluripotent stem cells produces liver organoids with rich cell type diversity, including hepatocytes, stellate cells, Kupffer cells, and other epithelial and mesenchymal cell lineages that make up the normal liver.

[0141] However, because the maintenance of pluripotent stem cells and the formation of downstream intermediates are sensitive, previous culture methods have typically involved the use of basement membrane matrix that mimics the niches that promote cell proliferation. Common basement membrane matrices used in laboratory settings are those isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. While these matrices offer significant utility in cell culture, their presence hinders their subsequent use in humans because they contain xenogeneic animal components and may also potentially carry pathogens. Therefore, there is an ongoing process to develop cell culture methods that do not involve the use of xenogeneic basement membrane matrices.

[0142] This article provides methods for using Matrigel without its own features. ® Methods for culturing organoids (such as liver organoids) in the presence of other xenogeneic basement membrane matrices are also disclosed. Other improvements for culturing liver organoids, such as methods for supplementing growth media and expanding cells for larger-scale manufacturing beyond the limitations of laboratory culture, are also disclosed.

[0143] In some implementations, human liver organoids (HLOs), such as those generated according to the methods disclosed herein, are used to model liver-related diseases and conditions. For example, HLOs generated according to the methods disclosed herein can be used to model hyperbilirubinemia by treating them with different concentrations of bilirubin.

[0144] {Once the claims are agreed upon / finalized, they will be updated with any further independent claims.} The incidence of liver diseases is increasing at an even faster rate. Neonatal hyperbilirubinemia (NH) is a condition that deteriorates the health of newborns. It affects 60% of all newborns and causes 114,000 deaths worldwide each year. Currently, the only treatment for NH involves 12 hours of light therapy or exchange transfusion, but these can cause other complications. Therefore, effective and scalable model systems for these liver diseases are now essential for understanding the underlying molecular mechanisms and developing potential therapies.

[0145] Currently, there are two main model organisms used for modeling NH: Gunn rats and UGT1A1 Knockout mice. However, these models lack key human proteins (OATP family) and epigenetic controls involved in bilirubin metabolism. Recent studies have revealed that many aspects of UGT1A1 regulation are not well translated between species. As the rate-limiting enzyme in bilirubin metabolism... UGT1A1 Genetic regulation of the gene differs significantly in humans. Gunn rats with UGT deficiency are quite difficult to maintain and require specific regulation. Furthermore, the UGT1 KO mouse model exhibits lethal hyperbilirubinemia and survives only a few weeks. Numerous treatments have been tested with these models, but most have failed.

[0146] Breeding model organisms such as mice and rats requires months of work and planning, and the chance of obtaining the desired genotype is relatively low. Furthermore, model organisms exhibit highly variable responses to biochemical perturbations across generations. These rodents also face the risk of losing the desired genotype during long-term breeding and require complex training and procedures to model diseases and evaluate the efficacy of treatments. Therefore, there is an urgent need to develop more measurable models to understand the dynamics of bilirubin metabolism.

[0147] HLOs, such as those produced according to the methods disclosed herein, are easy to use and exhibit very low batch-to-batch variability. Large quantities of HLOs can be produced within a few weeks. Utilizing these properties, several drugs were tested over short time spans to identify key pathways involved in bilirubin metabolism. Therefore, liver organoids are useful models for studying diseases and conditions associated with dysfunctional bilirubin metabolism, such as jaundice, Crigler-Najjar syndrome, Gilbert's syndrome, Dubin-Johnson syndrome, or Rotor syndrome.

[0148] These HLOs can be derived from patient-derived induced pluripotent stem cells (iPSCs), where the patients can be healthy or have a disease condition and are genetically identical to the corresponding patient. They express most liver markers expressed during the prenatal developmental stage. Furthermore, they are clonal and therefore respond similarly to external stimuli and biochemical perturbations. These HLOs are highly scalable and manageable, allowing screening methods to test a wide range of drugs and small molecules.

[0149] Although abnormal bilirubin metabolism can lead to disease, bilirubin is an important metabolite during early fetal development and acts as a metabolic hormone with antioxidant effects in adults. HLO models prepared by previous methods that do not involve the use of bilirubin resemble immature tissue and express fetal and intestinal markers. Therefore, as also disclosed herein, HLO maturation is induced by using low doses (mimicking normal fetal physiological levels) of bilirubin during the culture steps disclosed herein. Modulation of glucocorticoid receptor pathways in these HLOs, such as treatment with mifepristone and ketoconazole, improves bilirubin conjugation and metabolism.

[0150] Vitamin C is also essential for normal fetal development and is involved in the formation of the periportal region of the liver. L-gulonolactone oxidase (GULO) is a naturally occurring enzyme that synthesizes vitamin C, but this enzyme is nonfunctional in humans and some other animals such as guinea pigs, requiring exogenous vitamin C supplementation (usually through diet). As shown in guinea pig animal models, vitamin C deficiency causes significant metabolic disorders.

[0151] Compared to model organisms, iPSC cell lines are more readily genetically modified, and they can maintain longer lifecycles before differentiating into organoids. Leveraging this, iPSC-derived organoids expressing functional L-gulonolactone oxidase (GULO) (such as mouse GULO (mGULO)) were generated (those produced according to the methods disclosed herein). When both iPSCs and organoids are of human origin, expression of functional L-gulonolactone allows for ascorbic acid synthesis, which is normally inactive in humans. These mGULO organoids exhibit increased efficiency in bilirubin conjugation and improved activity upon treatment with bilirubin. Ascorbic acid production in mGULO organoids reduces oxidative stress in the organoids and drives NRF2 expression, a key regulator of cellular detoxification pathways, which in turn promotes the expression of UGT1A1, which catalyzes bilirubin conjugation. These mGULO organoids are genetically identical to the patients from which they are derived and encompass aspects of human bilirubin metabolism. Therefore, these organoids can serve as model systems for elucidating the mechanisms underlying liver diseases and conditions such as NH and for developing therapeutic treatments.

[0152] Definition of terminology In the following detailed description, reference is made to the accompanying drawings, which form part of this specification. In the drawings, similar symbols generally identify similar components unless the context otherwise requires. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter set forth herein. It will be readily understood that, as generally described herein and shown in the accompanying drawings, aspects of this disclosure can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0153] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains upon reading this disclosure. For the purposes of this disclosure, the following terms are explained as follows.

[0154] The disclosure herein uses affirmative language to describe multiple embodiments. This disclosure also includes embodiments that exclude, in whole or in part, the subject matter, such as substances or materials, methods, steps and conditions, schemes or procedures.

[0155] The article “a and an” as used in this article refers to one or more of the grammatical objects of the article (e.g., at least one). For example, “element” means one or more elements.

[0156] "Approximately" means a quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length that changes by up to 10% relative to a reference quantity, level, value, number, frequency, percentage, size, quantity, weight, or length.

[0157] The term “or” is used in the claims to mean “and / or” unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive; however, this disclosure supports the definition of referring only to alternatives and “and / or”. For example, “x, y, and / or z” can mean “x alone,” “y alone,” “z alone,” “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” In particular, x, y, or z can be explicitly excluded from the embodiments. As used herein, “another” can mean at least a second or more than one.

[0158] The term "one / kind" means more than one / kind.

[0159] As used herein, the term "multiple / species" can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more / species.

[0160] As used in this article, the term "set" refers to one or more items. For example, a set of items includes one or more items.

[0161] As used herein, the phrase “at least one” when used with a list of items means that different combinations of one or more items from the list may be used, and it may be necessary to use only one item from the list. An item can be a specific object, thing, step, action, process, or category. In other words, “at least one of…” means that any combination of items or the number of items from the list may be used, but it may be necessary to use all items from the list. 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 item C. In some cases, “at least one of item A, item B, or item C” means, but not limited to, two items A, one item B, and ten items C; four items B and seven items C; or some other suitable combination.

[0162] As used herein, “substantially” means sufficient for the intended purpose. Therefore, the term “substantially” allows for minor, insignificant variations in absolute or perfect condition, dimensions, measurements, results, etc., such as those expected by a person of ordinary skill in the art, that do not significantly affect overall performance. When used relative to numerical values ​​or parameters or characteristics that can be expressed as numerical values, “substantially” means within ten percent.

[0163] Throughout this specification, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” will be understood to imply inclusion of the stated steps or elements or groups of steps or elements, but do not exclude any other steps or elements or groups of steps or elements. “Comprising…” means including, but not limited to, whatever follows the phrase “comprising…”. Therefore, the phrase “comprising…” indicates that the listed elements are essential or necessary, and no other elements can be present. “Substantially comprising…” means encompassing any element listed following that phrase, and is limited to other elements that do not interfere with or facilitate the activities or actions specified for the listed elements in this disclosure. Therefore, the phrase “substantially comprising…” indicates that the listed elements are necessary or mandatory, but other elements are optional and may be present or absent depending on whether they substantially affect the activities or actions of the listed elements.

[0164] Throughout this specification, the terms "an embodiment," "an implementation," "a specific implementation," "a related implementation," "a particular implementation," "an additional implementation," or "another implementation," or combinations thereof, refer to a specific feature, structure, or characteristic described in connection with that implementation that is included in at least one embodiment of this disclosure. Therefore, the appearance of the foregoing phrase throughout this specification does not necessarily refer to the same implementation. Furthermore, specific features, structures, or characteristics can be combined in various embodiments in any suitable manner.

[0165] As used herein, the terms “individual,” “subject,” or “patient” have their common and conventional meaning as understood according to this specification, and refer to human or non-human mammals such as dogs, cats, mice, rats, cattle, sheep, pigs, goats, non-human primates, or birds such as chickens, and any other vertebrates or invertebrates. The term “mammal” is used in its usual biological sense. Thus, mammals specifically include, but are not limited to, primates, including apes (chimpanzees, monkeys) and humans, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, etc.

[0166] As used herein, the term "treatment" or similar terms relating to a disease or condition can refer to achieving a desired pharmacological and / or physiological effect. This effect can be preventative in terms of the complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of the partial or complete cure of the disease and / or side effects attributable to the disease. For example, treatment can include the implementation of a protocol that may include administering one or more medications to a patient in an effort to alleviate the signs or symptoms of a disease. The desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. Relief can occur before and after the signs or symptoms of a disease or condition appear. Therefore, "treatment" can include "prevention" of a disease or adverse condition. Furthermore, "treatment" does not require complete relief of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0167] As used herein, “treatment” can therefore cover any treatment of a disease in a subject, particularly a person, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e. causing the disease to subside and / or alleviating one or more symptoms of the disease. “Treatment” can also cover the delivery of a drug or the administration of a therapy to provide a pharmacological effect even in the absence of a disease or symptom.

[0168] As used throughout this application, the terms "therapeuticly effective" or "therapeuticly effective amount" can refer to an amount that effectively achieves the desired and / or beneficial effect, and / or any situation that promotes or enhances the health of a subject in relation to the medical treatment of a symptom. 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 amount can be administered in one or more doses. In these methods, a therapeutically effective amount is an amount appropriate for the therapeutic indication. A therapeutic indication means achieving any desired effect, such as alleviating, improving, stabilizing, reversing, slowing or delaying disease progression, improving quality of life, or prolonging lifespan, or one or more of these. Such achievements can be measured by any suitable method, such as measuring tumor size or blood cell count, or any other suitable measurement.

[0169] As used herein, the terms "effective amount" or "effective dose" have their common and customary meaning as understood according to this specification, and refer to the amount of the composition or compound that results in an observable effect. The actual dose level of the active ingredient in the active compositions of the subject matter disclosed herein can be varied to administer an amount of the active composition or compound that effectively achieves the desired response in a particular subject and / or application. 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 prior medical history of the subject being treated. In some embodiments, a minimum effective dose is administered, and the dose is gradually increased to the minimum effective amount in the absence of dose-limiting toxicity. This document considers the determination and adjustment of the effective dose, as well as the assessment of when and how such adjustments should be made.

[0170] As used in this article, the term "disease state" can generally refer to a condition that affects the structure or function of an organism. A disease state can include, for example, a stage in the progression of a disease.

[0171] As used herein, the term “evaluation” can include any form of measurement and includes determining the presence of an element. The terms “determine,” “measure,” “evaluate,” “assess,” and “determine” are used interchangeably and can include quantitative and / or qualitative determinations.

[0172] As used herein, unless otherwise stated or clear from the context of particular use, the terms “regulatory” or “modulation” or “control” or “differential control” can refer to upregulation (i.e., activation or stimulation, e.g., by excitation or enhancement) and downregulation (i.e., inhibition or suppression, e.g., by antagonism, reduction or inhibition).

[0173] As used herein, the terms “function” and “functional” have their common and conventional meanings as understood in accordance with this specification, and refer to biological function, enzymatic function, or therapeutic function.

[0174] As used herein, the term "marker" or "biomarker" can refer to any measurable substance taken as a sample from a subject whose presence indicates a phenomenon. Non-limiting examples of such phenomena can include disease states, symptoms, or exposure to compounds or environmental conditions. In the various embodiments described herein, biomarkers can be used for diagnostic purposes (e.g., diagnosing disease states, health states, asymptomatic states, symptomatic states, etc.). The term "biomarker" is used interchangeably with the term "marker." The term "marker" or "biomarker" can include biomolecules such as nucleic acids, peptides, proteins, hormones, etc., whose presence or concentration can be detected and associated with a known condition, such as a disease state. It can also be used to refer to differentially expressed genes, the expression patterns of which can be used as part of a predictive, prognostic, or diagnostic process in a health condition or disease state, or alternatively, in methods for identifying useful therapeutic or preventative therapies.

[0175] As used herein, the term “cell phenotype” can refer to any identifiable, observable, and / or measurable characteristic associated with a cell population.

[0176] As used herein, a “model” can include one or more in vitro or in vivo disease models; a model can also include algorithms, one or more mathematical techniques, one or more machine learning algorithms, or combinations thereof. According to the various embodiments disclosed herein, the model can be used in processes and / or applied to assays.

[0177] As used herein, “process” can include one or more steps involving one or more features of one or more models as disclosed herein.

[0178] As used herein, the terms “function” and “functional” have their common and conventional meanings as understood according to the specification, and may refer to biological function, enzymatic function, or therapeutic function.

[0179] As used herein, the term "inhibition" has its common and conventional meaning as understood in accordance with this specification, and can refer to a reduction or prevention of biological activity. Reduction can be a percentage of, about, at least, at least about, not exceeding or not exceeding about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount within a range defined by any two of the foregoing values. As used herein, the term "delay" has its common and conventional meaning as understood in accordance with this specification, and refers to slowing down, delaying, or postponing a biological event to a later time than otherwise expected. Delay can be a percentage delay of, that percentage being, about, at least, at least about, not exceeding or not exceeding about 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount within a range defined by any two of the foregoing values. The terms inhibition and delay do not necessarily represent 100% inhibition or delay. Partial inhibition or delay can be achieved.

[0180] As used herein, the term “separated” has its common and customary meaning as understood according to this specification, and refers to a substance and / or entity that has been (1) separated from at least some of the components that were originally associated with it (whether in nature and / or in an experimental setting), and / or (2) artificially produced, prepared and / or manufactured. A separated substance and / or entity may be equal to, about, at least, at least about, not exceeding or not exceeding about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100% or 100% of the other components originally associated with it (or includes and / or spans a range of the foregoing values). In some embodiments, the isolated reagent is, is about, is at least, is at least about, is not more than or does not exceed about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100% or 100% pure (or contains and / or spans a range of the foregoing values). As used herein, “isolated” material can be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” can refer to a cell not contained in a multicellular organism or tissue.

[0181] As used herein, “in vivo” is given its common and conventional meaning as understood in accordance with this specification, and refers to the execution of a method within a living organism (typically an animal, mammal, including humans and plants) rather than a tissue extract or a dead organism.

[0182] As used herein, “extracorporeal” is given its common and conventional meaning as understood in accordance with this specification, and refers to the execution of a method outside a living organism with virtually no change in natural conditions.

[0183] As used herein, “in vitro” is given its common and conventional meaning as understood in accordance with this specification, and refers to the execution of a method outside of biological conditions, such as in a petri dish or test tube.

[0184] As used herein, the term "nucleic acid" or "nucleic acid molecule" has its common and conventional meaning as understood according to this specification, and refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those naturally occurring in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by any ligation, breakage, endonuclease action, and exonuclease action. Nucleic acid molecules may consist of monomers of naturally occurring nucleotides (such as DNA and RNA) or analogs of naturally occurring nucleotides (e.g., enantiomers of naturally occurring nucleotides) or combinations thereof. Modified nucleotides may have alterations in the sugar moiety and / or in the pyrimidine or purine base moiety. Sugar modifications include, for example, replacing one or more hydroxyl groups with halogens, alkyl groups, amines, and azides, or the sugar may be functionalized into an ether or ester. Furthermore, the entire sugar moiety may be replaced by spatially and electronically similar structures (such as azahesaccharides and carbocyclic sugar analogs). Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogues of such linkages. Analogs of phosphodiester bonds include thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phosphoroanilothioate, phosphoranilidate, or phosphoramidate. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which consist of naturally occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded. "Oligonucleotide" is used interchangeably with nucleic acid and can refer to double-stranded or single-stranded DNA or RNA. One or more nucleic acids can be contained in nucleic acid vectors or nucleic acid constructs (e.g., plasmids, viruses, retroviruses, lentiviruses, bacteriophages, fosmids, phage particles, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or human artificial chromosomes (HACs)) that can be used to amplify and / or express one or more nucleic acids in various biological systems. Typically, the vector or construct will also contain, but is not limited to, the following elements: promoter, enhancer, terminator, inducer, ribosome binding site, translation initiation site, start codon, stop codon, polyadenylation signal, origin of replication, cloning site, multiple cloning site, restriction enzyme site, epitope, reporter gene, selection marker, antibiotic selection marker, targeting sequence, peptide purification tag or accessory gene, or any combination thereof.

[0185] Nucleic acids or nucleic acid molecules may include one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences may be contiguously linked within the same nucleic acid or nucleic acid molecule, or may have additional nucleic acids between them, such as adapters, repetitive sequences, or restriction enzyme sites, or may be of 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 any other sequence of any length within the range defined by any two of the aforementioned lengths. As used herein, the term "downstream" on nucleic acids has its common and conventional meaning as understood in this specification, and refers to a sequence following the 3' end of the preceding sequence on the strand containing the coding sequence (sense strand) when the nucleic acid is double-stranded. As used herein, the term "upstream" on nucleic acids has its common and conventional meaning as understood in this specification, and refers to a sequence preceding the 5' end of the subsequent sequence on the strand containing the coding sequence (sense strand) when the nucleic acid is double-stranded. As used herein, the term "grouping" on nucleic acids has its common and conventional meaning as understood in this specification, and refers to two or more sequences occurring directly nearby or between which additional nucleic acids (e.g., adapters, repetitive sequences, or restriction enzyme sites), or any other sequences, of length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. A sequence of 1, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or any length within the range defined by any two of the aforementioned lengths, but generally not containing a sequence that encodes a functional or catalytic polypeptide, protein, or protein domain.

[0186] The nucleic acids described in this article include nucleobases. The major, canonical, natural or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleobases include, but are not limited to, purine, pyrimidine, modified nucleobases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

[0187] As used herein, the terms “peptide,” “polypeptide,” and “protein” have their common and conventional meanings as understood from the specification and refer to macromolecules comprising 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, enzyme, structure, transport, defense, hormone, or signal transduction. Although chemical synthesis is also possible, peptides, polypeptides, and proteins are typically (but not always) biologically produced by ribosome complexes using nucleic acid templates. Using nucleic acid templates, peptides, polypeptides, and proteins can be mutated, such as by substitution, deletion, truncation, addition, duplication, or fusion of more than one peptide, polypeptide, or protein. These fusions of more than one peptide, polypeptide, or protein may be bound adjacently in the same molecule, or have additional amino acids (e.g., linkers, repeat sequences, epitopes, or tags) therebetween, or any other sequence having a length of, about, at least, at least about, no more than, or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases, or any length within the range defined by any two of the foregoing lengths. As used herein, the term “downstream” has its common and conventional meaning in polypeptides as understood from the specification, and refers to the sequence following the C-terminus of the preceding sequence. As used herein, the term "upstream" in polypeptides has its common and conventional meaning as understood from the specification, and refers to the sequence preceding the N-terminus of the subsequent sequence.

[0188] As used herein, the term "purity" for any given substance, compound, or material has its common and conventional meaning as understood in accordance with this specification, and refers to the actual abundance of the substance, compound, or material relative to an expected abundance. For example, the purity of a substance, compound, or material can be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, inclusive of all decimal places. Purity can be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, mediators, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process-related components, mycoplasma, pyrogens, bacterial endotoxins, and foreign substances. Purity can be measured using techniques including but not limited to: electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin-layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-Vis spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetric analysis or titration, or any combination thereof.

[0189] As used herein, the term "yield" for any given substance, compound, or material has its common and conventional meaning as understood from the description, 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 as a percentage of the expected total amount is, is about, is at least, is at least about, is no more than or no more than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, inclusive of all decimals. Yield can be affected by factors such as the efficiency of the reaction or process, excess side reactions, degradation, the quality of the input substance, compound, or material, or the loss of the desired substance, compound, or material at any stage of production.

[0190] As used herein, “pharmaceuticalally acceptable” has its common and customary meaning as understood in accordance with the product information leaflet, and refers to a carrier, excipient, and / or stabilizer that is non-toxic or has an acceptable level of toxicity to cells or mammals exposed thereto at the doses and concentrations used. As used herein, “pharmaceuticalally acceptable,” “diluent,” “excipient,” and / or “carrier” has its common and customary meaning as understood in accordance with the product information leaflet, and is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents compatible with administration to humans, cats, dogs, or other vertebrate hosts. Generally, pharmaceutically acceptable diluents, excipients, and / or carriers are those approved by federal, state, or other regulatory agencies, or listed in the United States Pharmacopeia or other recognized pharmacopoeias for use in animals (including humans and non-human mammals such as cats and dogs). The terms diluent, excipient, and / or “carrier” can refer to a diluent, adjuvant, excipient, or catalyst administered with a pharmaceutical composition. Such drug diluents, excipients, and / or carriers can be sterile liquids, such as water and oils, including those from petroleum, animal, vegetable, or synthetic sources. Water, saline solutions, and aqueous solutions of glucose and glycerol can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable drug diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. A non-limiting example of a physiologically acceptable carrier is a pH-buffered aqueous solution. Physiologically acceptable carriers may also include one or more of the following: antioxidants, such as ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin; gelatin; immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids; carbohydrates, such as glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and nonionic surfactants, such as TWEEN. ® Polyethylene glycol (PEG) and PLURONICS ® If desired, the composition may also contain small amounts of wetting agents, swelling agents, emulsifiers, or pH buffers. These compositions may be in the form of solutions, suspensions, emulsions, sustained-release formulations, etc. The formulation should be suitable for the method of application.

[0191] Cryoprotectants are additives to cell compositions that improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryoprotectants include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methylformamide, dimethylformamide, glyceryl 3-phosphate, proline, sorbitol, diethylene glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. Cryoprotectants can be used as part of a cryopreservation medium containing other components, such as nutrients (e.g., albumin, serum, bovine serum, fetal bovine serum [FCS]), to improve cell viability after thawing. In these cryopreservation media, at least one cryoprotectant may be found at concentrations of, about, at least, at least about, no more than or no more than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within the range defined by any two of the foregoing figures.

[0192] Other excipients with the desired 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, glucose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium tauride deoxycholate, magnesium stearate, octylphenol ethoxylate, benzyl chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be residual or contaminants from the manufacturing process, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components, or any combination thereof. The amount of excipients in the composition may be present in the composition as a percentage of, about, at least, at least about, not more than or not more than about 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, or any weight percentage within the range defined by any two of the above figures.

[0193] The term "pharmaceutically acceptable salt" has its common and conventional meaning as understood from the instructions for use, and refers to relatively non-toxic inorganic and organic acid or base addition salts comprising compositions or excipients, including but not limited to analgesics, therapeutic agents, other materials, etc. Examples of pharmaceutically acceptable salts include those derived from inorganic acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Examples of suitable inorganic bases for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, etc. Salts can also form with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, such organic bases in this category may include, but are not limited to, monoalkylamines, dialkylamines, and trialkylamines, including methylamine, dimethylamine, and triethylamine; monohydroxyalkylamines, dihydroxyalkylamines, or trihydroxyalkylamines, including monoethanolamine, diethanolamine, and triethanolamine; amino acids, including glycine, arginine, and lysine; guanidine; N-methylglucosamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenylethylamine; and tris(hydroxymethyl)aminoethane.

[0194] Appropriate formulations depend on the chosen route of administration. Techniques for formulating and administering the compounds described herein are known to those skilled in the art. Various techniques exist in the art for administering compounds, including but not limited to enteric, oral, rectal, topical, sublingual, oral, intraauricular, epidural, epidermal, aerosol, and parenteral delivery, encompassing intramuscular, subcutaneous, intraarticular, intravenous, portal venous, intra-articular, intradermal, intraperitoneal, intramedullary, intrathecal, direct intracardiac, intraperitoneal, intranasal, or intraocular injection. Pharmaceutical compositions are typically tailored to the specific intended route of administration.

[0195] As used herein, “carrier” has its common and conventional meaning as understood in accordance with the specification, and refers to a compound, particle, solid, semi-solid, liquid or diluent that facilitates the passage, delivery and / or incorporation of a compound into cells, tissues and / or organs of the body.

[0196] As used herein, “diluent” has its common and conventional meaning as understood according to the instructions for use, and refers to a component in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desired. For example, a diluent can be used to increase the volume of a potent drug that is too small to manufacture and / or administer. A diluent can also be a liquid used to dissolve a drug for administration by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate-buffered saline solution that mimics the composition of human blood.

[0197] As used herein, the terms “basement membrane matrix” or “extracellular matrix” have their common and conventional meaning as understood from the description and refer to any biological or synthetic compound, substance, or composition that enhances cell attachment and / or growth. Any extracellular matrix known in the art, and any analogues or derivatives thereof, may be used in the methods disclosed herein. Some examples of extracellular matrices or their analogues or derivatives include, but are not limited to, cell-based feeder layers, polymers, proteins, peptides, nucleic acids, carbohydrates, lipids, polylysine, polyornithine, collagen, collagen IV, gelatin, fibronectin, hylocinin, laminin, laminin-511, elastin, tendinin, heparan sulfate, inlinin, nestin, osteopontin, basement membrane glycans, fibrin, basement membrane, Matrigel. ® Hydrogels, PEI, WGA, or hyaluronic acid, or any combination thereof. Common basement membrane matrices used in the laboratory are those isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. However, these basement membrane matrices are derived from non-human animals and therefore contain xenogeneic components that prevent their use in humans. They are also not defined, which can lead to variability in manufacturing and the potential for pathogen contamination. Therefore, in some embodiments, methods for culturing cells may involve the synthesis and / or defined alternatives using these xenogeneic basement membrane matrices. The use of non-xenogeneic basement membrane matrices or their analogues or derivatives enables the manufacture of biological products more suitable for human use.

[0198] As used herein, the term "passage" has its common and conventional meaning as understood from the specification, and refers to a routine method in biological cell culture to maintain a viable cell population over a long period. Because cells in cell culture are typically proliferative, they undergo multiple mitotic cycles until they occupy available space, which is typically the surface of a cell culture vessel (e.g., a plate, dish, or flask) submerged in culture medium. For example, cells may grow as a monolayer on the surface of a cell culture vessel. If the growing cells occupy the entire available space on the surface, they cannot proliferate further and may exhibit senescence behavior. To allow cells to continue growing (which can be done to maintain cell viability and proliferative properties and / or increase cell numbers for downstream purposes), cells can be passaged by taking a portion of the cells and seeding this portion onto a fresh surface in the culture medium (e.g., a fresh surface of a cell culture vessel). This portion of cells will continue to proliferate and multiply until they occupy the available space on the new surface, where such passage can be repeated continuously.

[0199] The liver's microstructure consists of polygonal structures called "hepatic lobules." Classically, these lobules are hexagonal, but other geometries are observed depending on tissue specifications. Each lobular unit contains a plate or layer of hepatocytes enclosed by a bundle of blood vessels surrounding the internal central vein, known as the porta hepatis, which consists of the portal vein, hepatic artery, and bile duct. Hepatic activity occurs when blood flows from the peripheral porta hepatis through the hepatocytes and into the central vein to return to the circulatory system. Due to the asymmetrical organization of these lobules, the hepatocyte layer is divided into three regions. Cells in the "peri-portal region" (region 1) are closest to the porta hepatis and receive the most oxygenated blood, the peri-central region (region 3) is closest to the central vein and therefore receives the least amount of oxygenated blood, and the transitional region (region 2) lies between regions 1 and 3. Due to this separation, each region of the hepatocyte exhibits different activities. For example, hepatocytes in region 1 are involved in oxidative liver functions, such as gluconeogenesis and fatty acid oxidative metabolism, while hepatocytes in region 3 are involved in glycolysis, lipogenesis, and cytochrome P450-mediated detoxification. In some embodiments, the liver organoids disclosed herein exhibit periportal-like characteristics similar to those found in the periportal region of the liver lobule, including functional and cellular marker properties of the periportal region.

[0200] As used herein, the term "bilirubin" has its common and conventional meaning as understood according to the instructions, and refers to a naturally occurring metabolite produced by the normal catabolistic degradation of heme. Bilirubin is produced by biliverdin reductase catalyzing biliverdin. In the liver, bilirubin is conjugated to glucuronic acid by a family of enzymes called UDT-glucuronyltransferase (UGT). This conjugation makes bilirubin water-soluble, allowing it to be carried in bile to the small and colon, where it is further metabolized into waste products. Dysfunctional bilirubin metabolism, particularly due to abnormal UGT function that prevents bilirubin conjugation, leads to bilirubin accumulation and is associated with various diseases characterized by hyperbilirubinemia. However, it is worth noting that while excessive bilirubin is harmful, it also possesses antioxidant capabilities and may therefore have beneficial effects in reducing oxidative damage in cells.

[0201] As used herein, the term "hyperbilirubinemia" has its common and conventional meaning as understood according to the instructions, and refers to the condition of elevated bilirubin levels, a natural byproduct of heme metabolism. Bilirubin is filtered from the blood by the liver and converted into water-soluble intermediates, which are then released into the intestines in bile, metabolized by the microbiota, and excreted as waste. In newborns, the level of bilirubin initially cleared by the mother across the placenta may not be adequately cleared by the immature liver. Excessive levels of bilirubin can potentially cause serious neurological damage (kernicterus). In adults, hyperbilirubinemia can also be caused by diseases affecting the liver, such as hepatitis and cirrhosis. Neonatal hyperbilirubinemia is treated with phototherapy, or in extreme cases, with blood transfusions, while adult treatment targets the underlying cause.

[0202] As used herein, the terms “L-gulonolactone oxidase” and “GULO” have their common and conventional meanings as understood according to the specification, and refer to an enzyme that catalyzes the production of L-gulonolactone from L-gulonolactone and L-xylo-3-gulonolactone and hydrogen peroxide. L-xylo-3-gulonolactone is then spontaneously converted to ascorbic acid (vitamin C). Thus, this enzyme participates in the biosynthesis of vitamin C, an essential nutrient involved in many biological functions, such as acting as a cofactor for several important enzymes and as an antioxidant. It is noteworthy that humans and other primates of the suborder Alpha sauridae, certain species of bats, and guinea pigs have evolved to possess a non-functional GULO gene. Therefore, these organisms cannot synthesize ascorbic acid and require vitamin C from their diet or supplements, where vitamin C deficiency can lead to scurvy. As applied to the disclosure herein, a “functional GULO protein” is a GULO protein with L-gulonolactone catalytic activity to induce ascorbic acid production. Conversely, “inactive” or “non-functional” GULO proteins are proteins that lack the catalytic activity required to produce ascorbic acid. Human and human-derived cells contain non-functional GULO proteins and are not capable of synthesizing ascorbic acid. However, as disclosed herein, human cells can be engineered to express functional GULO proteins to achieve ascorbic acid synthesis. These functional GULO proteins can be expressed in human cells (or other cells that cannot synthesize ascorbic acid normally) using conventional cloning methods, such as genetically engineering cells to have the genetic sequence encoding a functional GULO protein.

[0203] As used herein, the terms “% w / w” or “% wt / wt” have their common and conventional meaning as understood according to the specification, and refer to a percentage of the weight of the component or reagent as a percentage of the total weight of the composition multiplied by 100. As used herein, the terms “% v / v” or “% vol / vol” have their common and conventional meaning as understood according to the specification, and refer to a percentage of the liquid volume of the compound, substance, component, or agent as a percentage of the total liquid volume of the composition multiplied by 100.

[0204] As used herein, the term "exogenous" has its common and conventional meaning as understood according to the specification, and refers to an external factor originating outside the biological sample (e.g., cells, cell populations, organoids, etc.) rather than being naturally present and / or generated by the biological sample itself. As used herein, exogenous components, reagents, and / or conditions 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 through endogenous function of the biological sample.

[0205] The terms “liver organoids” and “hepatocyte organoids” are used interchangeably herein and refer to a population of cells differentiated in vitro to form self-organizing structures, which are typically three-dimensional (3D) and include one or more functional cell types. Liver organoids differ from native liver tissue in many ways. For example, compared to native liver tissue, liver organoids may have structures with a single lumen and typically a spherical shape, and may include a non-native basement membrane. The single lumen of liver organoids contains 3D tissue but typically does not produce any lobular or cord-like structures, as is the case with native liver tissue. Liver organoids also typically lack hematopoietic tissue and acquired immune cell subsets, such as T cell lineages. Furthermore, liver organoids may have different efflux mechanisms compared to native liver tissue because they may have a three-dimensional structure with a lumen but no efflux mechanism. Additionally, liver organoids typically cannot accept dietary input because they lack tubular and connecting vascular channels.

[0206] Liver organoids can be derived from pluripotent stem cells (PSCs), including at least embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Liver organoids can also be formed from liver-derived stem cells. Typically, liver organoids can self-organize in a manner similar to in vivo processes through cell sorting and spatially restricted lineage shaping, but can also be directed in vitro by carefully introducing exogenous and / or endogenous differentiation factors and / or conditions as described herein, optionally through one or more directional steps, optionally involving the introduction of one or more components.

[0207] As used herein, the term "mature liver organoid" refers to a liver organoid that continues to develop from a liver organoid to include, in various embodiments, a luminal projection similar to a bile canal and / or a structure having a single lumen and generally a spherical shape. Mature liver organoids may exhibit lumines with smaller size and reduced roundness compared to the lumen of a liver organoid. In some embodiments, mature liver organoids can be produced by adding exogenous bilirubin and / or amino acid supplements as described herein. In some embodiments, mature liver organoids may be characterized by reduced expression levels of AFP, CDX2, and / or NANOG relative to liver organoids, and / or increased expression levels of ALB, SLC4A2, and / or HO-1 relative to liver organoids. In some embodiments, mature liver organoids may be characterized by expression of CYP2E1, CYP7A1, PROXI, MRP3, MRP3, and / or OATP2. In some embodiments, mature liver organoids may exhibit increased CYP3A4 and / or CYP1A2 protein levels and / or enzyme activity relative to liver organoids.

[0208] As used herein, the term "hyperbilirubinemia liver organoid" refers to a liver organoid that has been exposed to high concentrations of bilirubin (usually provided exogenously via single or multiple administrations) to mimic a hyperbilirubinemia state. In some embodiments, hyperbilirubinemia liver organoids contain genetic abnormalities that alter bilirubin metabolism, such as leading to increased levels of bilirubin anabolism and / or decreased levels of bilirubin catabolism. Hyperbilirubinemia liver organoids may be characterized by elevated levels of UGT1A1 and / or NRF2 compared to liver organoids not exposed to high concentrations of bilirubin.

[0209] As used herein, the term “tissue culture surface” has its common and conventional meaning as understood according to the specification, and refers to a substrate surface on which cells can aggregate and / or adhere to promote cell growth, differentiation and / or function.

[0210] As used herein, the term "engineered" refers to an artificially generated entity, including cells, nucleic acids, peptides, vectors, etc. In at least some cases, the engineered entity is synthetic and contains elements that are not naturally occurring or constructed in the manner used in this disclosure. In some embodiments, constructs and / or vectors are engineered using recombinant nucleic acid technology, and cells are engineered by transfection or transduction of engineered vectors. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic, or because the cells do not naturally express these proteins.

[0211] stem cells As used herein, the term "pluripotent stem cell" (also known as a universal stem cell) has its common and conventional meaning as understood from the description and refers to stem cells that can differentiate into embryonic cells and extraembryonic cells. These cells can form complete, viable organisms. These cells are formed from the fusion of an egg and a sperm cell. Cells produced from the first few divisions of a fertilized egg are also pluripotent.

[0212] As used herein, the term "embryonic stem cell (ESC)," often abbreviated as ES cell, has its common and conventional meaning as understood in the specification, and refers to pluripotent cells that are derived from the inner cell mass of the blastocyst (i.e., the early embryo).

[0213] As used herein, the term "pluripotent stem cell (PSC)" has its common and conventional meaning as understood according to the specification and encompasses any cell that can differentiate into almost any cell type in the body, namely, cells derived from any of the three germ layers (germinal epithelium), including the endoderm (internal stomach wall, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital organs), and ectoderm (epidermal tissue and nervous system). PSCs can be the progeny of inner cell mass cells implanted in the preblastocyst or obtained by inducing non-pluripotent cells, such as adult somatic cells, through forced expression of certain genes. Pluripotent stem cells can be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including humans, rodents, pigs, and cattle.

[0214] As used herein, the term "induced pluripotent stem cell (iPSC)," often abbreviated as iPS cells, has its common and conventional meaning as understood from the specification, and refers to a type of pluripotent stem cell artificially derived from normally non-pluripotent cells such as adult somatic cells through the induction of "forced" expression of certain genes. hiPSC refers to human iPSC. In some methods known in the art, iPSCs are obtained by transfecting certain stem cell-related genes into non-pluripotent cells such as adult fibroblasts. Transfection can be achieved through viral transduction using viruses such as retroviruses or lentiviruses. The transfected genes may contain major transcriptional regulators. October 3 / 4 ( POU5F1 )and Sox2 However, other genes can also enhance induction efficiency. After 3 to 4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically, and are typically isolated by morphological selection, doubling time, or by selection using reporter genes and antibiotics. As used in this article, iPSCs include first-generation iPSCs and second-generation iPSCs from mice, as well as human induced pluripotent stem cells. In some approaches, retroviral systems are used to utilize four key genes ( October 3 / 4 , Sox2 , Klf4 and c-Myc Human fibroblasts are converted into pluripotent stem cells. Among other methods, lentiviral systems are used to... OCT4 , SOX2 , NANOG and LIN28 Transformed somatic cells. Genes expressed in iPSCs that are induced include, but are not limited to, those expressed in iPSCs. October 3 / 4 ( POU5FI ); Sox Some members of the gene family (e.g., Sox1 , Sox2 , Sox3 and Sox 15 ), Klf Some members of the family (e.g., Klf1l , Klf2 , Klf4 and Klf5 ), Myc Some members of the 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.

[0215] As used herein, the term "precursor cell" has its common and conventional meaning as understood according to the specification and encompasses any cell that can be used in the methods described herein, by which one or more precursor cells acquire the ability to self-renew or differentiate into one or more specialized cell types. In some embodiments, the precursor cell is pluripotent or has the ability to become pluripotent. In some embodiments, the precursor cell is treated with an external factor (e.g., growth factor) to obtain pluripotency. In some embodiments, the precursor cell may be a totipotent (or pluripotent) stem cell; a pluripotent stem cell (induced or uninduced); a pluripotent stem cell; an oligopotent stem cell; and a unipotent stem cell. In some embodiments, the precursor cell may be derived from an embryo, an infant, a child, or an adult. In some embodiments, the precursor cell may be a treated somatic cell that is endowed with pluripotency through genetic manipulation or protein / peptide treatment. Precursor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), and ectodermal stem cells (EpiSCs).

[0216] In some embodiments, a step is to obtain pluripotent stem cells or stem cells that can be induced to become pluripotent. In some embodiments, pluripotent stem cells are derived from embryonic stem cells, which in turn are derived from totipotent cells of early mammalian embryos, and are capable of unrestricted undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst in early embryos. Methods for deriving embryonic stem cells from embryonic cells are well known in the art. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described in this application; however, those skilled in the art will understand that the methods and systems described herein are applicable to any stem cell.

[0217] Additional stem cells that may be used in embodiments according to this disclosure include, but are not limited to, databases hosted by the National Stem Cell Bank (NSCB) of the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF); the WISC Cell Bank of the Wi Cell Institute; the University of Wisconsin-Sinica Stem Cell and Regenerative Medicine Center (UW-SCRMC); Novocell, Inc. (San Diego, California); Cellartis AB (Gothenburg, Sweden); ES Cell International Pte Ltd (Singapore); Technion, Israel Institute of Technology (Haifa, Israel); and stem cell databases hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments according to 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); WA14 (H14). Exemplary human pluripotent cell lines include, but are not limited to, TkDA3-4, 1231A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34-1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF, or 317-12 cells.

[0218] In developmental biology, cell differentiation is the process by which less specialized cells transform into more specialized cell types. As used herein, the term "directed differentiation" describes a process by which less specialized cells become specific target cell types. The specificity of the target cell type can be determined by any applicable methods that can be used to define or alter the initial cell fate. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0219] In some embodiments, adenoviruses can be used to deliver the four essential genes, thereby generating iPSCs that are substantially identical to embryonic stem cells. Because adenoviruses do not combine any of their own genes with the target host, the risk of tumorigenesis is eliminated. In some embodiments, iPSCs are generated using non-viral technologies. In some embodiments, reprogramming can be accomplished via plasmids without any viral transfection system at all, although the efficiency is very low. In other embodiments, direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modifications. In some embodiments, it is possible to generate mouse iPSCs using a similar approach: repeated treatment of cells with certain proteins introduced into the cells via polyarginine anchors is 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.

[0220] As used herein, the term "feeder cell" has its common and conventional meaning as understood according to the specification, and refers to cells that support the growth of pluripotent stem cells, such as cells that support the growth of pluripotent stem cells by secreting growth factors into the culture medium or displaying them on the cell surface. Feeder cells are typically adherent cells and may be growth-arrested. For example, feeder cells may be growth-arrested due to 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 have to be growth-arrested. Feeder cells can be used for 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, the feeder cells are allogeneic or xenogeneic with the supported target stem cells, which may have implications for downstream applications. In some embodiments, the feeder cells are mouse cells. In some embodiments, the feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76 / 7 cells, human fibroblasts, human foreskin fibroblasts, human 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 feeder cells is used instead of or in combination with a feeder cell co-culture. In some embodiments, feeder cells are not used during the proliferation of target stem cells.

[0221] PSC differentiation Known methods for preparing downstream cell types (such as fixed endoderm, foregut endoderm, hindbrain endoderm, and / or liver lineage) 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 morulae. In some embodiments, the pluripotent stem cells are stem cells. The stem cells used in these methods may include, but are not limited to, embryonic stem cells or induced pluripotent stem cells. Embryonic stem cells may be derived from the inner cell mass of the embryo or from the embryonic gonadal crest. Embryonic stem cells may originate from a variety of animal species, including, but not limited to, various mammalian species including humans. In some embodiments, human embryonic stem cells are used to generate fixed endoderm or other downstream cell types, such as hindbrain, hindbrain endoderm, and / or liver lineage. In some embodiments, iPSCs are used to generate fixed endoderm or other downstream cell types, such as hindbrain, hindbrain endoderm, and / or liver lineage. In some implementations, human iPSCs (hiPSCs) are used to produce shaped endoderm or other downstream cell types, such as hindbrain, hindbrain endoderm, and / or liver lineage.

[0222] In some embodiments, PSCs, such as ESCs and iPSCs, undergo directed differentiation into embryonic germ layer cells, organ tissue progenitor cells, and then into tissues such as liver tissue or any other biological tissue. In some embodiments, directed differentiation is performed in a stepwise manner to obtain each of the differentiated cell types, wherein molecules (e.g., growth factors, ligands, agonists, antagonists) are added sequentially as differentiation progresses. In some embodiments, directed differentiation is performed in a non-stepwise manner, wherein molecules (e.g., growth factors, ligands, agonists, antagonists) are added simultaneously. In some embodiments, directed differentiation is achieved by selectively activating certain signaling pathways in PSCs or any downstream cells.

[0223] In some embodiments, embryonic stem cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a period of time, which is, about, at least, at least about, no more than or no more than about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours, or any time within the range defined by any two of the foregoing times (e.g., 6 hours to 300 hours, 24 hours to 120 hours, 48 ​​hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours). In some embodiments, more than one small molecule compound, activator, inhibitor, or growth factor is added. In these cases, more than one small molecule compound, activator, inhibitor, or growth factor may be added simultaneously or separately.

[0224] In some embodiments, embryonic stem cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors 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, etc. The concentration may be g / mL, 2000 ng / mL, 5000 ng / mL, 7000 ng / mL, 10000 ng / mL, or 15000 ng / mL, or any concentration within the range defined by any two of the foregoing concentrations (e.g., 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 the one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, the concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors varies during the treatment process. In some embodiments, more than one small molecule compound, activator, inhibitor, or growth factor is added. In these cases, the concentrations of the more than one small molecule compound, activator, inhibitor, or growth factor may vary.

[0225] In some embodiments, ESCs or iPSCs are cultured in a growth medium that supports stem cell growth. In some embodiments, ESCs or iPSCs are cultured in a stem cell growth medium. In some embodiments, the stem cell growth medium is RPMI 1640, DMEM, DMEM / F12, or advanced DMEM / F12. In some embodiments, the stem cell growth medium includes fetal bovine serum (FBS). In some embodiments, the stem cell growth medium comprises FBS at a concentration of, about, at least, at least about, no more than or no more than about 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any percentage within the range defined by any two of the foregoing concentrations (e.g., 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 xenogeneic components. In some embodiments, the growth medium comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0226] In some embodiments, pluripotent stem cells are prepared from somatic cells. In some embodiments, pluripotent stem cells are prepared from biological tissue obtained through 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 substrate. In some embodiments, PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate. In some embodiments, PBMCs are grown on an irradiated MEF feeder cell substrate.

[0227] In some embodiments, stem cells are treated with one or more growth factors to differentiate into well-defined endoderm cells. Such growth factors may include those from the TGF-β superfamily. In some embodiments, the one or more growth factors include the Nodal / activin and / or BMP subgroup of the TGF-β growth factor superfamily. In some embodiments, the one or more growth factors are selected from the group consisting of Nodal, activin A, activin B, BMP4, Wnt3a, or any combination of these growth factors. In some embodiments, the stem cells are contacted with activin A. In some embodiments, the stem cells are contacted with both activin A and BMP4.

[0228] In some embodiments, the defined endoderm (DE) may further undergo preendoderm patterning, foregut specialization, and morphogenesis, depending on FGF, Wnt, BMP, or retinoic acid, or any combination thereof. In some embodiments, human PSCs are effectively guided to differentiate into hepatic epithelium and mesenchyme in vitro. It should be understood that molecules such as growth factors can be added at any stage of development to promote the formation of specific types of liver tissue. In some embodiments, siRNAs and / or shRNAs targeting cellular components associated with FGF, Wnt, BMP, or retinoic acid signaling pathways are used to inhibit or activate these pathways.

[0229] Culture and expansion of shaped endoderm, foregut cells and downstream cell types Methods for preparing liver organoids have been previously explored in the following literature: Ouchi et al., “Modeling Steatohepatitis in Humans with Pluripotent Stem Cell-Derived Organoids” CellMetabolism (2019) 30(2):374-384; Shinozawa et al., “High-Fidelity Drug-Induced Fiver Injury Screen Using Human Pluripotent Stem Cell-Derived Organoids” Gastroenterology (2021) 160(3):831-846; PCT Publications WO 2018 / 085615, WO 2018 / 191673, WO 2018 / 226267, WO 2019 / 126626, WO 2020 / 023245, WO 2020 / 069285 and WO 2021 / 262676, each of which is hereby expressly incorporated by reference in its entirety. The disclosure of liver organoid compositions and methods of preparation thereof applies to the human liver organoids (HLOs) described herein.

[0230] In some embodiments, pluripotent stem cells, defined endoderm, hindbrain, hindbrain endoderm, foregut endoderm, and / or downstream hepatocyte types are cultured and expanded as described herein. In some embodiments, pluripotent stem cells, defined endoderm, hindbrain, hindbrain endoderm, and / or foregut endoderm are cultured and expanded as described herein. In some embodiments, pluripotent stem cells, defined endoderm, hindbrain and / or hindbrain endoderm cells are cultured and expanded as described herein. In some embodiments, foregut endoderm cells are cultured and expanded as described herein.

[0231] In some embodiments, pluripotent stem cells, defined endoderm, hindbrain, hindbrain endoderm, foregut endoderm, and / or downstream hepatocyte types are contacted with a TGF-β pathway inhibitor. In some embodiments, the TGF-β pathway inhibitor comprises one or more of A83-01, RepSox, LY365947, and SB431542. In some embodiments, cells are not treated with a TGF-β pathway inhibitor. The TGF-β pathway inhibitors provided herein can be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0232] In some embodiments, pluripotent stem cells, fixed endoderm, hindgut, hindgut endoderm, foregut endoderm, and / or downstream hepatocyte types are contacted with an FGF pathway activator. In some embodiments, the FGF pathway activator comprises an FGF protein. In some embodiments, the FGF protein comprises a recombinant FGF protein. In some embodiments, the FGF pathway activator comprises one or more of the following: FGF1, FGF2, FGF3, 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 an FGF pathway activator. The FGF pathway activator provided in this article can be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided in this article.

[0233] In some embodiments, pluripotent stem cells, fixed endoderm, hindbrain, hindbrain endoderm, foregut endoderm, and / or downstream hepatocyte types are contacted with a Wnt pathway activator. In some embodiments, the Wnt pathway activator comprises the Wnt protein. In some embodiments, the Wnt protein comprises recombinant Wnt protein. In some embodiments, the Wnt pathway activator comprises Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, or any combination thereof. In some embodiments, the Wnt pathway activator comprises an inhibitor of the GSK3 signaling pathway. In some embodiments, Wnt pathway activators include CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, alloxan, indirubin, aterabolone, tamparone, lithium chloride, TDZD 8, or TWS119, or any combination thereof. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, cells are not treated with a Wnt pathway activator. The Wnt pathway activators provided herein can be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0234] In some embodiments, pluripotent stem cells, morphological endoderm, hindbrain, hindbrain endoderm, foregut endoderm, and / or downstream hepatocyte types are contacted with a VEGF pathway activator. In some embodiments, the VEGF pathway activator includes one or more of VEGF or GS4012. In some embodiments, the cells are not treated with a VEGF pathway activator. The VEGF pathway activator provided herein can be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0235] In some embodiments, pluripotent stem cells, morphological endoderm, hindbrain, hindbrain endoderm, foregut endoderm, and / or downstream hepatocyte types are contacted with an EGF pathway activator. In some embodiments, the EGF pathway activator includes EGF. In some embodiments, the cells are not treated with an EGF pathway activator. The EGF pathway activator provided herein can be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0236] In some embodiments, pluripotent stem cells, defined endoderm, hindbrain, hindbrain endoderm, foregut endoderm, and / or downstream hepatocyte types are contacted with ascorbic acid. In some embodiments, the cells are not treated with ascorbic acid. The ascorbic acid provided herein can be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0237] In some embodiments, pluripotent stem cells, fixed endoderm, hindbrain, hindbrain endoderm, foregut endoderm, and / or downstream hepatocyte types are contacted with a BMP pathway activator or a 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 a BMP pathway activator or a BMP pathway inhibitor. The BMP pathway activators or BMP pathway inhibitors provided in this article can be used in combination with any of the other growth factors, pathway activators or pathway inhibitors provided in this article.

[0238] In some embodiments, pluripotent stem cells, morphological endoderm, hindbrain, hindbrain endoderm, foregut endoderm, and / or downstream hepatocyte types are contacted with a retinoic acid pathway activator. In some embodiments, the retinoic acid pathway activator includes retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, or AM580, or any combination thereof. In some embodiments, cells are not treated with a retinoic acid pathway activator. The retinoic acid pathway activators provided herein can be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0239] In some embodiments, pluripotent stem cells, defined endoderm, hindbrain, hindbrain endoderm, foregut endoderm, and / or downstream hepatocyte types are converted into hepatocyte types via a "one-step" process. For example, one or more molecules that can differentiate pluripotent stem cells into DE cultures (e.g., activin A) are combined with other molecules that can promote the directed differentiation of DE cultures (e.g., FGF4, CHIR99021, RA) to directly treat pluripotent stem cells.

[0240] In some embodiments, pluripotent stem cells, morphological endoderm, hindbrain, hindbrain endoderm, foregut endoderm, and / or downstream hepatocyte types are expanded in cell cultures. In some embodiments, pluripotent stem cells (e.g., ESCs and / or iPSCs), morphological endoderm, hindbrain, hindbrain endoderm, and / or downstream hepatocyte types are expanded in cell cultures. In some embodiments, pluripotent stem cells, morphological endoderm, hindbrain, hindbrain endoderm, and / or downstream hepatocyte types are expanded in basement membrane matrix. In some embodiments, pluripotent stem cells, morphological endoderm, hindbrain, hindbrain endoderm, and / or downstream hepatocyte types are expanded in Matrigel. ® In some embodiments, pluripotent stem cells, morphological endoderm, hindbrain, hindbrain 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, morphological endoderm, hindbrain, hindbrain endoderm, and / or downstream hepatocyte types are expanded in a non-xenogeneic basement membrane matrix. In some embodiments, pluripotent stem cells, morphological endoderm, hindbrain, hindbrain endoderm, and / or downstream hepatocyte types are not expanded in Matrigel. ® In some embodiments, pluripotent stem cells, morphological endoderm, hindbrain, hindbrain endoderm, and / or downstream hepatocyte types are expanded in laminin, collagen IV, nestin, basement membrane glycan, fibrin, and / or hydrogels. In some embodiments, pluripotent stem cells, morphological endoderm, hindbrain, hindbrain endoderm, and / or downstream hepatocyte types are expanded in cell cultures containing a ROCK inhibitor (e.g., Y-27632).

[0241] In some embodiments, pluripotent stem cells (e.g., ESCs and / or iPSCs) are differentiated into defined endoderm cells. In some embodiments, pluripotent stem cells are differentiated into defined endoderm cells by contacting them with activin A, BMP4, or both. In some embodiments, pluripotent stem cells are contacted with a concentration of activin A, which is, about, at least, at least about, no more than or no more than about 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, or 200 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations (e.g., 10 ng / mL to 200 ng / mL, 10 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, or 50 ng / mL to 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 a concentration of BMP4, wherein the concentration is, about, at least, at least about, no more than or no more than about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 1 Concentrations of 10 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, or 200 ng / mL, or any range defined by any two of the foregoing concentrations (e.g., 1 ng / mL to 200 ng / mL, 1 ng / mL to 100 ng / mL, 25 ng / mL to 200 ng / mL, 1 ng / mL to 80 ng / mL, or 25 ng / mL to 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.

[0242] This article provides a method for expanding hindgut cells. Exemplary methods are described in... Figure 5HThe method is not limited by specific characteristics, such as the time and growth conditions illustrated in the figure. In some embodiments, these methods include a) dissociating a motif-foregut cell monolayer into motif-foregut cells and / or motif-foregut endoderm cells; b) seeding motif-foregut cells and / or motif-foregut endoderm cells onto a tissue culture surface; and c) co-culturing motif-foregut cells and / or motif-foregut endoderm cells with TGF-β pathway inhibitors, FGF pathway activators, Wnt pathway activators, and VEGF pathway activators. In some embodiments, motif-foregut cells and / or motif-foregut endoderm cells are further cultured with ascorbic acid. In some embodiments, motif-foregut cells and / or motif-foregut endoderm cells are not co-cultured with ascorbic acid. In some embodiments, motif-foregut cells and / or motif-foregut endoderm cells are further cultured with EGF. In some embodiments, motif-foregut cells and / or motif-foregut endoderm cells are not co-cultured with EGF. In some embodiments, the foregut cells and / or foregut endoderm cells are further cultured with a ROCK inhibitor. In some embodiments, the foregut cells and / or foregut endoderm cells are not cultured with a ROCK inhibitor. In some embodiments, the foregut cell monolayer is dissociated into foregut cells and / or foregut endoderm cells using enzymatic dissociation and / or mechanical dissociation. In some embodiments, enzymatic dissociation may involve the use of any conventional enzymatic dissociation solution known in the art, such as Accutase, Accumax, trypsin, trypsin / EDTA, collagenase, dispersase, TrypEE Express, or TrypLE Select. In some embodiments, mechanical dissociation may involve using a pipette, microchannel, or other device with appropriately sized pores to disrupt the cells, mechanically shearing the cell mass without destroying individual cells. In some embodiments, the foregut cells and / or foregut endoderm cells are seeded onto the surface of a tissue container at cell densities of 1 × 10⁻⁶, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10 ... 5 1, 2×10 5 1, 3×10 5 1, 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 5 1×10 6 1, 2×10 6 1, 3×10 6 1, 4×10 6 One or 5×10 6 cells / cm 2The surface area of ​​the tissue culture surface, or the range defined by any two of the aforementioned cell densities (e.g., 1 × 10⁻⁶). 5 5×10 6 1×10 5 5×10 5 5×10 5 5×10 6 One or 3×10 5 7×10 5 cells / cm 2 The cell density is any number of cells (the surface area of ​​the tissue culture surface). In some embodiments, hindbrain cells and / or hindbrain endoderm cells are cultured at a density equal to or about 5 × 10⁻⁶. 5 cells / cm 2 The cell density of the tissue culture surface area is seeded onto the surface of the tissue container. In some embodiments, the tissue culture surface is coated with a basement membrane matrix or a component thereof. In some embodiments, the basement membrane matrix or a component thereof does not contain non-human animal components, such that the basement membrane matrix or a component thereof is not xenogeneic to humans. In some embodiments, the basement membrane matrix or a component thereof is not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the basement membrane matrix or a component thereof is not Matrigel... ® Cultrex ® Or Geltrex ®In some embodiments, the basement membrane matrix or its components comprise human adhesion proteins, collagen IV, nestin, basement membrane glycans, fibrin, and / or hydrogels or other non-human xenogeneic substances. In some embodiments, the basement membrane matrix or its components are or comprise laminin. In some embodiments, the basement membrane matrix or its components are or comprise laminin-511. In some embodiments, hindgut cells and / or hindgut endoderm cells are cultured until they spontaneously form three-dimensional (3D) spheroids. In some implementations, the hindbrain cells and / or hindbrain endoderm cells are cultured for a number of days, which is, about, at least, at least about, no more than or no more than about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days, or any number of days within a range defined by any two of the preceding days (e.g., 4 days–6 days, 2 days–35 days, 2 days–15 days, 20 days–35 days, or 10 days–20 days). In some implementations, the hindgut cells and / or hindgut 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 any number of days within a range defined by at least two of the preceding days (e.g., 4-6 days, 4-35 days, 4-15 days, 20-35 days, or 10-20 days).

[0243] 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, or about, at least, at least about, or not exceeding or not exceeding about 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, or 1000 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations (e.g., 100 nM-1000 nM, 100 nM-500 nM, 500 nM-1000 nM, or 300 nM-700 nM). In some implementations, the TGF-β pathway inhibitor is provided at a concentration equal to or about 500 nM.

[0244] In some embodiments of the methods provided herein, the FGF pathway activator is selected from the group consisting of: FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, the FGF pathway activator is FGF2. In some embodiments, the FGF pathway activator is provided at a concentration of, which is, about, at least, at least about, no more than or no more than about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, or 10 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations (e.g., 1 ng / mL-10 ng / mL, 1 ng / mL-5 ng / mL, 5 ng / mL-10 ng / mL, or 3 ng / mL-7 ng / mL). In some embodiments, the FGF pathway activator is provided at a concentration equal to or about 5 ng / mL.

[0245] In some embodiments of the methods provided herein, the Wnt pathway activator is selected from the group consisting of: Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, alloxan, indirubin, ateradon, tamparone, lithium chloride, TDZD 8, and TWS119. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the Wnt pathway activator is provided at a concentration of, or about, at least, at least about, no more than or no more than about 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, or 8 μM, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 1 μM-8 μM, 1 μM-3 μM, 3 μM-8 μM, or 2 μM-4 μM). In some embodiments, the Wnt pathway activator is provided at a concentration equal to or about 3 μM.

[0246] 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, or about, at least, at least about, not exceeding or not exceeding about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 1 ng / mL-20 ng / mL, 1 ng / mL-10 ng / mL, 10 ng / mL-20 ng / mL, or 5 ng / mL-15 ng / mL). In some implementations, the VEGF pathway activator is provided at a concentration equal to or about 10 ng / mL.

[0247] In some embodiments of the method provided herein, the hindbrain cells and / or hindbrain endoderm cells of step c) are cultured in a medium that also contains EGF. In some embodiments, EGF is provided at a concentration of, or about, at least, at least about, or not exceeding about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 10 ng / mL-30 ng / mL, 10 ng / mL-20 ng / mL, 20 ng / mL-30 ng / mL, or 15 ng / mL-25 ng / mL). In some embodiments, EGF is provided at a concentration equal to or about 20 ng / mL. In some implementations, the hindbrain cells and / or hindbrain endoderm cells of step c) are cultured in a medium that does not contain EGF.

[0248] In some embodiments of the method provided herein, the hindgut cells and / or hindgut endoderm cells of step c) are cultured in a medium further containing ascorbic acid. In some embodiments, ascorbic acid is provided at a concentration of, about, at least, at least about, no more than or no more than about 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 10 μg / mL-100 μg / mL, 10 μg / mL-50 μg / mL, 50 μg / mL-100 μg / mL, or 30 μg / mL-70 μg / mL). In some embodiments, ascorbic acid is provided at a concentration equal to or about 50 μg / mL. In some implementations, the hindbrain cells and / or hindbrain endoderm cells of step c) are cultured in a medium that does not contain ascorbic acid.

[0249] In some embodiments of the methods provided herein, the hindbrain cells and / or hindbrain endoderm cells of step c) are cultured in a medium that also contains the ROCK inhibitor. In some embodiments, the ROCK inhibitor is provided at a concentration of, or about, at least, at least about, no more than, or no more than about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 1 μM-20 μM, 1 μM-10 μM, 10 μM-20 μM, or 5 μM-15 μM). In some embodiments, the ROCK inhibitor is provided at a concentration equal to or about 10 μg / mL. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the hindbrain cells and / or hindbrain endoderm cells of step c) are cultured in a medium that does not contain the ROCK inhibitor.

[0250] In some embodiments, the hindbrain cells and / or hindbrain endoderm cells of the methods provided herein can be cultured for multiple generations. In some embodiments, steps a)-c) of the methods provided herein are repeated using the cells of step c). In some embodiments, these methods further include passage the cells of step c) once or multiple times. In some embodiments, the cells of step c) are passaged to hindbrain cells and / or hindbrain endoderm cells without spontaneously forming spheroids. In some embodiments, the hindbrain cells and / or hindbrain endoderm cells are passaged and cultured for a number of days, which is, about, at least, at least about, no more than or no more than about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or any number of days within the range defined by any two of the preceding days (e.g., 2 days-10 days, 2 days-4 days, 2 days-6 days, 4 days-10 days, 6 days-10 days, 4 days-6 days, or 3 days-7 days), and then the hindbrain cells and / or hindbrain endoderm cells are passaged again. In some embodiments, the foregut cells and / or 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 preceding 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), and then passaged again. In some embodiments, the foregut cells and / or foregut endoderm cells are passaged and cultured for 4, 5, or 6 days, and then passaged again. In some embodiments, no more than one, two, or three passages are performed. In some embodiments, the total yield of hindgut cells and / or hindgut endoderm cells (which may be in globular form) obtained according to the method provided herein involving multiple passages is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 times, or 180 times the total yield of hindgut cells and / or hindgut endoderm cells obtained in cell culture without passages. 0x, 190x, 200x, 210x, 220x, 230x, 240x, 250x, 260x, 270x, 280x, 290x, 300x, 350x, 400x, 450x, 500x, or 1000x, or any multiple within the range defined by any two of the aforementioned multiples (e.g., 50x-1000x, 50x-200x, 200x-1000x, or 100x-300x).

[0251] In some embodiments, these methods further include collecting hindbrain cells and / or hindbrain endoderm cells and differentiating the hindbrain cells and / or hindbrain endoderm cells into liver organoids. In some embodiments, hindbrain cells and / or hindbrain endoderm cells are cultured until they spontaneously form three-dimensional (3D) spheres, optionally wherein the spheres contain structures with a single lumen, and hindbrain cells and / or hindbrain endoderm cells are collected from the spheres. In some embodiments, these methods further include dissociating the spheres into clumps of individual hindbrain cells and / or hindbrain endoderm cells and / or hindbrain cells and / or hindbrain endoderm cells prior to the differentiation step. In some embodiments, hindbrain cells and / or hindbrain endoderm cells are collected from the hindbrain cell monolayer by dissociating the hindbrain cell monolayer into clumps of individual hindbrain cells and / or hindbrain endoderm cells and / or hindbrain cells and / or hindbrain endoderm cells prior to the differentiation step.

[0252] This document provides methods for expanding hindbrain cells and / or hindbrain endoderm and / or foregut endoderm cells in some embodiments. In some embodiments, these methods include a) dissociating a hindbrain cell monolayer into hindbrain cells and / or hindbrain endoderm cells; b) seeding the hindbrain cells and / or hindbrain endoderm cells onto a tissue culture surface; and c) co-culturing the hindbrain cells and / or hindbrain endoderm cells with a TGF-β pathway inhibitor, an FGF pathway activator, a Wnt pathway activator, and a VEGF pathway activator. In some embodiments, hindbrain cell monolayers are dissociated into hindbrain cells and / or hindbrain endoderm cells using enzymatic dissociation and / or mechanical dissociation. In some embodiments, enzymatic dissociation may involve using any conventional enzymatic dissociation solution known in the art, such as Accutase, Accumax, trypsin, trypsin / EDTA, collagenase, dispersase, TrypLE Express, or TrypLE Select. In some embodiments, mechanical dissociation may involve using a pipette, microchannel, or other device with appropriately sized pores to disrupt the cells, mechanically shearing the cell mass without destroying individual cells. In some embodiments, hindbrain cells and / or hindbrain endoderm cells are seeded onto the surface of a tissue container at cell densities of approximately 1 × 10⁻⁶, about 1 × 10⁻⁶, at least 1 × 10⁻⁶, at least about 1 × 10⁻⁶, or no more than about 1 × 10⁻⁶. 5 1, 2×10 5 1, 3×10 5 1, 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 51×10 6 1, 2×10 6 1, 3×10 6 1, 4×10 6 One or 5×10 6 cells / cm 2 The surface area of ​​the tissue culture surface, or the range defined by any two of the aforementioned cell densities (e.g., 1 × 10⁻⁶). 5 5×10 6 1×10 5 5×10 5 5×10 5 5×10 6 One or 3×10 5 7×10 5 cells / cm 2 The cell density is any number of cells (the surface area of ​​the tissue culture surface). In some embodiments, hindbrain cells and / or hindbrain endoderm cells are cultured at a density equal to or about 5 × 10⁻⁶. 5 cells / cm 2 The cell density of the tissue culture surface area is seeded onto the surface of the tissue container. In some embodiments, the tissue culture surface is coated with a basement membrane matrix or a component thereof. In some embodiments, the basement membrane matrix or a component thereof does not contain non-human animal components, such that the basement membrane matrix or a component thereof is not xenogeneic to humans. In some embodiments, the basement membrane matrix or a component thereof is not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the basement membrane matrix or a component thereof is not Matrigel... ® Cultrex ® Or Geltrex ®In some embodiments, the basement membrane matrix or its components comprise human adhesion proteins, collagen IV, nestin, basement membrane glycans, fibrin, and / or hydrogels or other non-human xenogeneic substances. In some embodiments, the basement membrane matrix or its components are or comprise laminin. In some embodiments, the basement membrane matrix or its components are or comprise laminin-511. In some embodiments, hindgut cells and / or hindgut endoderm cells are cultured until they spontaneously form three-dimensional (3D) spheroids. In some implementations, the hindbrain cells and / or hindbrain endoderm cells are cultured for a number of days, which is, about, at least, at least about, no more than or no more than about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days, or any number of days within a range defined by any two of the preceding days (e.g., 2 days to 35 days, 2 days to 15 days, 20 days to 35 days, or 10 days to 20 days). In some embodiments, the hindgut cells and / or hindgut 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 any number of days within a range defined by at least two of the preceding days (e.g., 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 with the cells from step c). In some embodiments, these methods further include passage the cells from step c) once or multiple times. In some embodiments, the cells passaged in step c) to hindgut cells and / or hindgut endoderm cells do not spontaneously form spheroids. In some embodiments, cell passages are performed no more than once, twice, or three times.In some embodiments, the total yield of hindgut cells and / or hindgut endoderm cells (which may be in globular form) obtained according to the method provided herein involving multiple passages is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 times, or 180 times the total yield of hindgut cells and / or hindgut endoderm cells obtained in cell culture without passages. 0x, 190x, 200x, 210x, 220x, 230x, 240x, 250x, 260x, 270x, 280x, 290x, 300x, 350x, 400x, 450x, 500x, or 1000x, or any multiple within the range defined by any two of the aforementioned multiples (e.g., 50x-1000x, 50x-200x, 200x-1000x, or 100x-300x). In some embodiments, Table 1 provides exemplary concentration ranges for each growth factor used in the method of the present invention. In embodiments of the method, any concentration or range of a growth factor under a certain “sub-implementation” may be used in combination with the concentration or range of other growth factors under the same or different “sub-implementations.” Therefore, combinations are not limited to combinations under the same “sub-implementation.” For example, the presence of each of the TGF-β pathway inhibitors, FGF pathway activators, Wnt pathway activators, and VEGF pathway activators as defined under any “Sub-Implementation” may be combined with the presence of EGF (EGF in any of the “Sub-Implementations” 1-3) or the absence of EGF (“Sub-Implementation” 4). This also applies to any combination of the listed growth factors, whether they are present or absent in the methods implemented herein. Exemplary non-limiting formulations are listed in Table 2. For each range provided in Tables 1 and 2, this should be interpreted as including any concentration within the defined range. For example, 100 nM-1000 nM TGF-β pathway inhibitors should be interpreted as including 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM TGF-β pathway inhibitors, or any concentration within the range defined by any two of the foregoing concentrations. Exemplary concentrations within the defined ranges are provided throughout this disclosure. In some embodiments, the TGF-β pathway inhibitor is A83-01. In some embodiments, the FGF pathway activator is FGF2. In some embodiments, the Wnt pathway activator is CHIR99021. In some embodiments, the VEGF pathway activator is VEGF. In some embodiments, the ROCK inhibitor is Y-27632.

[0253] Table 1. Exemplary combinations of growth factors for expanded foregut cells

[0254] Table 2: Exemplary Combinations

[0255] This article also discloses hindbrain cells and / or hindbrain endoderm cells generated by the methods provided herein.

[0256] Gene editing In some implementations, iPSCs, shaped endoderm cells, hindbrain globules, or organoids are genetically modified or edited according to methods known in the art. For example, gene editing using CRISPR nucleases such as Cas9 has been explored in PCT publications WO 2013 / 176772, WO 2014 / 093595, WO2014 / 0193622, WO 14 / 093655, WO 2014 / 993712, WO 2014-093661, WO 2014 / 204728, WO2014 / 2004729, WO 2015 / 071474, WO 2016 / 115326, WO 2016 / 141224, WO 2017 / 023803, and WO2017 / 070633, each of which is explicitly incorporated herein by reference in its entirety.

[0257] Methods and compositions for preparing liver organoids This document provides methods and compositions for differentiating hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm into liver organoids. In some embodiments, these methods include i) contacting hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm cells with a retinoic acid pathway activator, said hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm cells optionally in the form of spheroids, optionally in the form of individual cells or cell clusters detached from spheroids, and / or optionally as cells aggregated in micropores or other devices as described herein, optionally wherein the spheroids contain structures having a single lumen; and ii) contacting the cells of step i) with a culture medium (e.g., hepatocyte culture medium (HCM)) containing hepatocyte growth factor (HGF), oncogene M (OSM), and dexamethasone for a period of time, thereby differentiating the hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm cells into liver organoids. In some embodiments, the hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm cells can be any hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm cells disclosed herein. In some embodiments, the hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm cells are in the form of spheroids or as individual hindbrain cells and / or clumps of hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm cells derived from dissociated spheroids. In some embodiments, the hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm cells can be generated by methods that do not involve the use of xenogeneic basement membrane matrix. In some embodiments, the hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm cells can be those commonly known in the art.

[0258] In some embodiments of the methods provided herein, the retinoic acid pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580. In some embodiments, the retinoic acid pathway activator is retinoic acid. In some embodiments, the retinoic acid pathway activator is provided at a concentration of, or about, at least, at least about, not more than or not more than about 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, or 3.0 μM, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 1.0 μM-3.0 μM, 1.0 μM-2.0 μM, 2.0 μM-3.0 μM, or 1.5 μM-2.5 μM). In some implementations, the retinoic acid pathway activator is provided at a concentration equal to or about 2.0 μM.

[0259] 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, forgonidone, trevalevin, recombinant InlB321 protein, and an agonistic c-Met antibody, optionally LMH85. In some embodiments, the IL-6 family cytokines are selected from the group consisting of: IL-6, OSM, leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and cardiotrophin-like cytokines (CLC). In some embodiments, the corticosteroids are selected from the group consisting of: dexamethasone, beclomethasone, betamethasone, fluclomethasone, halometasone, and mometasone. In some embodiments, the culture medium (e.g., hepatocyte culture medium) is supplemented with HGF, OSM, and dexamethasone. In some implementations, the culture medium (e.g., hepatocyte culture medium) is supplemented with dexamethasone.

[0260] In some embodiments of the methods provided herein, HGF is provided at a concentration that is, is about, is at least, is at least about, does not exceed, or does not exceed about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 1 ng / mL-20 ng / mL, 1 ng / mL-10 ng / mL, 10 ng / mL-20 ng / mL, or 5 ng / mL-15 ng / mL). In some embodiments, HGF is provided at a concentration equal to or about 10 ng / mL.

[0261] In some embodiments of the methods provided herein, OSM is provided at a concentration of, or about, at least, at least about, not exceeding, or not exceeding about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 10 ng / mL-30 ng / mL, 10 ng / mL-20 ng / mL, 20 ng / mL-30 ng / mL, or 15 ng / mL-25 ng / mL). In some embodiments, OSM is provided at a concentration equal to or about 20 ng / mL.

[0262] In some embodiments of the methods provided herein, dexamethasone is provided at a concentration of, or about, at least, at least about, not exceeding, or not exceeding about 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 50 nM-200 nM, 50 nM-100 nM, 100 nM-200 nM, or 50 nM-150 nM). In some embodiments, dexamethasone is provided at a concentration equal to or about 100 nM.

[0263] In some embodiments of the methods provided herein, the cells of step i) and / or step ii) are contacted in a culture medium that also contains EGF. In some embodiments, EGF is provided at a concentration of, or about, at least, at least about, or not exceeding about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 10 ng / mL-30 ng / mL, 10 ng / mL-20 ng / mL, 20 ng / mL-30 ng / mL, or 15 ng / mL-25 ng / mL). In some embodiments, EGF is provided at a concentration equal to or about 20 ng / mL. In some implementations, the cells of step i) and / or step ii) are contacted in a culture medium that does not contain EGF.

[0264] In some embodiments of the method provided herein, the cells in 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 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 within a range defined by any two of the foregoing values, such as 7%-9%, 6%-10%, 5%-12%, 8%-14%, 10%-15%, 4%-15%, 15%-17%, 13%-19%, 12%-24%, or 10%-25%, etc. In some implementations, the supplemented growth medium contains approximately 6%-10%, 8%-14%, 10%-15%, 4%-15%, 15%-17%, 13%-19%, 12%-24%, or 10%-25% of non-essential amino acids by total volume, 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 essential amino acids within the range defined by any two of the foregoing values, such as 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% of essential amino acids by total volume.In some embodiments, the supplemented glycine is provided at a concentration of, wherein the concentration is, about, at least, at least about, or not more than or not more than about 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, or 24 mg / mL. The concentrations are 10 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, or 35 mg / mL, or any concentration within the range defined by any two of the foregoing concentrations (e.g., 19 mg / mL-21 mg / mL, 18 mg / mL-22 mg / mL, 16 mg / mL-24 mg / mL, 10 mg / mL-30 mg / mL, or 5 mg / mL-34 mg / mL). In some embodiments, the supplemented glycine is provided at a concentration equal to or about 18 mg / mL-22 mg / mL or 20 mg / mL. In some embodiments, the growth medium is any standard growth medium or composition thereof commonly used for cell culture. For example, the growth medium is Eagle's Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium with α-Modification (a-MEM), Eagle's Basal Medium (BME), Durbeco Modified Eagle's Medium (DMEM), or Hepatocyte Culture Medium (HCM). For illustrative purposes only, Table 3 depicts the standard non-essential and essential amino acid concentrations in MEM; and 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.

[0265] Table 3. Example amounts of amino acid supplements

[0266] In some embodiments of the method provided herein, the cells in step ii) are further contacted with a low / first concentration of bilirubin, wherein the resulting liver organoids are mature liver organoids. In some embodiments, the low / first concentration of bilirubin is the physiological concentration of bilirubin in a human fetus. In some embodiments, the low / first concentration of bilirubin is about, less than or less than about 0.1 mg / L to 1 mg / L, 0.5 mg / L to 1 mg / L, or 1 mg / L. In some embodiments, the low / first concentration of bilirubin is, or is about, less than or less than about 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, or 1 mg / L, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 0.1 mg / L to 1 mg / L, 0.1 mg / L to 0.5 mg / L, 0.5 mg / L to 1 mg / L, 0.3 mg / L to 0.7 mg / L, or 0.4 mg / L to 0.6 mg / L). In some embodiments, the low / first concentration of bilirubin is, or is about, less than or less than about 0.1 mg / L to 3 mg / L, 0.5 mg / L to 3 mg / L, or 3 mg / L. In some embodiments, the low / first concentration of bilirubin is, is about, less than or less than about 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, 1.25 mg / L, 1.5 mg / L, 1.75 mg / L, 2.0 mg / L, 2.25 mg / L, 2.5 mg / L, 2.75 mg / L or 3.0 mg / L, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 0.1 mg / L to 3 mg / L, 0.5 mg / L to 2.0 mg / L, 0.5 mg / L to 1.5 mg / L, 0.3 mg / L to 2.5 mg / L or 0.5 mg / L to 1.75 mg / L). In some embodiments, mature liver organoids exhibit luminal projections similar to bile canaliculi and / or have a single lumen and are typically spherical in shape. In some embodiments, compared to liver organoids not exposed to a low / first dose of bilirubin, mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof. In some embodiments, compared to liver organoids not exposed to a low / first dose of bilirubin, mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof. In some embodiments, mature liver organoids express CYP2E1, CYP7A1, PROX1, MRP3, MRP3, or OATP2, or any combination thereof.In some implementations, mature liver organoids exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids that have not been exposed to low-dose / first-dose bilirubin.

[0267] In some embodiments of the method provided herein, the cells in step ii) are further contacted with a high / second concentration of bilirubin, wherein the resulting liver organoids are hyperbilirubinemic liver organoids. In some embodiments, when used in the method disclosed herein, the liver organoids are, are about, are at least or are at least about 18 days old, 19 days old, 20 days old, 21 days old, 22 days old, 23 days old, 24 days old, 25 days old, 26 days old, 27 days old, 28 days old, 29 days old, 30 days old, 31 days old, 32 days old, 33 days old, 34 days old, or 35 days old, or a range defined by any two of the foregoing values, such as 18-35 days, 18-30 days, 20-25 days, or 18-25 days. In some implementations, the high / secondary concentration of bilirubin is approximately, greater than or greater than approximately 2 mg / L-10 mg / L, 5 mg / L-10 mg / L, 10 mg / L, or 20 mg / L. In some embodiments, the high / second concentration of bilirubin is, is about, greater than or greater than about 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L or 20 mg / L, or any concentration within a range defined by any two of the aforementioned concentrations (e.g., 2 mg / L to 20 mg / L, 2 mg / L to 10 mg / L, 10 mg / L to 20 mg / L, 5 mg / L to 15 mg / L or 8 mg / L to 12 mg / L). In some embodiments, liver organoids are exposed to high / secondary concentrations of bilirubin for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or a range defined by any two of the foregoing values, such as 1-10 days, 1-5 days, 3-8 days, 5-10 days, or 7-10 days, to form hyperbilirubinemic liver organoids. In some embodiments, compared to liver organoids not treated with high / secondary concentrations of bilirubin, hyperbilirubinemic liver organoids express elevated levels of UGT1A1 or NRF2, or both.

[0268] In some embodiments of the methods provided herein, liver organoids comprise a functional L-gulonolactone oxidase (GULO) protein and / or a gene or mRNA encoding a functional GULO protein, or both, wherein the liver organoids are 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-inducible system or any other system commonly known in the art for conditional expression. In some embodiments, liver organoids are genetically engineered to encode a functional GULO protein using CRISPR or any other genetic engineering method commonly known in the art. In some embodiments, the gene or mRNA encoding a functional GULO protein, or both, is introduced into mature liver organoids via transfection. In some embodiments, liver organoids containing a functional GULO protein express increased levels of NRF2 compared to liver organoids that do not contain a functional GULO protein. In some embodiments, liver organoids containing functional GULO proteins optionally express reduced levels of IL1B, IL6, or TNFα, or any combination thereof, when cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid, compared to liver organoids not containing functional GULO proteins. In some embodiments, liver organoids containing functional GULO proteins optionally exhibit reduced caspase-3 activity when cultured in ascorbic acid-depleted medium or in the absence of ascorbic acid, compared to liver organoids not containing functional GULO proteins. In some embodiments, liver organoids containing functional GULO proteins express increased levels of ALB, compared to liver organoids not containing functional GULO proteins. In some embodiments, liver organoids containing functional GULO proteins resemble periportal liver tissue and express periportal liver markers. In some embodiments, periportal markers include FAH, ALB, PAH, CPS1, HGD, or any combination thereof. In some embodiments, liver organoids containing functional GULO proteins exhibit increased CYP3A4 and CYP1A2 activity compared to liver organoids without functional GULO proteins. In some embodiments, liver organoids containing functional GULO proteins exhibit increased bilirubin conjugation activity compared to liver organoids without functional GULO proteins. In some embodiments, liver organoids containing functional GULO proteins exhibit increased viability in culture compared to liver organoids without functional GULO proteins.In some implementations, liver organoids have been differentiated from pluripotent stem cells containing a functional GULO protein and / or a gene or mRNA encoding the functional GULO protein, or both, thereby enabling the pluripotent stem cells to synthesize ascorbic acid.

[0269] In some embodiments of the methods provided herein, the liver organoids contain an inactive UGT1A1 gene, wherein the liver organoids are a model of Crigler-Najjar syndrome.

[0270] In some embodiments of the methods provided herein, these methods further include, prior to step i), the aggregation of hindforegut cells and / or hindforegut endoderm cells and / or foregut endoderm cells in microwells or other devices (e.g., Aggrewell). An exemplary schematic diagram for culturing liver organoids from, for example, hindforegut cells and / or hindforegut endoderm cells aggregated in microwells is illustrated in… Figure 5I In some embodiments, each aggregate of hindbrain cells and / or hindbrain endoderm cells and / or foregut endoderm cells comprises about 250, about 500, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 7500, about 8000, about 8500, about 9000, about 9500, or about 10000 hindbrain cells and / or hindbrain endoderm cells, or any number of hindbrain cells and / or hindbrain endoderm cells within any two of the aforementioned cell numbers. In some embodiments, aggregating hindforegut cells and / or hindforegut endoderm cells and / or foregut endoderm cells produces liver organoids of more uniform size. Further information regarding methods for aggregating hindforegut cells and / or hindforegut endoderm cells to produce liver organoids of more uniform size, such as using micropores or other devices (such as Aggrewell), can be found in PCT Publication WO 2021 / 030373, which is hereby expressly incorporated herein by reference in its entirety.

[0271] In some embodiments of the methods provided herein, the cells of step i) and / or step ii) are not cultured with basement membrane matrix or components thereof. In some embodiments, the cells of step i) and / or step ii) are not cultured with human xenogeneic basement membrane matrix or components thereof. In some embodiments, the cells of step i) and / or step ii) are not cultured with basement membrane matrix or components thereof isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. In some embodiments, the cells of step i) and / or step ii) are not cultured with Matrigel... ® Cultrex® Or Geltrex ® touch.

[0272] In some embodiments of the methods provided herein, the cells of step i) and / or step ii) are cultured in a non-static bioreactor. In some embodiments, the cells of step i) and / or step ii) are cultured in a rotating bioreactor.

[0273] In some embodiments of the methods provided herein, these methods also include cryopreservation of liver organoids. In some embodiments, cryopreservation of liver organoids includes slow freezing or vitrification. In some embodiments, the liver organoids are cryopreserved together with chroman 1, enrickasan, polyamines, and trans-ISRIB (CEPT). In some embodiments, chroman 1 is provided at a concentration equal to or about 50 nM. In some embodiments, enrickasan is provided at a concentration equal to or about 5 μM. In some embodiments, polyamines are provided at a concentration equal to or about 1:1000. In some embodiments, trans-ISRIB is provided at a concentration equal to or about 7 μM.

[0274] This article discloses liver organoids produced using the methods disclosed herein.

[0275] When applied to any of the cells disclosed herein, such as pluripotent stem cells, morphological endoderm, hindbrain, hindbrain 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 morphological endoderm, hindbrain, hindbrain endoderm, and / or liver organoids may be derived from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells.

[0276] How to use Methods for administering any of the liver organoids disclosed herein to a subject in need are also disclosed. Methods for treating liver-related diseases or conditions in subjects in need are also disclosed. In some embodiments, these methods include administering any of the liver organoids disclosed herein to a subject. In some embodiments, the liver organoids have been generated from cells derived from the subject. In some embodiments, the cells derived from the subject are induced pluripotent stem cells.

[0277] This document also discloses methods for screening. In some embodiments, these methods include contacting any liver organoids disclosed herein 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 are models of liver-related diseases or conditions, and evaluating the effect of the candidate compound or composition on the liver organoids includes evaluating the effect of the candidate compound or composition on the liver-related diseases or conditions. In some embodiments, the liver organoids have been generated 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 suffers from a liver-related disease or condition.

[0278] Liver-related diseases and symptoms The liver organoids disclosed herein can be used for the treatment and / or research or modeling of liver-related diseases and conditions. In some embodiments, these methods include administering any of the liver organoids or hepatocytes disclosed herein. The liver organoids or hepatocytes disclosed herein are also disclosed for the manufacture of medicaments for the treatment of liver-related diseases or conditions. Furthermore, the liver organoids or hepatocytes disclosed herein are disclosed for the treatment of liver-related diseases or conditions in subjects of need.

[0279] Liver-related diseases and conditions related to this disclosure may include conditions such as: liver dysfunction and / or failure (e.g., hyperammonemia and / or hyperbilirubinemia), hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, TT hepatitis and / or autoimmune hepatitis), viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, and metabolic diseases (e.g., liver diseases related to metabolic dysfunction). This includes liver organoids disclosed herein, which may be associated with other liver-related diseases and conditions such as MASLD, MetALD, non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), autoimmune liver diseases, Wilson's disease, metabolic-associated fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Najjar syndrome, urea cycle disorders, Wolman's disease, hepatocellular carcinoma, hepatoblastoma, drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic diseases, liver cysts, and / or alcohol-related liver diseases. Those skilled in the art will understand that the liver organoids disclosed herein may be associated with other liver-related diseases and conditions.

[0280] For example, liver organoids can be transplanted into subjects with liver dysfunction and / or failure, where the transplanted liver organoids are implanted into the subject's liver. After transplantation, subjects may have reduced serum bilirubin and / or ammonia levels, and / or increased serum protein levels, and / or improved symptoms of bile duct stricture and / or liver regeneration, and may also have increased survival rates.

[0281] For example, these liver organoids can be used in in vitro human model systems to study hepatocyte function and developmental divergences, investigate liver-related diseases, identify and / or screen therapeutic targets, and / or identify therapeutic compounds and / or compositions that are effective in treating liver-related diseases or conditions. Therefore, the liver organoids disclosed herein may allow for new developments in liver disease treatment and research.

[0282] Composition In some embodiments, compositions for carrying out any of the methods disclosed herein are also provided. In some embodiments, compositions produced according to processes provided in any of the methods disclosed herein are also provided. It is explicitly understood that in some embodiments, any method or composition described herein may be implemented relative to any other method or composition described herein, and different embodiments may be combined.

[0283] In some embodiments, this document provides compositions comprising foregut cells and / or foregut endoderm cells and / or liver organoids produced using any one or more methods provided herein. In some embodiments, this document provides compositions comprising foregut cells and / or foregut endoderm cells that have spontaneously formed three-dimensional (3D) spheroids, optionally wherein the spheroids comprise structures having a single lumen.

[0284] In some embodiments, the compositions provided herein are in vitro compositions generated outside a multicellular living organism. In some embodiments, the compositions provided herein are exogenous compositions that can be introduced into a multicellular living organism. In some embodiments, the compositions provided herein comprise exogenously provided components, reagents, and / or conditions that mimic the in vivo properties required to induce specific cell differentiation and / or organoid tissues.

[0285] In some embodiments, this document provides in vitro compositions comprising: hindbrain cells and / or hindbrain 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 compositions may further comprise endogenous TGF-β pathway inhibitors, FGF pathway activators, Wnt pathway activators, and / or VEGF pathway activators. In some embodiments, the compositions comprise hindbrain cells and / or hindbrain endoderm cells dissociated from monolayers and / or globules. In some embodiments, the compositions comprise hindbrain cells and / or hindbrain endoderm cells with a cell density greater than or equal to, exactly or about 1 × 10⁻⁶. 5 1, 2×10 5 1, 3×10 5 1, 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 5 1×10 6 1, 2×10 6 1, 3×10 6 1, 4×10 6 One or 5×10 6 cells / cm 2 The surface area of ​​the tissue culture surface, or any cell density within a range defined by any two of the aforementioned cell densities.

[0286] In some embodiments, this document provides compositions comprising a tissue culture surface coated with a basement membrane matrix or a component thereof. In some embodiments, the basement membrane matrix or a component thereof does not contain non-human animal components. In some embodiments, the basement membrane matrix or a component thereof does not contain non-human animal components that would make the basement membrane matrix or a component thereof xenogeneic to humans. In some embodiments, the basement membrane matrix or a component thereof is not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, and is not Matrigel... ® It's not Cultrex. ® And / or not Geltrex ® In some embodiments, the basement membrane matrix or its components comprise human adhesion proteins, collagen IV, nestin, basement membrane glycans, fibrin, and / or hydrogel.

[0287] In some embodiments, this document provides compositions comprising an exogenous TGF-β pathway inhibitor. In some embodiments, the exogenous TGF-β pathway inhibitor comprises, or is substantially composed of, A83-01, RepSox, LY365947, and / or SB431542. In some embodiments, the exogenous TGF-β pathway inhibitor comprises, or is substantially composed of, the TGF-β pathway inhibitor A83-01. In some embodiments, the composition comprises a TGF-β pathway inhibitor at a concentration equal to or about 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM, or at any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises a TGF-β pathway inhibitor at a concentration equal to or about 500 nM.

[0288] In some embodiments, this document provides compositions comprising exogenous FGF pathway activators. In some embodiments, the exogenous FGF pathway activator comprised, substantially comprised of, or comprised of the following: FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, 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 activator comprises, substantially comprises of, or comprises of FGF2. In some embodiments, the composition comprises an FGF pathway activator at a concentration equal to or about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, or 10 ng / mL, or any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises an FGF pathway activator at a concentration equal to or about 5 ng / mL.

[0289] In some embodiments, this document provides compositions comprising exogenous Wnt pathway activators. In some embodiments, the composition comprises an exogenous Wnt pathway activator comprising, substantially comprising, or comprising the following: Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloxin, indirubin, atporon, tamparone, lithium chloride, TDZD 8, and / or TWS119. In some embodiments, the composition comprises, is substantially composed of, or is composed of the exogenous Wnt pathway activator CHIR99021. In some embodiments, the composition comprises a Wnt pathway activator at a concentration equal to or about 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, or 8 μM, or at any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises a Wnt pathway activator at a concentration equal to or about 3 μM.

[0290] In some embodiments, this document provides compositions comprising an exogenous VEGF pathway activator. In some embodiments, the exogenous VEGF pathway activator comprised, substantially composed of, or composed of VEGF and / or GS4012. In some embodiments, the exogenous VEGF pathway activator comprised, substantially composed of, or composed of VEGF. In some embodiments, the composition comprises a VEGF pathway activator at a concentration equal to or about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL, or at any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises a VEGF pathway activator at a concentration equal to or about 10 ng / mL.

[0291] In some embodiments, this document provides compositions comprising exogenous EGF. In some embodiments, this document provides compositions not comprising exogenous EGF. In some embodiments, this document provides compositions comprising EGF at a concentration equal to or about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL, or at any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, this document provides compositions comprising EGF at a concentration equal to or about 20 ng / mL.

[0292] In some embodiments, this document provides compositions comprising exogenous ascorbic acid and / or genetically modified ascorbic acid. In some embodiments, this document provides compositions excluding exogenous ascorbic acid and / or genetically modified ascorbic acid. In some embodiments, this document provides compositions comprising ascorbic acid at concentrations equal to or about 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL, or at any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, this document provides compositions comprising ascorbic acid at concentrations equal to or about 50 μg / mL.

[0293] In some embodiments, this document provides compositions comprising a ROCK inhibitor. In some embodiments, this document provides compositions not comprising a ROCK inhibitor. In some embodiments, the ROCK inhibitor comprises, is substantially composed of, or is composed of Y-27632. In some embodiments, this document provides compositions comprising a ROCK inhibitor at a concentration equal to or about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM, or at any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, this document provides compositions comprising a ROCK inhibitor at a concentration equal to or about 10 μM.

[0294] In some embodiments, this document provides compositions comprising foregut cells and / or foregut endoderm cells that have differentiated from and / or are currently differentiating from stem cells. In some embodiments, this document provides compositions comprising foregut cells and / or foregut endoderm cells that have differentiated from and / or are currently differentiating from induced pluripotent stem cells. In some embodiments, this document provides compositions comprising foregut cells and / or foregut endoderm cells that have been passaged 1, 2, or 3 times. In some embodiments, this document provides compositions comprising foregut cells and / or foregut endoderm cells that have been passaged fewer than 4 times.

[0295] In some embodiments, this document provides compositions comprising A83-01, FGF2, CHIR99021, VEGF and / or Y-27632, and optionally also comprising iPSCs, PSCs and / or hindbrain cells and / or hindbrain endoderm cells.

[0296] In some embodiments, compositions are provided herein comprising: a) hindgut cells and / or hindgut 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, and wherein the composition optionally additionally comprises 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, substantially comprising, or comprising of: hepatocyte growth factor (HGF), PG-001, forgonide, trevallefime, recombinant InlB321 protein and / or an agonistic c-Met antibody (e.g., LMH85).

[0297] In some embodiments, this document provides compositions comprising IL-6 family cytokines. In some embodiments, the IL-6 family cytokines comprise, are substantially composed of, or consist of the following: IL-6, oncogene M (OSM), leukemia suppressor factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and / or cardiotrophin-like cytokines (CLC).

[0298] In some embodiments, this document provides compositions comprising corticosteroids. In some embodiments, the corticosteroid comprises, is substantially composed of, or is composed of: dexamethasone, beclomethasone, betamethasone, fluocinolone, halometasone, and / or mometasone.

[0299] In some embodiments, this document provides compositions comprising hepatocyte culture media supplemented with HGF, OSM, and / or dexamethasone. In some embodiments, this document provides compositions comprising hepatocyte culture media supplemented with dexamethasone. In some embodiments, this document provides compositions comprising hepatocyte culture media supplemented with HGF. In some embodiments, this document provides compositions comprising hepatocyte culture media supplemented with OSM.

[0300] In some embodiments, compositions comprising a retinoic acid pathway activator are provided herein. In some embodiments, the retinoic acid pathway activator comprises, is substantially composed of, or is composed of the following: retinoic acid, all-trans-retinoic acid, 9-cis-retinoic acid, CD437, EC23, BS 493, TTNPB, and / or AM580. In some embodiments, the retinoic acid pathway activator comprises, is substantially composed of, or is composed of retinoic acid. In some embodiments, the composition comprises a retinoic acid pathway activator at a concentration equal to or about 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, or 3.0 μM, or any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises a retinoic acid pathway activator at a concentration equal to or about 2.0 μM.

[0301] In some embodiments, the composition comprises HGF. In some embodiments, the composition comprises HGF at a concentration equal to or about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL, or any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises HGF at a concentration equal to or about 10 ng / mL.

[0302] In some embodiments, the composition comprises OSM. In some embodiments, the composition comprises OSM at a concentration equal to or about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL, or any concentration within the range defined by any two of the foregoing concentrations. In some embodiments, the composition comprises OSM at a concentration equal to or about 20 ng / mL.

[0303] In some embodiments, the composition comprises dexamethasone. In some embodiments, the composition comprises dexamethasone at a concentration equal to or about 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM, or any concentration within the range defined by any two of the aforementioned concentrations. In some embodiments, the composition comprises dexamethasone at a concentration equal to or about 100 nM.

[0304] In some embodiments, the composition comprises exogenous bilirubin. In some embodiments, the composition comprises both exogenous and endogenous bilirubin. In some embodiments, the composition comprises a low concentration of exogenous bilirubin. In some embodiments, the low concentration of exogenous bilirubin is at or near the physiological bilirubin concentration in human fetuses. Human fetal bilirubin levels are typically considered to be about 1 mg / L (0.1 mg / dL), which rises rapidly to 3 mg / L-10 mg / L (0.3 mg / dL-1.0 mg / dL) within 24 hours after birth. In some embodiments, the composition comprises exogenous and / or endogenous bilirubin in the form of, about, less than, or less than about: 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, 1.25 mg / L, 1.5 mg / L, 1.75 mg / L, 2.0 mg / L, 2.25 mg / L, 2.5 mg / L, 2.75 mg / L, or 3.0 mg / L, or a range defined by any two of the foregoing concentrations (e.g., 0.1 mg / L to 3 mg / L, 0.5 mg / L to 2.0 mg / L). Any concentration within the range of 0.1 mg / L, 0.2 mg / L, 0.3 mg / L to 2.5 mg / L, or 0.5 mg / L to 1.75 mg / L; or any concentration within the range defined by any two of the foregoing concentrations (e.g., 0.1 mg / L to 1 mg / L, 0.1 mg / L to 0.5 mg / L, 0.5 mg / L to 1 mg / L, 0.3 mg / L to 0.7 mg / L, or 0.4 mg / L to 0.6 mg / L).In some embodiments, the composition comprises a concentration of exogenous bilirubin, which is, is about, is less than, or is less than about: 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, 1.25 mg / L, 1.5 mg / L, 1.75 mg / L, 2.0 mg / L, 2.25 mg / L, 2.5 mg / L, 2.75 mg / L, or 3.0 mg / L, or a range defined by any two of the foregoing concentrations (e.g., 0.1 mg / L to 3 mg / L, 0.5 mg / L to 2.0 mg / L, ...). Any concentration within the range of 0.5 mg / L to 1.5 mg / L, 0.3 mg / L to 2.5 mg / L, or 0.5 mg / L to 1.75 mg / L; or any concentration within the range of 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, or 1 mg / L, or any range defined by any two of the foregoing concentrations (e.g., 0.1 mg / L to 1 mg / L, 0.1 mg / L to 0.5 mg / L, 0.5 mg / L to 1 mg / L, 0.3 mg / L to 0.7 mg / L, or 0.4 mg / L to 0.6 mg / L).

[0305] 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 having a single lumen and generally a spherical shape are provided herein. In some embodiments, compositions comprising mature liver organoids produced by 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, relative to liver organoids not contacted with a low dose of bilirubin are provided herein. In some embodiments, compositions comprising mature liver organoids expressing increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to liver organoids not contacted with a low dose of bilirubin are provided herein. 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 are provided, which, compared to liver organoids not exposed to a low dose of bilirubin, exhibit increased CYP3A4 and / or CYP1A2 activity. In some embodiments, compositions comprising mature liver organoids are provided herein, wherein the cells of the mature liver organoids are exposed to a low dose of exogenous bilirubin, and the mature liver organoids exhibit luminal projections similar to bile canaliculi, and / or have a single lumen and generally a spherical shape. In some embodiments, compositions comprising mature liver organoids are provided herein, wherein, compared to liver organoids in which the cells are not exposed to a low dose of bilirubin, the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof. In some embodiments, compositions comprising mature liver organoids are provided herein, wherein, compared to liver organoids in which the cells are not exposed to a low dose of bilirubin, the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof. In some embodiments, compositions comprising mature liver organoids are provided herein, the mature liver organoids expressing CYP2E1, CYP7A1, PROX1, MRP3, MRP3, or OATP2, or any combination thereof. In some embodiments, compositions comprising mature liver organoids are provided herein, the mature liver organoids exhibiting increased CYP3A4 and / or CYP1A2 protein levels and / or enzyme activity, relative to liver organoids in which cells have not been exposed to low doses of bilirubin.

[0306] In some embodiments, this document provides compositions comprising hyperbilirubinemia liver organoids, wherein the hyperbilirubinemia liver organoid cells are contacted with a high concentration and / or a second concentration of bilirubin. In some embodiments, this document provides compositions comprising hyperbilirubinemia liver organoids, wherein the high concentration and / or second concentration of bilirubin is, is about, greater than, or greater than about: 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L, or a range defined by any two of the foregoing concentrations (e.g., 2 mg / L to 20 mg / L, 2 mg / L to 10 mg / L, 10 mg / L to 20 mg / L). Any concentration within the range of 4 mg / L, 5 mg / L to 15 mg / L or 8 mg / L to 12 mg / L; or any concentration within the range of 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L or 20 mg / L, or any concentration within the range defined by any two of the foregoing concentrations (e.g., 4 mg / L to 20 mg / L, 2 mg / L to 10 mg / L, 10 mg / L to 20 mg / L, 5 mg / L to 15 mg / L or 8 mg / L to 12 mg / L). In some embodiments, this document provides compositions comprising hyperbilirubinemic liver organoids, wherein, relative to liver organoids not treated with high / secondary concentrations of bilirubin, the hyperbilirubinemic liver organoids express elevated levels of UGT1A1 or NRF2 or both.

[0307] In some embodiments, this document also provides compositions comprising foregut cells and / or foregut endoderm cells, liver organoids and / or mature liver organoids engineered to contain a functional L-gulonolactone oxidase (GULO) protein and / or a gene or mRNA encoding a functional GULO protein, or both, wherein the foregut cells and / or foregut endoderm cells, liver organoids and / or mature liver organoids are capable of synthesizing ascorbic acid. In some embodiments, this document provides compositions comprising foregut cells and / or foregut endoderm cells, liver organoids and / or mature liver organoids engineered to express a functional GULO protein, wherein 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-inducible system.

[0308] In some embodiments, this document provides compositions comprising foregut cells and / or foregut endoderm cells, liver organoids, and / or mature liver organoids engineered using CRISPR-mediated knock-in engineering to contain a gene encoding a functional GULO protein. In some embodiments, this document provides compositions comprising foregut cells and / or foregut endoderm cells, liver organoids, and / or mature liver organoids containing a functional GULO-encoding gene or mRNA, or both, encoding a functional GULO protein, wherein the functional gene is introduced into the foregut cells and / or foregut endoderm cells, liver organoids, mature liver organoids, and / or precursor cells via transfection. In some embodiments, this document provides compositions comprising foregut cells and / or foregut endoderm cells, liver organoids, and / or mature liver organoids engineered using adenovirus-mediated gene transfection engineering to contain a gene encoding a functional GULO protein. In some embodiments, this document provides compositions comprising hindgut cells and / or hindgut endoderm cells, liver organoids and / or mature liver organoids engineered using adeno-associated virus-mediated gene transfection to contain genes encoding functional GULO proteins.

[0309] In some embodiments, the compositions provided herein comprise liver organoids and / or mature liver organoids containing a functional GULO protein, wherein, relative to liver organoids and / or mature liver organoids not containing a functional GULO protein, the liver organoids and / or mature liver organoids express increased levels of NRF2. In some embodiments, the compositions provided herein comprise liver organoids and / or mature liver organoids containing a functional GULO protein, wherein, relative to liver organoids and / or mature liver organoids not containing a functional GULO protein, these liver organoids and / or mature liver organoids express decreased levels of IL1β, IL6, or TNFα, or any combination thereof. In some embodiments, liver organoids and / or mature liver organoids containing a functional GULO protein exhibit decreased caspase-3 activity relative to liver organoids and / or mature liver organoids not containing a functional GULO protein. In some embodiments, liver organoids containing functional GULO proteins and / or mature liver organoids express increased levels of ALB compared to liver organoids and / or mature liver organoids that do not contain functional GULO proteins. In some embodiments, liver organoids and / or mature liver organoids containing functional GULO proteins resemble periportal liver tissue and / or express periportal liver markers. In some embodiments, periportal liver markers include FAH, ALB, PAH, CPS1, HGD, or any combination thereof, 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 proteins exhibit increased CYP3A4 and / or CYP1A2 protein levels and / or enzyme activity compared to liver organoids and / or mature liver organoids that do not contain functional GULO proteins. In some embodiments, liver organoids containing functional GULO proteins and / or mature liver organoids exhibit increased bilirubin conjugation activity compared to liver organoids and / or mature liver organoids that do not contain functional GULO proteins. In some embodiments, liver organoids containing functional GULO proteins and / or mature liver organoids exhibit increased viability in culture compared to liver organoids and / or mature liver organoids that do not contain functional GULO proteins. In some embodiments, the liver organoids and / or mature liver organoids have been differentiated from pluripotent stem cells containing functional GULO proteins and / or genes or mRNA encoding functional GULO proteins, or both, thereby enabling the pluripotent stem cells to synthesize ascorbic acid.

[0310] In some embodiments, compositions according to Table 1, Table 2 or Table 3 are also provided herein.

[0311] In some embodiments, compositions comprising a liquid component of supplemental amino acids are provided herein, the liquid component comprising exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by volume of a solution of non-essential amino acids (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), by volume A solution containing exactly or approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of essential amino acids (containing exactly or approximately 6320 mg / L arginine, 1200 mg / L cysteine, 2100 mg / L histidine, 2620 mg / L isoleucine, 2620 mg / L leucine, 3625 mg / L lysine, 755 mg / L methionine, 1650 mg / L phenylalanine, 2380 mg / L threonine, 510 mg / L tryptophan, 1800 mg / L tyrosine, and 2... 340 mg / L valine) and hepatocyte culture medium (HCM) at exactly or approximately 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% by volume, supplemented with exactly or approximately 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, or 11 mg / mL. , 12mg / mL, 13mg / mL, 14mg / mL, 15mg / mL, 16mg / mL, 17mg / mL, 18mg / mL, 19mg / mL, 20mg / mL, 21mg / mL, 22mg / mL, 23mg / mL, 2 4mg / mL, 25mg / mL, 26mg / mL, 27mg / mL, 28mg / mL, 29mg / mL, 30mg / mL, 31mg / mL, 32mg / mL, 33mg / mL, 34mg / mL or 35mg / mL glycine.

[0312] In some embodiments, this document provides compositions comprising a liquid component of supplemental amino acids, the liquid component comprising exactly or about 14% of a solution of non-essential amino acids (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% by volume of a solution of essential amino acids (containing exactly or about 6320 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% by volume of a solution of essential amino acids (containing exactly or about 6320 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). The composition contains arginine (1200 mg / L cysteine, 2100 mg / L histidine, 2620 mg / L isoleucine, 2620 mg / L leucine, 3625 mg / L lysine, 755 mg / L methionine, 1650 mg / L phenylalanine, 2380 mg / L threonine, 510 mg / L tryptophan, 1800 mg / L tyrosine, and 2340 mg / L valine) and hepatocyte culture medium (HCM) at exactly or about 80% by volume, supplemented with exactly or about 20 g / L glycine. In some embodiments, the composition provided herein may have a pH between about 6 and 8, or between 6.5 and 7.5, or exactly or about 7.0. In some embodiments, the composition provided herein contains hepatocyte growth factor (HGF), oncogene M, dexamethasone, and / or ascorbic acid.

[0313] In some embodiments, the compositions provided herein comprise liver lineage-typed cells differentiated from defined endoderm cells using retinoic acid. In some embodiments, the compositions provided herein comprise liver lineage-typed cells characterized as liver organoids. In some embodiments, the compositions provided herein comprise liver organoids characterized as secreting increased levels of albumin and / or urea relative to liver organoids contained in HCMs without amino acid supplementation. In some embodiments, the compositions provided herein comprise liver organoids characterized as expressing increased levels of liver maturation-related gene expression relative to liver organoids contained in HCMs without amino acid supplementation. In some embodiments, the compositions provided herein comprise liver organoids characterized as expressing decreased levels of vimentin relative to liver organoids contained in HCMs without amino acid supplementation. In some embodiments, the compositions provided herein explicitly do not contain non-human animal basement membrane matrix or components thereof. In some embodiments, the compositions provided herein explicitly do not contain mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, Matrigel... ® Cultrex ® and / or Geltrex ® .

[0314] In some embodiments, this document also provides hyperbilirubinemic liver organoids comprising naturally occurring and / or engineered mutations of UDP-glucuronyltransferase family 1 member A1 (UGT1A1). In some embodiments, this document provides hyperbilirubinemic liver organoids produced by exogenous bilirubin exposure of precursor cells, precursor liver organoids, and / or precursor mature liver organoids to at least two rounds. In some embodiments, this document provides hyperbilirubinemic liver organoids produced from clonal-derived cells and / or iPSCs.

[0315] In some embodiments, this document provides compositions for cryopreservation of liver organoids, chroman I, enledgeridine, polyamines, and trans-ISRIB (CEPT). In some embodiments, this document provides compositions for cryopreservation of mature liver organoids, chroman I, enledgeridine, polyamines, and trans-ISRIB (CEPT). In some embodiments, this document provides compositions for cryopreservation of hyperbilirubinemia liver organoids, chroman I, enledgeridine, polyamines, and trans-ISRIB (CEPT).

[0316] Pharmaceutical compositions according to various embodiments of this disclosure may include one or more additional pharmaceutically acceptable components that may include carriers, excipients, and / or stabilizers that are non-toxic or have acceptable levels of toxicity to cells or mammals exposed thereto at the doses and concentrations employed. As used herein, “pharmaceutically acceptable,” “diluent,” “excipient,” and / or “carrier” have their common and conventional meanings as understood from the specification and are intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents compatible with administration to human, cat, dog, or other vertebrate hosts. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are those approved by federal, state, or other regulatory agencies, or listed in the United States Pharmacopeia or other recognized pharmacopoeias for use in animals (including humans and non-human mammals such as cats and dogs). The terms diluent, excipient, and / or “carrier” may refer to a diluent, adjuvant, excipient, or catalyst administered with the pharmaceutical composition. Such drug diluents, excipients, and / or carriers can be sterile liquids, such as water and oils, including those from petroleum, animal, vegetable, or synthetic sources. Water, saline solutions, and aqueous solutions of glucose and glycerol can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable drug diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. A non-limiting example of a physiologically acceptable carrier is a pH-buffered aqueous solution. Physiologically acceptable carriers may also include one or more of the following: antioxidants, such as ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin; gelatin; immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids; carbohydrates, such as glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and nonionic surfactants, such as TWEEN. ® Polyethylene glycol (PEG) and PLURONICS ® If desired, the composition may also contain small amounts of wetting agents, swelling agents, emulsifiers, or pH buffers. These compositions may be in the form of solutions, suspensions, emulsions, sustained-release formulations, etc. The formulation should be suitable for the method of application.

[0317] Other excipients with the desired 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, glucose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium tauride deoxycholate, magnesium stearate, octylphenol ethoxylate, benzyl chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be residual or contaminants from the manufacturing process, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components, or any combination thereof. The amount of excipients in the composition may be present in the composition as a percentage of, about, at least, at least about, not more than or not more than about 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, or any weight percentage within the range defined by any two of the above figures.

[0318] Pharmaceutical compositions may include one or more "pharmaceutically acceptable salts," which may include relatively non-toxic inorganic and organic acid or base addition salts of the composition or excipient, including but not limited to analgesics, therapeutic agents, other materials, etc. Examples of pharmaceutically acceptable salts include those derived from inorganic acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Examples of suitable inorganic bases for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, etc. Salts may also form with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, such organic bases in this category may include, but are not limited to, monoalkylamines, dialkylamines, and trialkylamines, including methylamine, dimethylamine, and triethylamine; monohydroxyalkylamines, dihydroxyalkylamines, or trihydroxyalkylamines, including monoethanolamine, diethanolamine, and triethanolamine; amino acids, including glycine, arginine, and lysine; guanidine; N-methylglucosamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenylethylamine; and tris(hydroxymethyl)aminoethane.

[0319] Appropriate formulations depend on the chosen route of administration. Techniques for formulating and administering the compounds described herein are known to those skilled in the art. Various techniques exist in the art for administering compounds, including but not limited to enteric, oral, rectal, topical, sublingual, oral, intraauricular, epidural, epidermal, aerosol, and parenteral delivery, encompassing intramuscular, subcutaneous, intraarticular, intravenous, portal venous, intra-articular, intradermal, intraperitoneal, intramedullary, intrathecal, direct intracardiac, intraperitoneal, intranasal, or intraocular injection. Pharmaceutical compositions are typically tailored to the specific intended route of administration.

[0320] As used herein, “carrier” has its common and conventional meaning as understood in accordance with the specification, and may refer to a compound, particle, solid, semi-solid, liquid or diluent that facilitates the passage, delivery and / or incorporation of a compound into cells, tissues and / or organs of the body.

[0321] As used herein, “diluent” has its common and conventional meaning as understood according to the instructions for use, and may refer to a pharmaceutical composition component that lacks pharmacological activity but may be pharmaceutically necessary or desired. For example, a diluent may be used to increase the volume of a potent drug that is too small to manufacture and / or administer. A diluent may also be a liquid used to dissolve a drug for administration by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate-buffered saline solution that mimics the composition of human blood.

[0322] Dosage and route of administration Embodiments of this disclosure may include methods of administering or treating animals, which may involve administering a controlled dose of at least one treatment that produces a desired physiological effect in an organism that has or is suspected of having or is susceptible to a disease, symptom, or condition. In some embodiments, the disease, symptom, or condition may be a liver-related disease or condition.

[0323] In some embodiments, at least one treatment may include a composition or pharmaceutical composition that may be administered to an animal (e.g., a mammal, primate, monkey, or human) in an amount of about 0.005 mg / kg body weight to about 50 mg / kg body weight, about 0.01 mg / kg body weight to about 15 mg / kg body weight, about 0.1 mg / kg body weight to about 10 mg / kg body weight, about 0.5 mg / kg body weight 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. Under certain conditions, the dosage may be approximately 0.5 mg / kg of human body weight or approximately 6.5 mg / kg of human body weight. In some cases, some subjects (e.g., mammals, mice, rabbits, cats, pigs, or dogs) may be administered at doses of approximately 0.005 mg / kg of body weight to approximately 50 mg / kg of body weight, approximately 0.01 mg / kg of body weight to approximately 15 mg / kg of body weight, approximately 0.1 mg / kg of body weight to approximately 10 mg / kg of body weight, approximately 0.5 mg / kg of body weight to approximately 7 mg / kg of 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. Of course, those skilled in the art will understand that many concentrations can be used in the methods of this disclosure, and that any number of concentrations can be adjusted and tested in part using the guidance provided herein in order to find the concentration that achieves the desired results under given conditions. In some embodiments, the dose or therapeutically effective dose of the compound disclosed herein will be a dose sufficient to achieve plasma concentrations of the compound or its active metabolites within the range set forth herein, such as about 1 nM-10 nM, 10 nM-100 nM, 0.1 μM-1 μM, 1 μM-10 μM, 10 μM-100 μM, 100 μM-200 μM, 200 μM-500 μM, or even 500 μM-1000 μM, preferably about 1 nM-10 nM, 10 nM-100 nM, or 0.1 μM-1 μM.

[0324] In other implementations, the treatment may be administered in combination with one or more other therapeutic agents for a given disease, symptom, or condition.

[0325] The compounds and pharmaceutical compositions are preferably prepared and administered in dosage units. Solid dosage units are tablets, capsules, and suppositories. For the treatment of a subject, different daily doses may be used depending on the activity of the compound, the route of administration, the nature and severity of the disease or symptom, the subject's age, and weight.

[0326] However, in some cases, higher or lower daily doses may be appropriate. Daily doses can be administered by a single dose as a single dose unit or several smaller dose units, or by administering the dose in multiple smaller doses at specific intervals.

[0327] Treatment can be administered topically or systemically at a therapeutically effective dose. Of course, the effective dose for this purpose will depend on the severity of the disease or condition, as well as the subject's weight and overall health. Generally, in vitro dosages can provide useful guidance on the amount of a pharmaceutical composition that can be used for in situ administration, and animal models can be used to determine the effective dose for treating a specific condition.

[0328] Various considerations, such as those outlined in Langer, 1990, Science The following descriptions can be found in Goodman and Gilman's (eds.), 1990, Id., each incorporated herein by reference and used for all purposes. The parenteral dose of an active pharmaceutical agent can be converted to the corresponding oral dose by multiplying the parenteral dose by an appropriate conversion factor. For general applications, the parenteral dose in mg / mL multiplied by 1.8 equals the corresponding oral dose in milligrams (“mg”). For oncology applications, the parenteral dose in mg / mL multiplied by 1.6 equals the 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., (WB Saunders Co.), pp. 1708 and 1651.

[0329] However, it should be understood that the specific dose level for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, overall health status, sex, diet, time of administration, route of administration, excretion rate, drug combination, and the severity of the specific disease being treated.

[0330] In some embodiments, administration may include a combination of one or more therapeutic unit doses with a pharmaceutically acceptable carrier, and may additionally include other pharmaceutical reagents, agents, carriers, adjuvants, diluents, and excipients. In some embodiments, the carrier, medium, or excipient may facilitate the administration, delivery, and / or improve the preservation of the composition. In other embodiments, the one or more carriers include, but are not limited to, aqueous saline solutions such as physiological saline, Ringer's solution, PBS (phosphate-buffered saline), and mixtures of various salts, including potassium and phosphate salts, with or without sugar additives such as glucose. The carrier may include aqueous and non-aqueous sterile injectable solutions containing antioxidants, buffers, bacteriostatic agents, bactericidal antibiotics, and solutes that make the formulation isotonic with the intended recipient's body fluids; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickeners. In other embodiments, the one or more excipients may include, but are not limited to, water, saline, dextran, glycerol, ethanol, and combinations thereof. Non-toxic excipients, such as wetting agents, buffers, or emulsifiers, may also be added to the composition. Oral formulations may include commonly used excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.

[00275] Depending on the specific application and the potency of the active ingredient, the amount of the active ingredient in a unit dose formulation may vary or be adjusted between 0.1 mg and 10,000 mg, more typically 1.0 mg and 1,000 mg, and most typically 10 mg and 500 mg. The composition may also contain other compatible therapeutic agents if desired.

[0331] Treatment can be administered to the subject through a variety of suitable routes of administration or formulations. Treatments, such as immunotherapy, can also be used to treat a variety of diseases in the subject. Subjects include, but are not limited to, mammals, primates, monkeys (e.g., macaques, rhesus monkeys, or pig-tailed macaques), humans, dogs, cats, cattle, pigs, poultry (e.g., chickens), mice, rabbits, and rats. In the specific embodiments described herein, the subject is a human.

[0332] The route of administration of the therapeutic compounds described herein can be any suitable route. Routes of administration can be, but are not limited to, oral, parenteral, skin, nasal, rectal, vaginal, and ocular routes. In other embodiments, the route of administration can be parenteral, mucosal, intravenous, subcutaneous, local, intradermal, oral, sublingual, intranasal, or intramuscular. The choice of route of administration can depend on the characteristics of the compound (e.g., the physical and chemical properties of the compound), the age and weight of the animal, the specific disease (e.g., cancer type), and the severity of the disease (e.g., cancer stage or severity). Of course, combinations of routes of administration can be used as needed.

[0333] Some embodiments of this disclosure include a method for providing treatment to a subject, the method comprising administering one or more compositions once or multiple times; if more than one administration is present, the compositions may be the same or different.

[0334] toxicity The ratio between the toxicity and therapeutic effect of a particular treatment is its 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 can be used to formulate a range of doses for human use. The dose of such compounds is preferably within a range of plasma concentrations with low or no toxicity, including ED50. The dose may vary within this range depending on the dosage form used and the route of administration employed. See, for example, Fingl et al., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Chapter 1, page 1, 1975. The exact formulation, route of administration, and dose can be selected by an individual practitioner based on the patient's condition and the specific method of using the compound. For in vitro formulations, the exact formulation and dose can be selected by an individual practitioner based on the patient's condition and the specific method of using the compound.

[0335] Reagent test kit In some embodiments, kits providing tools for performing any of the methods described herein are also disclosed. In some embodiments, kits comprising any of the compositions described herein or tools for generating such compositions are also disclosed.

[0336] In some embodiments, the kit may be prepared from readily available components and reagents. For example, such a kit may contain any one or more of the following components and / or reagents: enzymes, reaction tubes, buffers, detergents, 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, components and reagents may be packaged together in any combination and / or may be packaged individually. In some embodiments, the kit may include components and reagents concentrated to a higher working concentration than disclosed herein or provided herein. In some embodiments, individual components may also be provided in the kit at a concentrated amount; in some aspects, components are provided individually at the same concentration as they are in solutions containing other components. In some embodiments, the concentration of the components may be provided at 1x, 2x, 5x, 10x, or 20x or more. In some embodiments, the kit may contain components that can be individually packaged or placed in a container such as a tube, bottle, vial, syringe, or other suitable container tool.

[0337] In some implementations, the kit is contained in a container. The kit may also include instructions for using the kit to assess cell expression and / or differentiation. The reagents 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 assessing the expression of biomarkers suitable for classifying cell states.

[0338] In some implementations, the kits are produced using Good Manufacturing Practices (GMP) and comply with GMP.

[0339] Having described the embodiments in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the embodiments as defined in the appended claims. Furthermore, it should be understood that all examples in this disclosure are provided as non-limiting examples.

[0340] Example The following non-limiting embodiments are provided to further illustrate the embodiments disclosed herein. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent methods that have been found to work well in the practice of the embodiments, and therefore can be considered as embodiments constituting a pattern of their practice. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the particular embodiments disclosed herein without departing from the spirit and scope of the embodiments disclosed herein, and still obtain the same or similar results.

[0341] Example 1. A scheme for freezing liver organoids Using CELLBANKER ® 1. Cryopreservation scheme Cellbanker contains 50 nM chromanol, 5 μM enricane, 1:1000 polyamine and 7 μM trans-ISRIB (CEPT). ® 1 (AMSBIO) Keep on ice or at 4°C.

[0342] Human liver organoids (HLOs) collected from 6-well plates between days 20 and 24 were filtered through a 150 μM sieve into 50 mL conical tubes (total volume should not exceed approximately 25 mL). Approximately 20 mL of cold PBS was added to achieve a final volume of approximately 45 mL. The suspension was pipetted up and down five times to wash the organoids. The tubes were then centrifuged at 300 × g for 5 minutes at 4 °C to precipitate the HLO. The supernatant was aspirated and washed again with 10 mL–20 mL of fresh PBS, centrifuged, and the aspiration was repeated. The washed HLO was resuspended in a cellbanker. ® In 1+CEPT frozen medium, two frozen vials were used per well of a 6-well plate, with each vial containing 0.75 mL of frozen medium suspension. The frozen vials were then transferred to a -80°C freezer for 24 hours, and then transferred to liquid nitrogen for longer storage.

[0343] Cryopreservation solution using Cell Reservoir One Vitrify Keep Cell Reservoir One Vitrify (Nacalai) containing CEPT on ice or at 4°C.

[0344] Perform the same HLO collection and washing steps as described above. Resuspend the washed HLO in Cell ReservoirOne Vitrify+CEPT cryopreservation medium and rapidly transfer it to cryopreservation tubes. Using tweezers, immerse the tubes to 2 / 3 of their height in liquid nitrogen for 10 seconds, then fully immerse them. This step should be performed within 60 seconds or less. Then transfer the tubes to a liquid nitrogen storage tank for long-term storage.

[0345] CELLBANKER ® 1. Thawing procedure The standard thawing procedure can be followed. In short, immerse the frozen vial in 37°C water for up to 2 minutes, until a small amount of solid culture medium remains in the vial. Add the preheated culture medium to the vial and transfer the contents to a conical tube containing fresh culture medium. Pipette the suspension to wash the thawed HLO, then centrifuge at 300×g for 5 minutes at room temperature. Aspirate the culture medium and resuspend the HLO in a solution containing CEPT (and optional Matrigel). ® 1) in fresh culture medium.

[0346] Cell Reservoir One Vitrify thawing solution Preheat 10 mL of cell culture medium in a centrifuge tube at 37°C. Also preheat the sterile plate in a 37°C incubator. Remove the cryopreservation tube containing the frozen cells from the liquid nitrogen reservoir, remove the cap, and discard all liquid nitrogen from the tube. Thaw the HLO rapidly by adding more than 800 μL of preheated cell culture medium to the tube and pipetting several times. The larger the volume used, the faster the sample thaws. The appropriate volume of culture medium should be added according to the size of the tube. Then transfer the thawed cell suspension to a sterile centrifuge tube. Wash the cryopreservation tube with fresh cell culture medium and add the thawed cell suspension. Centrifuge the HLO at 300 × g for 5 minutes at room temperature. Aspirate the culture medium and resuspend the HLO in a solution containing CEPT (and optional Matrigel). ® In fresh culture medium.

[0347] Example 2. Freezing and thawing of liver organoid cultures Human liver organoids (on day 22 of culture) were frozen in Cellbanker. ® 1 or Vitrify (at -80°C for 24 hours, then transferred to liquid nitrogen storage). An exemplary scheme is provided in Example 1.

[0348] For testing, organoids were thawed and immediately placed in hepatocyte culture medium. Live / dead staining was performed on the same day as thawing. Additionally, culture medium was collected on the day of thawing and 48 hours later to analyze albumin secretion. Figure 1A A diagram illustrating this situation is provided.

[0349] Figure 1B The results of live / dead staining of thawed liver organoids are shown. Organoids were stained with calcein AM (which labels live cells), ethidium homodimer-1 (which labels dead cells), and NucBlue (which labels cell nuclei). Human liver organoids showed less damage after short-term freezing, suggesting that freezing is a feasible method for transporting organoids. Organoids subjected to vitrification (e.g., using Cell Reservoir One Vitrify) had a slightly higher survival rate than those subjected to slow freezing (e.g., using CELLBANKER). ®1) Survival rate of cryopreserved organoids.

[0350] Figure 1C The results show 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. Liver organoids that underwent slow freezing or vitrification did not exhibit albumin secretion immediately after thawing. However, liver organoids that underwent vitrification showed up to 70% recovery of ALB secretion function compared to the unfrozen control after 3 days of recovery culture.

[0351] Example 3. Enhanced maturation of 2D and 3D liver organoids Methods for promoting the maturation of liver organoids in cultures were explored. Previous methods for liver organoid growth used hepatocyte culture medium, and the supplementation of this medium with additional amino acids was tested. The amino acid (AA) supplemented 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 culture medium (HCM) (Hepatocyte Culture Medium Bullet Kit; Lonza, Walkersville, MD), which was 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 factors, including but not limited to hepatocyte growth factor (HGF), oncogene M and / or dexamethasone.

[0352] According to conventional methods, liver organoids are prepared by differentiation of the fixed endoderm and induction of liver lineage using retinoic acid, but after retinoic acid induction, they are cultured in AA-supplemented medium for 8, 10 or 12 days.

[0353] Liver organoids cultured in HCM without AA supplementation were used as controls, and the liver-specific gene albumin of these liver organoids was quantified by RT-qPCR. ALB ), cytochrome P450 family 3 subfamily A member 4 ( CYP3A4 ), phosphoenolpyruvate carboxylase 1 ( PCK1 ) and glucose-6-phosphate catalytic subunit ( G6PCThe expression of all these genes was enhanced (Figure 2A). Liver organoids cultured in AA-supplemented medium for 8, 10, and 12 days showed enhanced expression of all these genes compared to control liver organoids.

[0354] Compared to liver organoids cultured in HCM without AA supplementation as a control, ALB secretion and urea production in liver organoids cultured in AA-supplemented medium for 8, 10, and 12 days (and 14 days for urea quantification) after retinoic acid induction were also quantified (Figure 2B). Prolonged exposure to AA-supplemented medium (12 or 14 days) resulted in enhanced albumin secretion and urea production compared to control liver organoids.

[0355] Pluripotent stem cells were engineered with a luciferase reporter factor driven by the PCK1 promoter to enable measurement of gluconeogenesis and differentiation into liver organoids. An exemplary PCK1 luciferase reporter factor can be found in PCT Publication WO 2021 / 262676, which is hereby explicitly incorporated herein by reference in its entirety. PCK1 expression in these liver organoids having this construct can be quantified by luciferase activity, either in live cells or after lysis. Figure 2C A diagram illustrating this process is provided.

[0356] Liver organoids were grown in AA-supplemented medium or in HCM without AA supplementation as a control. Figure 2D Bright-field images of these organoids are shown in Figure 2E. As measured by live-cell and lysed luciferase activity, liver organoids grown in AA-supplemented medium showed increased PCK1 expression compared to controls (Figure 2E).

[0357] The use of AA-supplemented medium can also be extended from 3D organoids to two-dimensional (2D) hepatocyte cultures derived from pluripotent stem cell differentiation. To test the effect of the medium on 2D hepatocyte cultures, HCMs without AA supplementation were replaced with AA-supplemented medium at different time points after liver induction (counted from the start of initial pluripotent stem cell differentiation, day 12, day 14, day 16, day 18, day 21, or day 34 of culture). A schematic diagram of this process is shown in Figure 3A, where A is HCM only, B is HCM+AA starting from day 12, and CG are HCM+AA replacements at days 14, 16, 18, 21, and 34, respectively.

[0358] Bright-field images of 2D hepatocyte cultures grown on AA-supplemented medium at different time points are shown below. Figure 3B In all conditions, the cells exhibited normal hepatocyte morphology.

[0359] Lactate production in 2D hepatocyte cultures grown in AA-supplemented medium or in HCM without AA supplementation was compared. Figure 3C For each time point, the culture medium was replaced with fresh medium the previous day, and samples were taken for testing 24 hours later. Hepatocytes grown in AA-supplemented medium showed reduced lactate levels, indicating a shift in metabolism from glucose-dependent to pyruvate-dependent compared to cells grown in standard hepatocyte growth medium.

[0360] Quantitative analysis of albumin secretion in 2D hepatocyte cultures after HCM with AA-supplemented medium replaced with AA-supplemented medium at different time points. Figure 3D Hepatocytes cultured for 12 days with added AA supplementation showed the highest albumin secretion. Increased albumin secretion relative to control hepatocytes was also observed at even longer culture durations. Figure 3E ).

[0361] ALB E-cadherin ( E-cad ), CYP3A4 , G6PC pyruvate kinase M1 / 2 ( PKM )and PCK1 Gene expression was quantified in 2D hepatocytes grown on day 58 in HCM without AA supplementation or in AA-supplemented medium at different culture times (Fig. 3F). Overall, AA supplementation enhanced liver maturation and gene expression. Interestingly, PCK1 and G6PC Expression (which is inhibited by insulin that is usually added to the culture medium) decreased with increasing duration of AA supplementation, suggesting that the addition of AA normalizes the metabolism of liver organoids.

[0362] Pluripotent stem cells were engineered to express mScarlet, a reporter factor acting as gluconeogenesis, under the PCK1 promoter, and the pluripotent stem cells differentiated into hepatocytes in 2D cultures. Hepatocytes were grown in HCM without AA supplementation or in AA-supplemented medium. Cells were also optionally cultured for 24 hours without insulin (in DMEM / F12 supplemented with 0.2% BSA) prior to quantitative fluorescence. Figure 3G Images of mScarlet fluorescence from these hepatocytes are shown. Cells grown in AA-supplemented medium showed increased mScarlet expression driven by the PCK1 promoter after 24 hours of insulin starvation, indicating increased metabolism in these cells compared to controls.

[0363] Detection of EpCAM (epithelial marker), vimentin (mesenchymal marker), and DAPI or hepatocyte nuclear factor 4α (HNF4α) (nuclear marker) in 2D hepatocyte cultures grown in HCM without AA supplementation or with AA-supplemented medium on day 12 at day 57. Figure 3H Although hepatocytes differentiated from pluripotent stem cells cultured in HCM without AA supplementation exhibit vimentin-positive mesenchymal cells, this vimentin signal is absent in cells cultured in AA-supplemented medium, suggesting that culturing cells in AA-supplemented medium potentially reduces mesenchymal cells and enriches hepatocytes.

[0364] Example 4. Without Matrigel ® The production of embedded human organs A brief exploration of Matrigel without the use of heterologous components was conducted in PCT Publication WO 2018 / 085615. ® The culture of 3D human liver organoids, the PCT disclosure of which is hereby expressly incorporated herein by reference in its entirety. Figure 4A The diagram illustrates this approach. Haptoforegut cells and / or aptoforegut 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, oncogene M, and dexamethasone). These three-dimensional structures can be pipetted from the original cell layer and further cultured to grow into liver organoids. Figure 4B ). In the absence of Matrigel ® These liver organoids grown under specific conditions exhibit normal organoid morphology. Figure 4C ) and expected liver function, such as albumin secretion ( Figure 4D )and ALB Alpha-fetoprotein (AFP) AFP ), HNF4α Retinol-binding protein 4 ( RBP4 ) and α1 antitrypsin ( AAT The expression of () Figure 4E ).

[0365] Not using Matrigel ® Grown organoids are advantageous because they are not grown using xenogeneic animal components and therefore can be used for human applications. Furthermore, they can grow to relatively large sizes that are easier to handle. However, these organoids may be less efficient than those grown in Matrigel. ® Organoids grown in medium exhibit greater size variation and may display anomalous morphology (because Matrigel...). ®It helps maintain the 3D form and may not be well-suited for cryopreservation (which could reduce scaling up for manufacturing purposes). Additionally, standard protocols involve the use of Matrigel during culture steps other than organoid maturation, such as pluripotent stem cell culture. ® .

[0366] To overcome one or more of these potential drawbacks, methods for using Matrigel in the absence of Matrigel were explored. ® An alternative method for culturing organoids in cases involving other basement membrane matrices with heterogeneous components.

[0367] A method for culturing foregut cells after initial induction using a combination of FGF2, EGF, VEGF, CHIR99201, and A83-01 was briefly explored in PCT Publication WO 2018 / 191673. Modified forms of this combination, with or without EGF, can be used to expand foregut cells derived from the differentiation of well-defined endoderm cells in the presence of FGF pathway activators (e.g., FGF4) and Wnt pathway activators (e.g., CHIR99021).

[0368] exist Figure 5A The diagram illustrates the expansion of foregut cells prior to their subsequent differentiation into the liver lineage. After the fixed endoderm is induced into the foregut, the cells are dissociated into single cells using a standard enzyme dissociation solution (such as Accutase) and then 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).

[0369] The study explored the effects of these dissociated foregut cells on the division of Matrigel. ® The ability to grow on substrates other than those used for amplification purposes. Culture plates coated with Matrigel. ® Or laminin, which is a basement membrane matrix component that can be manufactured and obtained without animal products. A basement membrane matrix-like coating is preferred for culture because foregut cells would otherwise not be able to properly adhere to the plate for growth. In Matrigel... ® Foregut cells expanded on plates coated with laminin grew appropriately during 7 days of culture and began to form 3D spheroids. Figure 5B After further culture (days 8-10), fully formed spheroids were observed, which could further mature into liver organoids. Figure 5CAlthough there was no significant difference in initial cell adhesion rates, the spheroid formation efficiency with laminin coating was higher than that with Matrigel. ® The coating is better.

[0370] The number of dissociated foregut cells used for seeding the plate for expansion was also tested. 1.0 × 10⁶ cells were used. 6 One or 5.0×10 6 One foregut cell was seeded into a laminin-coated 6-well plate (9.6 cm per well). 2 Surface area) and let it grow for 5 days ( Figure 5D As the number of inoculated cells increases, spheroid formation begins earlier (e.g., inoculated with 5.0 × 10⁶ cells). 6 (A plate of individual cells is seen in a larger spheroid at each time point). Therefore, spheroid formation depends on cell density.

[0371] Because the expanded foregut cells spontaneously form spheroids, they can be used as a source for manufacturing large-scale spheroids. Spontaneously formed 3D spheroids were collected from foregut cell culture on day 12 and used for liver organoid maturation. Additional spheroids were formed over a further 23 days after spheroid collection (until day 35 of culture). Figure 5E These newly formed spheroids exhibit the same ability to mature into liver organoids.

[0372] After initial dissociation, plate seeding, and recovery of foregut cells, further passages of proliferating foregut cells can be performed. Four passages (passed every 10 days) of the initial day 8 foregut culture differentiated from pluripotent stem cells were tested. Globule formation occurred by the third passage, but less than the fourth (Fig. 5F). In the fourth passage, mesenchymal cells were predominantly proliferating. Passaging from the initial foregut culture three times can increase the cell number by approximately 200-fold, thus providing a significant advantage for scaling up. Tail-type homology frames 2 (CHOFCs) were performed on foregut cells from each passage. CDX2 ), Fork head frame A2 ( FOXA2 ), AFP vimentin ( VIM SRY-Box transcription factor 17 ( SOX17 ), HNF4α and ALB Quantitative expression (Figure 5G).

[0373] CDX2 and SOX17 The expression was maintained in the 4th generation. FOXA2 and HNF4α The expression of ...

[0374] In general, to obtain continuous formation of human liver organoids from single-cell passages of foregut cells, dissociated foregut cells can be grown on a laminin (e.g., laminin-511) coating in EP medium (containing FGF4) with a dense plate seeding number (e.g., 5 × 10⁻⁶). 6 Cells / 6-well plate [9.6cm] 2 Foregut cells should be passaged a maximum of three times, as mesenchymal cells primarily grow in subsequent passages, and globular formation ceases. Figure 5H The diagram illustrates the formation of liver organoids starting from pluripotent stem cells and including foreguinal cell passage for large-scale processing. "Expandable foreguinal organoids" refer to spontaneously formed foreguinal globules, which are collected for maturation into liver organoids. The "organoid re-formation" step represents the continuous passage of foreguinal cells and the spontaneous formation of additional foreguinal globules, which are then collected and matured into liver organoids.

[0375] This process of expanding foregut cells to increase liver organoid production can be further refined using microplates (e.g., Aggrewell plates (StemCell Technologies)) or other devices (such as molding plates) designed to aggregate foregut cells to form more uniform organoids. These methods of using devices to produce uniform organoids are explored in PCT Publication WO 2021 / 030373, which is hereby expressly incorporated by reference in its entirety. Figure 5I The illustration shows liver organoid formation starting from pluripotent stem cells and including foreguinal cell passage for large-scale processing, as well as a schematic diagram of foreguinal cell aggregation using a device.

[0376] Example 5. Scalable 3D bioreactor liver organoid formation No Matrigel ® Liver organoids are difficult to maintain in static culture. This study explored the use of 3D rotational culture to maintain freely suspended liver organoids. Figure 6A A schematic diagram of the process is provided. Globules (e.g., those formed on a basement membrane matrix or its components that do not contain non-human animal components as described herein) induced by the method described in the previous embodiment are collected and grown in a hepatocyte culture medium via rotational culture. Under these rotational culture conditions, the globules mature into liver organoids over a period of 15 days. Figure 6B Quantitative analysis of liver organoids (3D) grown in rotational culture. AFP , HNF4α , FOXA2 , ALB , CDX2 and VIMGene expression was observed and compared with first-generation (P1) foregut cells (Fig. 6C). Liver organoids exhibited... ALB and AFP High expression of this substance indicates mature hepatocytes. CDX2 and HNF4α The expression of [the substance] was not significantly different from that of foregut cells. Therefore, this demonstrates that when grown in rotational culture, [the substance] can be produced without the need for Matrigel. ® Liver organoids can be cultured in the presence of other basement membrane matrices.

[0377] Example 6. Additional Materials and Methods animal All animal experiments were conducted with the approval of the Institutional Review Board and the Institutional Animal Protection and Use Committee. Adult Gunn (Gunn-Ugt1a1j / BluHsdRrrc) rats (breeding pairs, 9–12 weeks old) were obtained from the Rat Resource Research Center (RRRC, Columbia, MO). Rats were housed in standard rat cages with wood shavings bedding, maintained at a temperature of 20–24°C and a relative humidity of 45–55%, under a 12:12 hour light:dark cycle. Prior to the study, all animals had free access to standard food (Cincinnati Lab Supply, Cincinnati, OH). All animals were handled in accordance with the institution’s guidelines and regulations.

[0378] Maintenance of PSC Human iPSC line 72.3 (RRID: CVCL_A1BW) was obtained from the pluripotent stem cell facility at Cincinnati Children's Hospital Medical Center (CCHMC), under the joint supervision of CN. Mayhew and JM. Wells. Undifferentiated hiPSCs were cultured on laminin-511 E8 fragment (Nippi)-coated culture dishes in StemFit medium (Ajinomoto Company) with 100 ng / mL basic fibroblast growth factor (FGF; R&D Systems) at 37°C in 5% CO2 and 95% air.

[0379] Production of human liver organoids (HLOs) pluripotent stem cells were used at a rate of 2×10 5Cells were seeded at a density of 10 cells / well in 24-well plates coated with laminin iMatrix-511Silk 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 containing activin A and BMP4 to produce a defined endoderm. On day 4, the medium was replaced with RPMI, activin A, and 0.2% dFBS, with the 0.2% dFBS being replaced with 2% dFBS on day 5. From day 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-amphoteric acid) to induce the hindgut. On day 9, cells were dissociated into a single-cell suspension using Accutase treatment. This single-cell suspension was then mixed with 50% Matrigel. ® The cells were mixed with 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 in 50 μl droplets in 6-well plates. These cells were fed EP medium every 48 hours for 4 days to generate organoids. The organoids were then treated with advanced DMEM and retinoic acid (RA) every 48 hours for 4 days to define the liver lineage. The organoids were then fed hepatocyte culture medium (HCM), hepatocyte growth factor (HGF), oncogene M, and dexamethasone every 3–4 days to generate HLO.

[0380] HLO ripening with low doses of bilirubin For ripening induction in HLO, organoids were fed complete HCM with supplements in addition to a low dose of bilirubin (1 mg / L) on day 15. This treatment continued until day 30, at which time organoids were harvested. Bright-field images were captured on a KEYENCE BZ-X710 fluorescence microscope (Keyence) and analyzed using the ImageJ suite. RNA was isolated using the Rneasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. qPCR was performed on a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific Inc.) using the TaqMan Gene Expression Master Mixture (Applied Biosystems). All primer and probe information for each target gene was obtained from the Universal Probe Library Assay Design Center website (available at lifescience.roche.com / en_us / brands / universal-probe-library.html). For fully mounted 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 on a Nikon A1 inverted confocal microscope.

[0381] Bilirubin and drug treatment On day 27, mature organoids were treated for 5 days with bilirubin (1 mg / L–10 mg / L), doxycycline (100 ng / mL; added 3 days before gene expression activation), and / or additional drugs such as hydrocortisone, dexamethasone, ketoconazole, and mifepristone (1 μM–2 μM each), and then harvested for downstream assays. Bilirubin assays were performed using a colorimetric kit (ab235627 from Abcam). RNA was isolated using the Rneasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. qPCR was performed on a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific Inc.) using the TaqMan Gene Expression Master Mixture (Applied Biosystems). All primer and probe information for each target gene was obtained from the Universal Probe Library Design Center (available at lifescience.roche.com / en_us / brands / universal-probe-library.html). Images were captured using a KEYENCE BZ-X710 fluorescence microscope (Keyence).

[0382] Organoid transplantation to the portal vein HLOs were harvested on day 27 and dissociated into organoid fragments via repeated pipetting. The fragments were washed with PBS and resuspended in HCM containing a mixture of 2% FBS and CEPT (50 nM chromanol, 5 μM enrikacin, 1:1000 polyamine, and 7 μM trans-ISRIb) to enhance viability. Four days prior to transplantation, recipient rats were treated with a single dose of retrorsine (5 mg / kg) and tacrolimus (0.8 mg / kg). A midline incision was made, the intestine was pushed to one side, and bleeding was controlled by clamping the portal vein posteriorly using a 32 g 1-inch needle. 200 μL of the 3 × 10⁻⁶ treasury solution was infused overnight. 3 organoids (approximately 5 × 10) 5(1 cell) was injected into the portal vein. Excessive blood loss was prevented by applying SURGICEL SnoW absorbable hemostatic agent (Ethicon). The animal was then sutured with 5-0 vicryl-coated surgical sutures (Ethicon) and GLUture (Zoetis Inc.), and buprenorphine (0.1 mg / kg) was administered as an analgesic. The animal was then maintained with doxycycline (2 mg / kg) and tacrolimus every 3-4 days until the day of harvest. Blood was collected periodically via the retroorbital method as needed.

[0383] Live cell imaging and functional assays For in vivo imaging of organoids, Celldiscoverer 7 (Zeiss) was used to image every 30 minutes for 7 days. Visualization of bilirubin conjugation was achieved using 5 μM fluorescent UnaG, which was incubated with HLO medium and imaged for 2 days.

[0384] 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 on a QuantStudio 5 Real-Time PCR System (Applied Biosystems) using the TaqMan Gene Expression Master Mixture (Applied Biosystems). All samples were amplified using the TaqMan Gene Expression Assay and normalized with an 18S rRNA endogenous control. For RNA sequencing, the quality of extracted RNA was evaluated using an Agilent 2100 Bioanalyzer (Agilent). Sequence libraries were prepared using the TruSeq Stranded mRNA Kit (Illumina) and sequenced using a NovaSeq 6000 (Illumina). Reads were aligned to the human genome assembly hg38 and quantified using the quasi-mapper Salmon (v1.8.0). Gene expression analysis was performed using the RBioconductor package DESeq2 (v1.36.0). The read count matrix was normalized using a size factor, and a variance stabilization transformation (VST) was applied to the normalized representation data. The data was visualized using the clusterProfiler (v.4.4.1) and pheatmap (v1.0.12) packages.

[0385] Alternatively, whole transcriptome RNA sequencing (HLO) of isolated total RNA (including those treated with bilirubin and those treated with mifepristone) was performed using the Illumina NovaSeq platform service of Novogene Co., Ltd. (Beijing, China). RNA sequencing parameters were 150 bp paired-end sequencing at a 20M read depth per sample. Fastq read files for each sample were obtained and aligned using Salmon, a quasi-mapping tool for aligning and quantifying transcripts using RNA-seq data. Raw transcript counts and normalized transcripts per million (TPM) values ​​were obtained, and differential expression was analyzed using DESeq2. Statistical and biological significance were defined as P < 0.05, FDR < 0.05, and fold change > 1, with at least 3 transcript counts in 3 out of 6 samples. hclust and pheatmap were used for heatmap visualization and hierarchical cluster analysis, respectively. Finally, pathway analysis was performed in R version 4.0.3 using biomaRt and org.Hs.eg.db.

[0386] mGULO editor The mouse GULO (L-gulonolactone oxidase) (mGULO) cDNA sequence was retrieved from NCBI. A 5' adapter and Kozak sequence were added to the beginning of the sequence, and an HA tag was added to the end. Additionally, P2A-mCherry was added to the far end after the HA tag and 3' adapter. The custom gene was then synthesized and cloned into the pAAVS1-Ndi-CRISPRi(Gen1)PCSF#117 vector using restriction sites AflII and Agel. The vector had the TetON system and was then inserted into Neo using Gateway technology. r Choose a marker.

[0387] Generation and maintenance of mGULO iPSC Then, using lentivirus-mediated CRISPR / Cas9, the PCSF#117 vector containing the modified GULO sequence was inserted into the AAVS1 locus of a 72.3 iPSC cell line. Correct clones were then selected using G418. PCR was used to verify the correct insertion, random insertion, and copy number of surviving clones, and DNA sequencing was also performed for verification. The edited iPSC plates were then seeded onto cell culture plates coated with laminin iMatrix-511 Silk and maintained in StemFitBasic04 complete medium containing Y-27632. Cells were passaged with Accutase every 4–7 days until passage 40 (p40). mGULO HLOs were generated according to the HLO generation protocol described herein. mGULO protein expression was verified using a GLUO ELISA kit (MBS2890737 from MyBioSource, San Diego, CA).

[0388] mGULO HLO treatment and visualization of bilirubin On day 24, mGLUO HLO was treated with doxycycline (Dox) (100 ng / ml) to induce mGULO expression. Subsequently, on day 27, mature organoids were treated with bilirubin and Dox for 5 days and then harvested for downstream assays. Bilirubin was measured and visualized using a colorimetric kit (ab235627) and UnaG (a green-to-dark light-converting fluorescent protein that fluoresces only when bound to bilirubin). Images were captured on a KEYENCE BZ-X710 fluorescence microscope (Keyence).

[0389] ChIP-PCR and ChIP-qPCR ChIP experiments were performed using a high-sensitivity ChIP kit (Abcam). In short, organoids were fixed with PFA, then whole chromatin was prepared and sonicated to an optimal size of 300 bp, confirmed by gel electrophoresis. The chromatin was used for immunoprecipitation with either EP300 antibody or an IgG1 isotype control. DNA fragments were amplified using custom primers for PCR and qPCR, and the enrichment fold data were normalized relative to the immunoprecipitation of the IgG control.

[0390] Protein expression assay Albumin secretion was measured by collecting 200 μL of supernatant from an HLO cultured in a HCM and stored at -80°C until use. The supernatant was analyzed using a human albumin ELISA quantitative kit (BethylLaboratories) according to the manufacturer's instructions.

[0391] For mouse GULO expression assays, organoids were dissociated and washed with PBS. Cells were then lysed with RIPA lysis and extraction buffer and a mixture of Halt protease and phosphatase inhibitors (Thermo Scientific) to extract total protein, which was then measured using a mouse GULO / L-gulonolactone oxidase ELISA kit (MyBioSource.com) according to the manufacturer's instructions.

[0392] Metabolite assay Bilirubin levels were measured by collecting supernatant from bilirubin-treated HLO and serum from rats. The supernatant and serum were measured using a bilirubin assay kit (Total and Direct, Colorimetric) (abcam) and a bilirubin assay kit (Sigma-Aldrich) according to the manufacturer's instructions.

[0393] Cellular antioxidant levels were measured by harvesting HLO, washing it in PBS, and then seeding the plate into a 96-well assay plate. The levels were then quantified using the Cellular Antioxidant Assay Kit (abcam) according to the manufacturer's instructions.

[0394] Activity assay HLO was harvested, washed in PBS, plated into 96-well assay plates, and subjected to rifampin and omeprazole for 24 hours for CYP3A4 and CYP1A2 assays, respectively. The assays were then performed 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.

[0395] Apoptosis was assessed by lysing HLO and measuring the lysate using a caspase-3 assay kit (colorimetric) (abcam) according to the manufacturer's instructions.

[0396] Rat serum was measured using an aspartate aminotransferase (AST) activity assay kit and an alanine aminotransferase (ALT) activity assay kit (Sigma-Aldrich).

[0397] Quantitative and statistical analysis Statistical analyses were performed using R software v4.2.0 with unpaired two-tailed Student's t-test, Dunn-Holland-Wolfe test, or Welch test. Statistical analyses of stiffness measurements were performed using nonparametric Kruskal-Wallis and post-hoc Dunn-Holland-Wolfe tests. For comparisons between two unpaired groups, a nonparametric Brunner-Munzel test was performed unless otherwise specified, provided the groups were independent and their variances were unequal. A p-value <0.05 was considered statistically significant. N-values ​​refer to biologically independent replicates. Image analyses were unblinded.

[0398] Example 7. Low doses of bilirubin promote the maturation of fetal-like liver organoids. The role of bilirubin in liver development was studied using a human liver organoid model. Figure 7A An exemplary schematic diagram depicts the preparation of liver organoids treated with a low concentration of bilirubin (e.g., 1 mg / L) similar to that of human fetuses (approximately 1 / 10 of the physiological concentration in adults). Bilirubin is added to early organoids differentiating into the liver lineage. Exemplary methods for generating liver organoids have previously been explored, for example, in PCT disclosures WO 2018 / 085615, WO 2018 / 191673, WO 2018 / 226267, WO 2019 / 126626, WO 2020 / 023245, WO 2020 / 069285, WO 2020 / 243613, WO 2021 / 030373, and WO 2021 / 262676, each of which is hereby explicitly incorporated herein by reference in its entirety. Based on these exemplary methods, low concentrations of bilirubin are added to liver organoids formed after retinoic acid induction and foregut endoderm formation. Liver organoids can be cultured in standard hepatocyte culture medium (HCM) with bilirubin. For example, the hepatocyte culture medium can be supplemented with hepatocyte growth factors, including but not limited to hepatocyte growth factor (HGF), oncogene M, and / or dexamethasone. The liver organoids should be contacted with a growth medium containing 1 mg / L bilirubin for at least 5–10 days to promote liver organoid maturation.

[0399] Figure 7B The liver organoids matured with 1 mg / L bilirubin exhibited luminal projections similar to bile canaliculi, natural structures found in liver tissue. Compared to the lumen of the control liver organoids not treated with bilirubin, the resulting mature liver organoids showed luminal projections with smaller size and reduced roundness (Fig. 7C).

[0400] Quantitative gene expression analysis of bilirubin-treated liver organoids using RT-...

Claims

1. A method for expanding hindgut cells and / or hindgut endoderm cells, the method comprising: a) Dissociate the hind-foregut cell monolayer and / or the hind-foregut endoderm cell monolayer into hind-foregut cells and / or hind-foregut endoderm cells; b) Seeding the hindgut cells and / or the hindgut endoderm cells onto a tissue culture surface; and c) The hindbrain cells and / or the hindbrain endoderm cells are cultured together with TGF-β pathway inhibitors, FGF pathway activators, Wnt pathway activators and VEGF pathway activators.

2. The method according to claim 1, wherein the hindbrain cell monolayer is dissociated into the hindbrain cells and / or the hindbrain endoderm cells using enzymatic dissociation and / or mechanical dissociation.

3. The method according to claim 1 or 2, wherein the hindbrain cells and / or the hindbrain endoderm cells are prepared at a ratio equal to or about 1 × 10⁻⁶. 5 1, 2×10 5 1, 3×10 5 1, 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 5 1×10 6 1, 2×10 6 1, 3×10 6 1, 4×10 6 One or 5×10 6 cells / cm 2 The cell density of the surface area of ​​the tissue culture surface, or any cell density having a range defined by any two of the aforementioned cell densities, is seeded onto the surface of the tissue container.

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 a component thereof.

5. The method of claim 4, wherein the basement membrane matrix or its components do not contain non-human animal components that make the basement membrane matrix or its components xenogeneic to humans, optionally wherein the basement membrane matrix or its components are not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein 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 comprise human adhesion protein, collagen IV, nestin, basement membrane glycan, fibrin and / or hydrogel.

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

8. The method according to any one of claims 1 to 7, wherein the hindbrain cells and / or the hindbrain 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, optionally 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 equal to or about 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the TGF-β pathway inhibitor is provided at a concentration equal to 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, optionally FGF2.

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

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

14. The method according to any one of claims 1 to 13, wherein the Wnt pathway activator is provided at a concentration equal to or about 1 µM, 1.5 µM, 2 µM, 2.5 µM, 3 µM, 3.5 µM, 4 µM, 4.5 µM, 5 µM, 5.5 µM, 6 µM, 6.5 µM, 7 µM, 7.5 µM or 8 µM, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the Wnt pathway activator is provided at a concentration equal to 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, optionally VEGF.

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

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

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

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

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

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

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

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

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

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

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

27. The method of claim 26, wherein the hindbrain cells and / or the hindbrain endoderm cells are cultured until they spontaneously form three-dimensional (3D) spheroids, and the hindbrain cells and / or the hindbrain endoderm cells are collected from the spheroids, optionally the method further comprising dissociating the spheroids into individual clumps of hindbrain cells and / or hindbrain endoderm cells and / or hindbrain cells and / or hindbrain endoderm cells prior to the differentiation step, optionally wherein the spheroids comprise structures having a single lumen, and / or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells.

28. The method of claim 26, wherein prior to the differentiation step, the hindbrain cells and / or the hindbrain endoderm cells are collected from the hindbrain cell monolayer by dissociating the hindbrain cell monolayer into individual hindbrain cells and / or hindbrain endoderm cells and / or clumps of hindbrain cells and / or hindbrain endoderm cells.

29. A method for differentiating hindbrain cells and / or hindbrain endoderm cells into liver organoids, the method comprising: i) Contacting hindbrain cells and / or hindbrain endoderm cells with a retinoic acid pathway activator, wherein the hindbrain cells and / or hindbrain endoderm cells are optionally in the form of spheroids, optionally in the form of individual cells or cell clusters detached from the spheroids, and / or optionally are cells aggregated in micropores or other devices described herein, wherein optionally the spheroids contain structures having a single lumen, and / or wherein the spheroids do not contain hematopoietic tissue and acquired immune cells; as well as ii) Contact the cells of step i) with a culture medium for a period of time, thereby causing the hindbrain cells and / or the hindbrain endoderm cells to differentiate into liver organoids, optionally wherein the culture medium is a hepatocyte culture medium.

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

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

32. The method of claim 30 or 31, wherein the IL-6 family cytokines are selected from the group consisting of: IL-6, oncogene M (OSM), leukemia inhibitory factor (LIF), myocardial trophoprotein-1, ciliary neurotrophic factor (CTNF), and myocardial trophoprotein-like cytokines (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, fluocinolone, halometasone, and mometasone.

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

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

36. The method according to any one of claims 29 to 35, wherein the hindbrain cells and / or the hindbrain endoderm cells are 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 hindbrain cells and / or the hindbrain endoderm cells are in the form of spheroids or individual hindbrain cells and / or hindbrain endoderm cells, and / or in the form of clumps of hindbrain cells and / or hindbrain endoderm cells derived from the dissociated spheroids, optionally wherein the spheroids comprise structures having a single lumen, and / or wherein the spheroids do 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, BS 493, TTNPB and AM580, optionally 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 µM, 1.1 µM, 1.2 µM, 1.3 µM, 1.4 µM, 1.5 µM, 1.6 µM, 1.7 µM, 1.8 µM, 1.9 µM, 2.0 µM, 2.1 µM, 2.2 µM, 2.3 µM, 2.4 µM, 2.5 µM, 2.6 µM, 2.7 µM, 2.8 µM, 2.9 µM, or 3.0 µM, or at any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the retinoic acid pathway activator is provided at a concentration equal to or about 2.0 µM.

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

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

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

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

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

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

46. ​​The method according to claim 44 or 45, 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 essential amino acids, or essential amino acids within the range defined by any two of the foregoing values, optionally wherein the supplemented growth medium contains about 4%-10%, 6%-12%, 10%-16%, 12%-15%, 13%-19%, or about 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15% or 16% of essential amino acids, by total volume.

47. The method according to any one of claims 44 to 46, wherein the supplemented glycine is at or above 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, or more. The glycine supplemented therein may be provided at concentrations of 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, or 35 mg / mL, or at any concentration within the range defined by any two of the foregoing concentrations, optionally wherein the supplemented glycine is provided at a concentration equal to or about 18 mg / mL to 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 contacted with a low concentration / first concentration of bilirubin, wherein the liver organoids formed are mature liver organoids.

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

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

51. The method according to any one of claims 48 to 50, wherein the mature liver organoid exhibits a luminal protrusion similar to a bile canal, and / or has a single lumen and generally has a spherical shape, and / or wherein 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, relative to liver organoids not exposed to a low dose / first dose of bilirubin, the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof.

53. The method according to any one of claims 48 to 52, wherein the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to liver organoids not 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 relative to liver organoids not exposed to 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 contacted with a high / second concentration of bilirubin, wherein the liver organoids formed are hyperbilirubinemic liver organoids.

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

58. The method according to claim 56 or 57, wherein, relative to liver organoids not treated with high / secondary concentrations of bilirubin, the hyperbilirubinemic liver organoids express elevated levels of UGT1A1 or NRF2 or both.

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, wherein the liver organoid is capable of synthesizing ascorbic acid.

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

61. The method of claim 59 or 60, wherein a tetracycline-inducible system is optionally used to conditionally express the gene encoding the functional GULO protein.

62. The method according to any one of claims 59 to 61, wherein the liver organoid is genetically engineered using CRISPR to encode the functional GULO protein.

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

64. The method according to any one of claims 59 to 63, wherein the liver organoids containing the functional GULO protein express an increased level of NRF2 compared to liver organoids not containing the functional GULO protein.

65. The method according to any one of claims 59 to 64, wherein, relative to liver organoids not containing the functional GULO protein, the liver organoids containing the functional GULO protein optionally express reduced levels of IL1B, IL6, or TNFα, or any combination thereof, when 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, relative to liver organoids not containing the functional GULO protein, the liver organoids containing the functional GULO protein optionally exhibit reduced caspase-3 activity when cultured in an ascorbic acid-depleted medium or in the absence of ascorbic acid.

67. The method according to any one of claims 59 to 66, wherein the liver organoids containing the functional GULO protein express increased levels of ALB compared to liver organoids not containing the functional GULO protein.

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

69. The method of claim 68, wherein the periportal liver markers include FAH, ALB, PAH, CPS1, HGD, or any combination thereof.

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

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

72. The method according to any one of claims 59 to 71, wherein the liver organoids containing the functional GULO protein exhibit 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 has been differentiated from pluripotent stem cells, the pluripotent stem cells comprising a functional GULO protein and / or a gene or mRNA encoding the functional GULO protein or both, thereby the pluripotent stem cells being capable of synthesizing ascorbic acid.

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

75. The method according to any one of claims 29 to 74, the method further comprising, prior to step i), assembling the hindbrain cells and / or the hindbrain endoderm cells in micropores or other devices (e.g., Aggrewell), wherein the assembling of the hindbrain cells and / or the hindbrain endoderm cells produces liver organoids of 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 basement membrane matrix or components thereof, optionally wherein the cells of step i) and / or step ii) are not cultured with human xenogeneic basement membrane matrix or components thereof, optionally wherein the cells of step i) and / or step ii) are not cultured with basement membrane matrix or components thereof isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein the cells of step i) and / or step ii) are not cultured with Matrigel ® Cultrex ® Or Geltrex ® touch.

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

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

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

80. The method of claim 79, wherein cryopreservation of the liver organoids comprises slow freezing or vitrification cryopreservation, optionally wherein the liver organoids are cryopreserved with chroman I, enricane, polyamine and trans-ISRIB (CEPT).

81. The method according to any one of claims 1 to 80, wherein the hindbrain cells and / or the hindbrain endoderm cells have been 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 hindbrain cells and / or the hindbrain endoderm cells have been derived from a subject, optionally a subject suffering from a liver-related disease or condition.

83. The method according to any one of claims 1 to 82, wherein the method is applicable to processes conforming to Good Manufacturing Practices (GMP).

84. Haptomigrium cells and / or aptomigrium endoderm cells or liver organoids produced by the method according to any one of claims 1 to 83.

85. An in vitro composition comprising: pluripotent stem cells, shaped endoderm, hindbrain, hindbrain endoderm and / or downstream hepatocyte types, 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 of claim 85, wherein the composition comprises hindbrain cells and / or hindbrain endoderm cells, and the hindbrain cells and / or the hindbrain endoderm cells are dissociated hindbrain cells and / or hindbrain endoderm cells.

87. The in vitro composition according to claim 85 or 86, wherein the hindbrain cells and / or the hindbrain endoderm cells are greater than or equal to, exactly or about 1 × 10⁻⁶. 5 1, 2×10 5 1, 3×10 5 1, 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 5 1×10 6 1, 2×10 6 1, 3×10 6 1, 4×10 6 One or 5×10 6 cells / cm 2 The cell density of the surface area of ​​the tissue culture surface, or any cell density within a range defined by any two 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 a component thereof.

89. The in vitro composition of claim 88, wherein the basement membrane matrix or its components do not contain non-human animal components that make the basement membrane matrix or its components xenogeneic to humans, optionally wherein the basement membrane matrix or its components are not isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells, optionally wherein 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 comprise human adhesion protein, collagen IV, nestin, basement membrane polysaccharide, 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 hindbrain cells and / or the hindbrain endoderm cells are spontaneously formed three-dimensional (3D) spheres, optionally wherein the spheres comprise 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; optionally wherein the TGF-β pathway inhibitor comprises A83-01 or A83-01.

93. The in vitro composition according to any one of claims 85 to 92, wherein the TGF-β pathway inhibitor has a concentration equal to or about 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1000 nM, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the TGF-β pathway inhibitor has a concentration equal to 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, optionally wherein the FGF pathway activator comprises FGF2 or FGF2.

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

96. The in vitro composition according to any one of claims 85 to 95, wherein the Wnt pathway activator is selected from the group consisting of: Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, alloxan, indirubin, ateradon, tamparone, lithium chloride, TDZD 8 and TWS119, wherein the Wnt pathway activator optionally comprises CHIR99021 or CHIR99021.

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

99. The in vitro composition according to any one of claims 85 to 98, wherein the VEGF pathway activator has a concentration equal to or about 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the VEGF pathway activator has a concentration equal to 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 wherein the composition does not comprise exogenous EGF.

101. The in vitro composition according to any one of claims 85 to 100, wherein the EGF has a concentration equal to or about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL or 30 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the EGF has a concentration equal to 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 ascorbic acid and / or genetically modified ascorbic acid, or wherein the composition does not comprise exogenous ascorbic acid and / or genetically modified ascorbic acid.

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

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

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

106. The in vitro composition according to any one of claims 85 to 105, wherein the hindbrain cells and / or the hindbrain endoderm cells, shaped endoderm, hindbrain endoderm and / or downstream hepatocytes are differentiated from stem cells, optionally wherein the hindbrain cells and / or the hindbrain endoderm cells, shaped endoderm, hindbrain endoderm and / or downstream hepatocytes are differentiated from induced pluripotent stem cells.

107. The in vitro composition according to any one of claims 85 to 107, wherein the hindbrain cells and / or the hindbrain endoderm cells, the fixed endoderm, the hindbrain endoderm and / or the downstream hepatocytes have been passaged less than 4 times.

108. The in vitro composition according to claim 108, wherein the cells comprise or are substantially composed of hindbrain cells and / or hindbrain 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. Liver organoids produced by the method according to any one of claims 29 to 83.

111. An in vitro composition comprising: a) hindbrain cells and / or hindbrain endoderm cells, liver organoids and / or mature liver organoids, and b) a culture medium, wherein the culture medium optionally comprises hepatocyte culture medium and optionally supplemented with cMET tyrosine kinase receptor agonists, IL-6 family cytokines and corticosteroids, and wherein the composition optionally additionally comprises c) a retinoic acid pathway activator.

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

113. The in vitro composition according to any one of claims 111 to 112, wherein the IL-6 family cytokines are selected from the group consisting of: IL-6, oncogene M (OSM), leukemia inhibitory factor (LIF), myocardial trophoprotein-1, ciliary neurotrophic factor (CTNF), and myocardial trophoprotein-like cytokines (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, fluocinolone, halometasone, 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, BS 493, TTNPB and AM580, optionally wherein the retinoic acid pathway activator comprises 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 has a concentration of 1.0 µM, 1.1 µM, 1.2 µM, 1.3 µM, 1.4 µM, 1.5 µM, 1.6 µM, 1.7 µM, 1.8 µM, 1.9 µM, 2.0 µM, 2.1 µM, 2.2 µM, 2.3 µM, 2.4 µM, 2.5 µM, 2.6 µM, 2.7 µM, 2.8 µM, 2.9 µM or 3.0 µM, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the retinoic acid pathway activator has a concentration equal to or about 2.0 µM.

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

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

121. The in vitro composition according to any one of claims 115 to 120, wherein the dexamethasone has a concentration equal to or about 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM or 200 nM, or any concentration within the range defined by any two of the aforementioned concentrations, optionally wherein the dexamethasone has a concentration equal to 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, wherein the in vitro composition further comprises a low concentration of exogenous bilirubin, optionally wherein the low concentration of bilirubin is at or near the physiological bilirubin concentration of human fetus.

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

125. The in vitro composition of claim 124, wherein the composition comprises a mature liver organoid, and wherein the mature liver organoid exhibits a luminal projection similar to a bile canal, and / or has a single lumen and generally has a spherical shape, and / or wherein the mature liver organoid does not contain hematopoietic tissue and acquired immune cells.

126. The in vitro composition of claim 125, wherein the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof relative to liver organoids not exposed to low doses of bilirubin.

127. The in vitro composition according to claim 125 or 126, wherein the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof, relative to liver organoids not exposed to 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 relative to liver organoids not exposed to low doses of bilirubin.

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

131. The in vitro composition of claim 130, wherein, relative to the liver organoids in which said cells have not been contacted with a low dose of bilirubin, the mature liver organoids express reduced levels of AFP, CDX2, NANOG, or any combination thereof.

132. The in vitro composition according to claim 130 or 131, wherein, relative to liver organoids in which said cells have not been contacted with low doses of bilirubin, the mature liver organoids express increased levels of ALB, SLC4A2, or HO-1, or any combination thereof.

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 relative to liver organoids in which the cells have not been exposed to a low dose of bilirubin.

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

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

137. The in vitro composition according to claim 135 or 136, wherein the hyperbilirubinemic liver organoids express elevated levels of UGT1A1 or NRF2 or both, relative to liver organoids not treated with high / secondary concentrations of bilirubin.

138. The in vitro composition according to any one of claims 111 to 137, wherein the hindbrain cells and / or the hindbrain endoderm cells, liver organoids and / or mature liver organoids comprise a functional L-gulonolactone oxidase (GULO) protein and / or a gene or mRNA encoding the functional GULO protein or both, wherein the hindbrain cells and / or the hindbrain 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 a tetracycline-inducible system is optionally used to conditionally express the gene encoding the functional GULO protein.

141. The in vitro composition according to any one of claims 138 to 139, wherein the hindbrain cells and / or the hindbrain endoderm cells, liver organoids and / or mature liver organoids are engineered using CRISPR to contain a 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 are introduced into the liver organoid by transfection.

143. The in vitro composition according to any one of claims 138 to 142, wherein the liver organoids and / or mature liver organoids containing the functional GULO protein express increased levels of NRF2 relative to liver organoids and / or mature liver organoids not containing the functional GULO protein.

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

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

146. The in vitro composition according to any one of claims 138 to 145, wherein the liver organoids and / or mature liver organoids containing the functional GULO protein express increased levels of ALB compared to liver organoids and / or mature liver organoids that do 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 resemble periportal liver tissue and expresses periportal liver markers.

148. The in vitro composition of 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 organoids and / or mature liver organoids containing the functional GULO protein exhibit increased CYP3A4 and CYP1A2 activity relative to liver organoids and / or mature liver organoids not containing the functional GULO protein.

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

151. The in vitro composition according to any one of claims 138 to 150, wherein the liver organoids and / or mature liver organoids containing the functional GULO protein exhibit increased viability in culture compared to liver organoids and / or mature liver organoids 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 has been differentiated from pluripotent stem cells, the pluripotent stem cells comprising a functional GULO protein and / or a gene or mRNA encoding the functional GULO protein or both, thereby the pluripotent stem cells being capable of synthesizing ascorbic acid.

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

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

155. The method of claim 153 or 154, wherein the liver organoid has been generated from cells derived from the subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells.

156. The method according to any one of claims 154 to 155, wherein administration comprises 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 condition includes one or more types of liver dysfunction and / or failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic disease, autoimmune liver disease, Wilson's disease, metabolic-associated fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Najjar syndrome, urea cycle disorder, Wolman's disease, liver cancer, hepatoblastoma, metabolic dysfunction-associated liver disease (MASLD), MetALD, metabolic dysfunction-associated fatty liver disease (MASH), drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic disease, liver cysts, 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 failure includes hyperammonemia and / or hyperbilirubinemia; or wherein the metabolic disease includes non-alcoholic fatty liver disease (NAFLD); or wherein the non-alcoholic fatty liver disease (NAFLD) includes metabolic dysfunction-associated steatohepatitis (MASH); or wherein the hepatitis includes hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, TT hepatitis, and / or autoimmune hepatitis.

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

160. The method according to any one of claims 154 to 159, wherein the subject has improved symptoms of 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 is implanted onto the liver of the subject.

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

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

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

166. The method according to any one of claims 154 to 165, wherein the method is applicable to processes conforming to Good Manufacturing Practices (GMP).

167. A method for screening, the method comprising contacting liver organoids according to 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 organoids.

168. The method of claim 167, wherein the liver organoid is a model of a liver-related disease or condition, and assessing the effect of the candidate compound or composition on the liver organoid includes assessing the effect of the candidate compound or composition on the liver-related disease or condition.

169. The method of claim 167 or 168, wherein the liver organoid has been generated from cells derived from the subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells.

170. The method of claim 169, wherein the subject suffers from a liver-related disease or condition.

171. The method according to any one of claims 167 to 170, wherein the method is applicable to processes conforming to Good Manufacturing Practices (GMP).

172. A composition comprising a liquid component of supplementary amino acids according to Table 3.

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

174. A composition comprising a liquid component for supplementing amino acids, said liquid component comprising, by volume, exactly or about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of a solution of non-essential amino acids (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), by volume A solution of essential amino acids at exactly or approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% (containing exactly or approximately 6320 mg / L arginine, 1200 mg / L cysteine, 2100 mg / L histidine, 2620 mg / L isoleucine, 2620 mg / L leucine, 3625 mg / L lysine, 755 mg / L methionine, 1650 mg / L phenylalanine, 2380 mg / L threonine, 510 mg / L tryptophan, 1800 mg / L tyrosine, and 23... 40 mg / L valine and hepatocyte culture medium (HCM) at exactly or approximately 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% by volume, supplemented with exactly or approximately 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, or 11 mg / mL. , 12mg / mL, 13mg / mL, 14mg / mL, 15mg / mL, 16mg / mL, 17mg / mL, 18mg / mL, 19mg / mL, 20mg / mL, 21mg / mL, 22mg / mL, 23mg / mL, 2 4mg / mL, 25mg / mL, 26mg / mL, 27mg / mL, 28mg / mL, 29mg / mL, 30mg / mL, 31mg / mL, 32mg / mL, 33mg / mL, 34mg / mL or 35mg / mL glycine.

175. The composition according to any one of claims 172 to 174, wherein the composition comprises a liquid component for supplementing amino acids, the liquid component for supplementing amino acids comprising exactly or about 14% of a solution of non-essential amino acids (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% by volume of a solution of essential amino acids (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), comprising exactly or about 6% by volume of a solution of essential amino acids (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). The medium contains approximately 6320 mg / L arginine, 1200 mg / L cysteine, 2100 mg / L histidine, 2620 mg / L isoleucine, 2620 mg / L leucine, 3625 mg / L lysine, 755 mg / L methionine, 1650 mg / L phenylalanine, 2380 mg / L threonine, 510 mg / L tryptophan, 1800 mg / L tyrosine, and 2340 mg / L valine, and is prepared with hepatocyte culture medium (HCM) at exactly or approximately 80% by volume, supplemented with exactly or approximately 20 g / L glycine.

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

0.

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

178. The composition according to any one of claims 172 to 177, wherein the composition further comprises liver lineage-defined cells differentiated from defined endoderm cells using retinoic acid.

179. The composition of claim 178, wherein the liver lineage-typed cells are characterized as liver organoids.

180. The composition of claim 179, wherein the liver organoid is characterized as having increased levels of albumin and urea secretion relative to liver organoids contained in HCM without amino acid supplementation.

181. The composition according to claim 179 or 180, wherein the liver organoid is characterized as expressing an increased level of liver maturation-related gene expression relative to liver organoids contained in HCM without amino acid supplementation.

182. The composition according to any one of claims 179 to 181, wherein the liver organoid is characterized as expressing reduced levels of vimentin relative 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 that make the basement membrane matrix or its components xenogeneic to humans.

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

185. An in vitro hyperbilirubinemia liver organoid, said in vitro hyperbilirubinemia liver organoid comprising UDP-glucuronyltransferase family 1 member A1 ( UGT1A1 Naturally occurring and / or engineered mutations in ( ).

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

187. The in vitro hyperbilirubinemia liver organoids according to claim 185 or 186, wherein the hyperbilirubinemia liver organoids are clonal and / or derived from iPSCs.

188. A cryopreservation composition comprising liver organoids, chroman I, enricane, polyamines, and trans-ISRIB (CEPT).

189. A cryopreservation composition comprising mature liver organoids, chroman1, enricane, polyamines, and trans-ISRIB (CEPT).

190. A cryopreserved composition comprising hyperbilirubinemia liver organoids, chroman I, enricane, polyamines, and trans-ISRIB (CEPT).

191. A kit comprising tools for performing the method according to any one of claims 1 to 83 or 153 to 171.

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

193. The use of the method, composition or kit according to any one of claims 1 to 192 as a medicine, a tool for treating and / or preventing disease, a diagnostic tool and / or for medical research.

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