Culture media and conditions for in vitro expansion and / or maturation of hepatocytes

Abstract

Provided herein are fully defined 2D culture conditions, including culture supplements and requirements for extracellular matrix, as well as culture conditions and media for hepatocyte maturation, that promote the proliferation and long-term maintenance of primary human hepatocytes.

Description

[Technical field]

[0001] The present disclosure relates generally to methods for culturing, expanding, and maturing hepatocytes. [Background technology]

[0002] Liver disease (e.g., liver disease) is any condition that negatively affects the normal, healthy performance of the liver. Impairment of liver function leads to illness. For example, liver failure can result in the accumulation of toxins (e.g., nitrogenous waste compounds) in the blood. These toxins can travel to the brain and affect the nervous system. The CDC reports that 4.5 million Americans have been diagnosed with liver disease. Organ replacement therapy can restore failed native liver function, but the demand far exceeds availability. A viable alternative is to transplant a population of primary human hepatocytes (PHH), which are in short supply.

[0003] Therefore, there is a need for improved methods of culturing and propagating PHH. Summary of the Invention

[0004] The present invention provides methods for the long-term maintenance, expansion, and maturation of primary human hepatocytes (PHH), which may be useful for generating grafts for transplantation in human recipients and for replenishing or restoring native liver function.

[0005] In one aspect, the disclosure provides a method of culturing PHH, the method comprising culturing one or more hepatocytes in contact with an extracellular matrix (ECM) in the presence of a growth medium comprising a basal medium for human cells supplemented with one or more (e.g., 2, 3, 4, or 5) activators of Wnt signaling, one or more (e.g., 2, 3, 4, or 5) receptor tyrosine kinase ligands, and one or more (e.g., 2, 3, 4, or 5) epithelial phenotype stabilizers.

[0006] In some embodiments of the above aspect, one or more (e.g., 2, 3, 4, or 5) of the Wnt signaling activators are R-spondin1, R-spondin2, R-spondin3, R-spondin4, Wnt3a, or any combination of the above. In some embodiments, the one or more Wnt signaling activators include R-spondin1 and Wnt3a.

[0007] In some embodiments of the foregoing aspects, one or more (e.g., two, three, four, or five) of the receptor tyrosine kinase ligands are epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF), or any combination of the foregoing. In some embodiments, the EGF is human EGF, the FGF is human fibroblast growth factor 7 (FGF-7) or human fibroblast growth factor 10 (FGF-10), the HGF is human HGF, or the TGF is transforming growth factor alpha (TGFα) (e.g., human TGFα). In some embodiments, the one or more receptor tyrosine kinase ligands include human EGF, FGF-7, FGF-10, HGF, and TGFα.

[0008] In some embodiments of the foregoing aspects, one or more (e.g., two, three, four, or five) of the epithelial phenotype stabilizers are transforming growth factor beta (TGFβ) inhibitors. In some embodiments, the TGFβ inhibitor is an activin receptor-like kinase 5 (ALK5) inhibitor (e.g., A83-01). In some embodiments, one of the epithelial phenotype stabilizers is a corticosteroid (e.g., hydrocortisone).

[0009] In some embodiments of the foregoing aspects, the growth medium further comprises one or more cell survival agents and / or one or more cell proliferation agents. In some embodiments, the growth medium comprises serum (e.g., fetal bovine serum). In some embodiments, the growth medium does not comprise serum (e.g., fetal bovine serum).

[0010] In some embodiments of the foregoing aspects, the growth medium comprises a serum replacement component. In some embodiments, the serum replacement component is KNOCKOUT™ serum replacement (KOSR), human platelet lysate, human serum, or bovine serum. In some embodiments, the v / v of the serum replacement component is 1% during culture. In some embodiments, the v / v of the serum replacement component is increased with a gradient of 1-5-10% during culture. In some embodiments, the v / v of the serum replacement component is increased with a gradient of 5-10% during culture. In some embodiments, the growth medium does not comprise a serum replacement component.

[0011] In some embodiments of the foregoing aspects, the growth medium comprises a Rho kinase inhibitor. In some embodiments, the Rho kinase inhibitor is Y-27632. In some embodiments, the growth medium does not contain a Rho kinase inhibitor.

[0012] In some embodiments of the foregoing aspects, the ECM comprises collagen (e.g., collagen I or collagen IV) or laminin (e.g., laminin 111, laminin 211, laminin 221, laminin 332, laminin 411, laminin 421, laminin 511, or laminin 521). In some embodiments, the ECM comprises both collagen and laminin. In some embodiments, the ECM comprises collagen I. In some embodiments, the ECM comprises collagen IV. In some embodiments, the ECM comprises laminin 111. In some embodiments, the ECM comprises laminin 511. In some embodiments, the ECM comprises laminin 521. In some embodiments, the ECM comprises a combination of collagen I, collagen IV, laminin 111, laminin 511, and laminin 521. In some embodiments, the ECM does not comprise a hydrogel (e.g., MATRIGEL™). In some embodiments, the ECM comprises a hydrogel (eg, MATRIGEL™).

[0013] In some embodiments of the foregoing aspects, the culturing step is performed on a surface (e.g., a two-dimensional surface). In some embodiments, the surface is coated with an ECM. In some embodiments, the PHHs are adhesively attached to the surface during the culturing step. In some embodiments, the expanded PHHs are dissociated, aggregated, and maintained in culture to promote further proliferation and / or maturation.

[0014] In some embodiments of the foregoing aspects, the growth medium further comprises a B27 supplement and / or an N2 supplement. In some embodiments, the B27 supplement does not contain vitamin A.

[0015] In some embodiments of the foregoing aspects, the growth medium further comprises an amino acid supplement. In some embodiments, the amino acid supplement is a non-essential amino acid (NEAA) supplement. In some embodiments, the NEAA supplement comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. In some embodiments, the growth medium does not comprise an amino acid supplement.

[0016] In some embodiments of the foregoing aspects, the growth medium does not contain a Notch inhibitor or a Notch agonist. In some embodiments, the growth medium does not contain gastrin.

[0017] In some embodiments, the growth medium further comprises a serum replacement component, hi some embodiments, the serum replacement component comprises KNOCKOUT™ serum replacement (KOSR), human platelet lysate, human serum, or bovine serum.

[0018] In some embodiments, the culturing comprises culturing the cells under hypoxic conditions or in the presence of a hypoxic mimetic. In some embodiments, culturing is performed under hypoxic conditions. Hypoxic conditions can include, for example, an oxygen concentration of less than 20%. In some embodiments, culturing under hypoxic conditions includes culturing the cells at an oxygen concentration of 1% to 19% (e.g., 1% to 10%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some embodiments, the oxygen concentration is 1% to 10%. In some embodiments, the oxygen concentration is 5%.

[0019] In some embodiments, the culturing step includes expanding the plated cells (step P0) and first passaging the expanded cells (step P1). In some embodiments, the P0 step has a duration of 7 to 16 days (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days). In some embodiments, the P0 step has a duration of 11 days. In some embodiments of the foregoing aspects, the P0 step has a duration of 13 days.

[0020] In some embodiments, the P1 step has a duration of 7 to 20 days (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments of the foregoing aspects, the P1 step has a duration of 11 days. In some embodiments, the P1 step has a duration of 13 days.

[0021] In some embodiments, the P0 step comprises administering hepatocytes at a concentration of 200 to 13,333 cells / cm. 2 , e.g., 200 cells / cm 2 ~1,000 cells / cm 2 (e.g., 200 cells / cm 2 , 300 cells / cm 2 , 400 cells / cm 2 , 500 cells / cm 2 , 600 cells / cm 2 , 700 cells / cm 2, 800 cells / cm 2 , 900 cells / cm 2 , or 1,000 cells / cm 2 ), 1,000 cells / cm 2 ~10,000 cells / cm 2 (e.g., 1,000 cells / cm 2 , 2,000 cells / cm 2 , 3,000 cells / cm 2 , 4,000 cells / cm 2 , 5,000 cells / cm 2 , 6,000 cells / cm 2 , 7,000 cells / cm 2 , 8,000 cells / cm 2 , 9,000 cells / cm 2 , or 10,000 cells / cm 2 ), or 10,000 cells / cm 2 ~13,333 cells / cm 2 (e.g., 10,000 cells / cm 2 , 11,000 cells / cm 2 , 12,000 cells / cm 2 , 13,000 cells / cm 2 , or 13,333 cells / cm 2 In some embodiments, the P0 step comprises seeding at a density of 667 cells / cm. 2 In some embodiments, the P1 step includes seeding hepatocytes at a density of 333 to 13,333 cells / cm. 2 In some embodiments, the P1 step comprises seeding hepatocytes at a density of 1,333 cells / cm. 2 The method includes seeding hepatocytes at a density of 100-200 μg / ml.

[0022] In some embodiments, the growth medium comprises a serum replacement component, and the concentration of the serum replacement component is varied over the duration of the culture step. In some embodiments, the concentration of the serum replacement component is 1% (v / v) on day 0 of the P0 step.

[0023] In some embodiments, the concentration of serum replacement is increased to 5% (v / v) (i) when the cell density reaches 15%-30% (e.g., 15%, 20%, 25%, or 30%) confluency, or (ii) between days 3-7 (e.g., days 3, 4, 5, 6, or 7) of the P0 step. In some embodiments, the concentration of serum replacement is increased to 5% (v / v) on day 5 of the P0 step. In some embodiments, the concentration of serum replacement component is 5% (v / v) on day 0 of the P0 step. In some embodiments, the concentration of serum replacement component is 5% (v / v) on day 0 of P0 and remains at 5% (v / v) until the concentration of serum replacement component is increased. In some embodiments, the concentration of serum replacement is increased to 10% (v / v) (i) when the cell density reaches 40%-60% (e.g., 40%, 45%, 50%, 55%, or 60%) confluency, or (ii) between days 7 and 13 (e.g., days 7, 8, 9, 10, 11, 12, or 13) of the P0 step. In some embodiments, the concentration of serum replacement is increased to 10% (v / v) on day 9 of the P0 step.

[0024] In some embodiments, the concentration of serum replacement components is 1% (v / v) on day 0 of the P1 step. In some embodiments, the concentration of serum replacement is increased to 5% (v / v) (i) when the cell density reaches 15%-30% (e.g., 15%, 20%, 25%, or 30%) confluency, or (ii) between days 3-7 (e.g., days 3, 4, 5, 6, or 7) of the P1 step. In some embodiments, the concentration of serum replacement is increased to 5% (v / v) on day 5 of the P1 step.

[0025] In some embodiments, the concentration of the serum replacement component is 5% (v / v) on day 0 of the P1 step. In some embodiments, the concentration of the serum replacement component is 5% (v / v) on day 0 of the P1 step and remains at 5% (v / v) until the concentration of the serum replacement component is increased.

[0026] In some embodiments, the concentration of serum replacement is increased to 10% (v / v) (i) when the cell density reaches 40%-60% (e.g., 40%, 45%, 50%, 55%, or 60%) confluency, or (ii) between days 5-13 (e.g., days 5, 6, 7, 8, 9, 10, 11, 12, or 13) of the P1 step. In some embodiments of the foregoing aspects, the concentration of serum replacement is increased to 10% (v / v) on day 7 of the P1 step. In some embodiments, the concentration of serum replacement is increased to 10% (v / v) on day 9 of the P1 step.

[0027] In some embodiments of the foregoing aspects, the method further comprises, after the culturing step, determining an expression profile of the PHH. In some embodiments of the aforementioned aspects, the duration of the culture step is 3 to 120 days (e.g., about 15 to about 120 days, about 16 to 119 days, about 17 to about 118 days, about 18 to about 117 days, about 19 to about 116 days, about 20 to about 115 days, about 25 to about 110 days, about 30 to about 100 days, about 40 to about 90 days, about 50 to about 80 days, about 60 to about 70 days, about 65 days or more).

[0028] In some embodiments of the foregoing aspects, the culturing step comprises culturing a 9.5 cm 2 ~10,000cm 2 In some embodiments of the above aspects, the culturing step is carried out using a multi-well plate or flask or vessel having a surface area of ​​500 cm or less. 2 ~10,000cm 2 For example, in some embodiments, the surface area is 9.5 cm 2 ~500cm 2 (For example, 9.5 cm 2 , 100cm 2 , 200cm 2 , 300cm 2 , 400cm 2 , or 500cm2 ), 500cm 2 ~1,000cm 2 (For example, 500 cm 2 , 600cm 2 , 700cm 2 , 800cm 2 , 900cm 2 , or 1,000 cm 2 ) or 1,000 cm 2 ~10,000cm 2 (For example, 1,000 cm 2 , 2,000cm 2 , 3,000cm 2 , 4,000cm 2 , 5,000cm 2 , 6,000cm 2 , 7,000cm 2 , 8,000cm 2 , 9,000cm 2 , or 10,000 cm 2 In some embodiments, the culturing step is at 636 cm 2 ~6360cm 2 The procedure is carried out using a flask or vessel with a surface area of

[0029] In some embodiments of the foregoing aspects, after the culturing step, the expression profile of the PHHs comprises expression of one or more (e.g., 2, 3, 4, or 5) proteins selected from hepatocyte nuclear factor 4 alpha (HNF4α), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), keratin 18 (CK18), and albumin by at least 80% (e.g., at least 85%, 90%, or 95%) of the PHHs.

[0030] In some embodiments of the foregoing aspects, after the culturing step, the expression profile of the PHHs comprises expression of Ki67 by up to 15% (e.g., up to 10%, 5%, 4%, 3%, 2%, or 1%) of the PHHs.

[0031] In some embodiments of the foregoing aspects, after the culturing step, the expression profile of the PHHs comprises expression of Ki67 by at least 15% of the PHHs (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more).

[0032] In some embodiments of the foregoing aspects, after the culturing step, the expression profile of the PHHs comprises downregulation of one or more (e.g., 2, 3, 4, or 5) proteins selected from mature hepatocyte markers (e.g., Cyp3a4, Cyp1a2, NR1I2, urea cycle enzymes ABCG2, ABCC2, ABCB11, SR-B1, or SLC10A1) in at least 10% (e.g., at least 11%, 12%, 13%, 14%, 15%, or 20%) of control PHHs plated overnight.

[0033] In some embodiments of the foregoing aspects, after the culturing step, the expression profile of the PHHs comprises upregulation of one or more (e.g., 2, 3, 4, or 5) proteins selected from fetal / liver progenitor / bile duct cell markers (e.g., AFP, Cyp3a7, EPCAM, LGR5, KRT7, KRT19, or AQP1) in at least 10% (e.g., at least 11%, 12%, 13%, 14%, 15%, or 20%) of control PHHs plated overnight.

[0034] In some embodiments, the expanded PHHs require additional maturation steps to upregulate urea cycle enzymes, proteins characteristic of a mature cell state, and to downregulate fetal / hepatic progenitor / cholangiocyte markers.

[0035] In some embodiments of the foregoing aspects, after the culturing step, the expression profile of the PHHs comprises expression of one or more (e.g., 2, 3, 4, or 5) proteins selected from HNF4α, LGR5, CK18, and ALB by at least 80% (e.g., at least 85%, 90%, or 95%) of the PHHs.

[0036] In some embodiments of the foregoing aspects, after the culturing step, the expression profile of the expanded PHHs comprises expression of Ki67 by up to 15% (e.g., up to 10%, 5%, 4%, 3%, 2%, or 1%) of the PHHs.

[0037] In some embodiments of the foregoing aspects, after the culturing step, the expression profile of the expanded PHHs comprises expression of Ki67 by at least 15% of the PHHs (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more).

[0038] In some embodiments of the foregoing aspects, after the culturing step, the PHH continue to express and secrete albumin at a lower level than non-growing PHH. In some embodiments of the foregoing aspects, after the culturing step, the expression profile of the PHH comprises increased expression of urea upon maturation of the PHH.

[0039] In some embodiments of the foregoing aspects, after the culturing step, the hepatocyte yield is greater than or equal to 1 cm 2 Win, at least 5 x 10 3 (e.g., at least 5 × 10 3 , 5×10 4 , 5×10 5 , 5×10 6 , or 5x10 7 ).

[0040] In some embodiments of the aforementioned aspects, after the culturing step, the hepatocyte yield is increased by at least 2-fold (e.g., at least 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 30-fold) within 14-28 days (e.g., within 15-27 days, within 16-26 days, within 17-25 days, within 18-24 days, within 19-23 days, within 20-21 days, or within 22 days) of culture.

[0041] In some embodiments of the foregoing aspects, after the culturing step, the hepatocyte yield increases by at least 500-fold (e.g., at least 1000-fold) within 30 days (e.g., within 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, or 14 days) of culture. In some embodiments of the foregoing aspects, after the culturing step, the hepatocyte yield increases by at least 500-fold within 24 days of culture.

[0042] In some embodiments of the foregoing aspects, following the culturing step, hepatocyte yield is increased by 500-fold to 2000-fold, e.g., 1000-fold to 2000-fold (e.g., 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 1,100-fold, 1,200-fold, 1,300-fold, 1,400-fold, 1,500-fold, 1,600-fold, 1,700-fold, 1,800-fold, 1,900-fold, or 2,000-fold) within 30 days (e.g., 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, or 14 days) of culture. In some embodiments of the foregoing aspects, after the culturing step, the hepatocyte yield is increased by 500-fold to 2000-fold, e.g., 1000-fold to 2000-fold (e.g., 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 1,100-fold, 1,200-fold, 1,300-fold, 1,400-fold, 1,500-fold, 1,600-fold, 1,700-fold, 1,800-fold, 1,900-fold, or 2,000-fold) within 24 days of culture.

[0043] In some embodiments of any of the aforementioned aspects, the method further includes maturing the hepatocytes in a maturation medium comprising a basal medium for human cells supplemented with one or more hepatocyte maturation supplements.

[0044] In another aspect, the disclosure provides a method of maturing a population of hepatocytes, the method comprising maturing an expanded population of hepatocytes in the presence of a maturation medium comprising a basal medium for human cells supplemented with one or more hepatocyte maturation supplements. In some embodiments of the aforementioned aspect, prior to maturation, the hepatocytes are expanded by culturing them in contact with an ECM in the presence of a growth medium comprising a basal medium, an activator of Wnt signaling, one or more receptor tyrosine kinase ligands, and one or more epithelial phenotype stabilizers. In some embodiments of the aforementioned aspect, the expanded hepatocytes have an immature phenotype.

[0045] In some embodiments of any of the preceding aspects, the maturation step begins immediately after hepatocyte proliferation. In some embodiments of any of the preceding aspects, the maturation step does not begin immediately after hepatocyte proliferation. In some embodiments of any of the preceding aspects, the maturation step has a duration of 3 to 12 days (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days). In some embodiments of any of the preceding aspects, the maturation step is 7 days.

[0046] In some embodiments of any of the preceding aspects, the maturation basal medium is LONZA™ HCM™, William E, or HepatoZYME-SFM. In some embodiments of any of the preceding aspects, the one or more maturation supplements include an antibiotic, HEPES, GLUTAMAX™, ITS, a Notch inhibitor, an EGFR inhibitor, oncostatin M, an antioxidant, a glucocorticoid, a pregnane X receptor (PXR) activator, a bile acid, cAMP or a cAMP analog, cholesterol, a thyroid hormone, a serum replacement component, or any combination of the foregoing.

[0047] In some embodiments of the aforementioned aspects, the Notch inhibitor is Compound E, gamma secretase inhibitor XX, or a combination thereof. In some embodiments of the aforementioned aspects, the antibiotic is penicillin, streptomycin, or a combination thereof. In some embodiments of the aforementioned aspects, the EGFR inhibitor is erlotinib hydrochloride. In some embodiments of the aforementioned aspects, the antioxidant is vitamin C. In some embodiments of the aforementioned aspects, the glucocorticoid is dexamethasone, hydrocortisone, or a combination thereof. In some embodiments of the aforementioned aspects, the PXR activator is vitamin K2. In some embodiments of the aforementioned aspects, the bile acid is lithocholic acid, ursodeoxycholic acid, or a combination thereof. In some embodiments of the aforementioned aspects, the cAMP analog is 8-bromo cAMP, forskolin, or a combination thereof. In some embodiments of the aforementioned aspects, the thyroid hormone is T3. In some embodiments of the aforementioned aspects, the serum replacement component is insulin transferrin selenium (ITS), KOSR, Trace Elements A, Trace Elements B, or a combination thereof.

[0048] In some embodiments of any of the foregoing aspects, the maturation medium lacks one or more of R-spondin1 / Wnt3a, epidermal growth factor (EGF), transforming growth factor alpha (TGFα), N-acetylcysteine, nicotinamide, B27 supplement, N2 supplement, fibroblast growth factor 7 (FGF7), and fibroblast growth factor 10 (FGF10).

[0049] In another aspect, the present disclosure provides a kit comprising a growth medium comprising a basal medium for human cells supplemented with one or more activators of Wnt signaling, one or more receptor tyrosine kinase ligands, and one or more epithelial phenotype stabilizers, the kit further comprising a package insert instructing a user of the kit to culture one or more hepatocytes according to a method according to any one of the preceding embodiments.

[0050] In another aspect, the disclosure provides a growth medium comprising a basal medium for human cells to which is added one or more (e.g., 2, 3, 4, or 5) activators of Wnt signaling, one or more (e.g., 2, 3, 4, or 5) receptor tyrosine kinase ligands, and one or more (e.g., 2, 3, 4, or 5) epithelial phenotype stabilizers.

[0051] In some embodiments of any of the above aspects, one or more (e.g., 2, 3, 4, or 5) of the Wnt signaling activators are R-spondin1, R-spondin2, R-spondin3, R-spondin4, Wnt3a, or any combination of the above. In some embodiments, the one or more Wnt signaling activators include R-spondin1 and Wnt3a.

[0052] In some embodiments of any of the foregoing aspects, one or more (e.g., two, three, four, or five) of the receptor tyrosine kinase ligands are epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF), or any combination of the foregoing. In some embodiments, the EGF is human EGF, the FGF is human fibroblast growth factor 7 (FGF-7) or human fibroblast growth factor 10 (FGF-10), the HGF is human HGF, or the TGF is transforming growth factor alpha (TGFα) (e.g., human TGFα). In some embodiments, the one or more receptor tyrosine kinase ligands include human EGF, FGF-7, FGF-10, HGF, and TGFα.

[0053] In some embodiments of the foregoing aspects, the growth medium comprises a Rho kinase inhibitor. In some embodiments, the Rho kinase inhibitor is Y-27632. In some embodiments, the growth medium does not contain a Rho kinase inhibitor.

[0054] In some embodiments of any of the foregoing aspects, one or more (e.g., two, three, four, or five) of the epithelial phenotype stabilizers are transforming growth factor beta (TGFβ) inhibitors. In some embodiments, the TGFβ inhibitor is an activin receptor-like kinase 5 (ALK5) inhibitor (e.g., A83-01). In some embodiments, one of the epithelial phenotype stabilizers is a corticosteroid (e.g., hydrocortisone).

[0055] In some embodiments of any of the aforementioned aspects, the growth medium further comprises one or more cell survival agents and / or one or more cell proliferation agents. In some embodiments of any of the foregoing aspects, the ECM comprises collagen (e.g., collagen I or collagen IV) or laminin (e.g., laminin 111, laminin 211, laminin 221, laminin 332, laminin 411, laminin 421, laminin 511, or laminin 521). In some embodiments, the ECM comprises collagen I. In some embodiments, the ECM comprises collagen IV. In some embodiments, the ECM comprises laminin 111. In some embodiments, the ECM comprises laminin 511. In some embodiments, the ECM comprises laminin 521. In some embodiments, the ECM comprises a combination of collagen I, collagen IV, laminin 111, laminin 511, and laminin 521.

[0056] In some embodiments of any of the foregoing aspects, the culturing step is performed on a surface (e.g., a two-dimensional surface). In some embodiments, the surface is coated with an ECM. In some embodiments, the PHHs are adhesively attached to the surface during the culturing step. In some embodiments, the expanded PHHs are dissociated, aggregated, and maintained in culture to promote further proliferation and / or maturation.

[0057] In some embodiments of any of the aforementioned aspects, the growth medium further comprises a B27 supplement and / or an N2 supplement. In some embodiments, the B27 supplement does not contain vitamin A.

[0058] In some embodiments of any of the preceding aspects, the growth medium does not contain a Notch inhibitor or a Notch agonist. In some embodiments, the growth medium does not contain gastrin.

[0059] In some embodiments of the foregoing aspects, the growth medium further comprises an amino acid supplement. In some embodiments, the amino acid supplement is a non-essential amino acid (NEAA) supplement. In some embodiments, the NEAA supplement comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. In some embodiments, the growth medium does not comprise an amino acid supplement.

[0060] In another aspect, the disclosure provides a tissue culture vessel comprising a growth medium described herein (eg, of any of the above embodiments). In another aspect, the present disclosure provides an incubator that maintains a tissue cell container under hypoxic conditions. Hypoxic conditions can include, for example, an oxygen concentration of less than 20%. In some embodiments, the incubator maintains an oxygen concentration of 1%-19% (e.g., 1%-10%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some embodiments, the oxygen concentration is 1%-10%. In some embodiments, the oxygen concentration is 5%.

[0061] In another aspect, the present disclosure provides a kit comprising a maturation medium comprising a basal medium for human cells supplemented with one or more maturation supplements, the kit further comprising a package insert instructing a user of the kit to mature one or more hepatocytes according to a method according to any one of the preceding embodiments.

[0062] In another aspect, the disclosure provides a maturation medium comprising a basal medium for human cells supplemented with one or more hepatocyte maturation supplements. In some embodiments of the foregoing aspects, the maturation basal medium is LONZA™ HCM™, William E, or HepatoZYME-SFM. In some embodiments of the foregoing aspects, the one or more maturation media include an antibiotic, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), GLUTAMAX™, ITS, a Notch inhibitor, an EGFR inhibitor, oncostatin M, an antioxidant, a glucocorticoid, a pregnane X receptor (PXR) activator, a bile acid, cAMP or a cAMP analog, cholesterol, a thyroid hormone, a serum replacement component, or any combination of the foregoing.

[0063] In some embodiments of the aforementioned aspects, the Notch inhibitor is Compound E, gamma secretase inhibitor XX, or a combination thereof. In some embodiments of the aforementioned aspects, the antibiotic is penicillin, streptomycin, or a combination thereof. In some embodiments of the aforementioned aspects, the EGFR inhibitor is erlotinib hydrochloride. In some embodiments of the aforementioned aspects, the antioxidant is vitamin C. In some embodiments of the aforementioned aspects, the glucocorticoid is dexamethasone, hydrocortisone, or a combination thereof. In some embodiments of the aforementioned aspects, the PXR activator is vitamin K2. In some embodiments of the aforementioned aspects, the bile acid is lithocholic acid, ursodeoxycholic acid, or a combination thereof. In some embodiments of the aforementioned aspects, the cAMP analog is 8-bromo-cAMP, forskolin, or a combination thereof. In some embodiments of the aforementioned aspects, the thyroid hormone is T3. In some embodiments of the aforementioned aspects, the serum replacement component is ITS, KOSR, Trace Elements A, Trace Elements B, or a combination thereof.

[0064] In some embodiments of any of the foregoing aspects, the maturation medium lacks one or more of R-spondin1 / Wnt3a, epidermal growth factor (EGF), transforming growth factor alpha (TGFα), N-acetylcysteine, nicotinamide, B27 supplement, N2 supplement, fibroblast growth factor 7 (FGF7), and fibroblast growth factor 10 (FGF10). [Brief description of the drawings]

[0065] [Figure 1]Brightfield image of primary human hepatocyte (PHH) cells cultured with growth medium referenced as Expand 1.0 formulation, which contained modified Dulbecco's modified Eagle's medium / Ham's nutrient mixture F-12 supplemented with Wnt signaling activators R-spondin1 and Wnt3a; receptor tyrosine kinase ligands recombinant epidermal growth factor, recombinant transforming growth factor, recombinant human fibroblast growth factor 7, recombinant human fibroblast growth factor 10, and recombinant human hepatocyte growth factor; epithelial phenotype stabilizer A83-01; N-acetylcysteine, nicotinamide, Rho kinase inhibitor Y-27632; vitamin A-free B27 supplement, and N2 supplement; -2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), GLUTAMAX™, penicillin, and streptomycin. PHH cells are shown after plating and culturing for 6 days in a T-75 culture flask (passage "P" 0). [Diagram 2] FIG. 1 is a series of photomicrographs showing brightfield images of cells at days 2, 9, and 14 of growth, and immunostaining of markers at day 13 of growth, for hepatic phenotype in PHHs cultured in Growth Medium using a hepatocyte lot (Lot D in Table 3) grown as a homogenous culture of hepatic lineage cells. PHHs were cultured on collagen I extracellular matrix (ECM) in the presence of Growth Medium. Growth Medium was as described in FIG. 1. At day 13 of culture, PHHs express HNF4α, albumin, and LGR5. [Diagram 3] Figure 3 is a series of photomicrographs showing brightfield images of cells at days 2, 10, and 13 of expansion, as well as immunostaining for markers at day 13 of expansion for the hepatic phenotype in PHH grown in laminin 521 ECM with the growth medium described in Figure 1 using the hepatocyte cell lot described in Figure 2. At day 13 of P0 culture, PHH express HNF4α, albumin, and LGR5. [Figure 4]1 is a series of photomicrographs showing brightfield images of expanded PHHs cultured in the presence of the growth medium described in FIG. 1 using a hepatocyte lot (Lot A in Table 3) grown as a heterogeneous culture composed of hepatic islets in a background of mesenchymal-like cells at day 20 (passage ("P") 0), day 76 (P3), day 89 (P4), and day 113 (P5), respectively. [Diagram 5] Figure 3 is a series of photomicrographs showing immunostaining for markers of hepatic and proliferation phenotypes in PHHs cultured in the presence of growth medium described in Figure 1. The images depict colony tips staining positive for selected markers during long-term culture of the PHH lot described in Figure 4. At day 68, PHHs express HNF4α, albumin, LGR5, and Ki67. [Figure 6] FIG. 3 is a series of photomicrographs showing stages of growing PHHs cultured in the presence of growth medium as described in FIG. 1 for the PHH lots described in FIG. 2. Cells progressively grow in culture, reaching confluence on day 9 of P0, and further progress to a confluent state with well-defined cell edges by day 17 of P0. [Figure 7] Graph showing quantification of fold expansion at P0 after 17 days of culture based on the number of PHH cells seeded and cultured in the presence of growth medium described in FIG. 1 for the PHH lots described in FIG. 2. [Figure 8] FIG. 3 is a graph showing secreted human albumin in NSG mice implanted with grafts containing expanded PHHs of the hepatocyte lots described in FIG. 2. Unexpanded PHHs served as reference controls. Unexpanded PHHs and PHHs cultured in the presence of growth medium described in FIG. 1 for 10 days were dissociated, aggregated with fibroblasts, and encapsulated in fibrin hydrogel to prepare grafts, then implanted in NSG mice. Blood was collected every 2 weeks and secreted human albumin was quantified by ELISA. Human albumin is shown at days 4, 8, 13, 18, 22, 27, 32, 36, 41, 46, 50, 55, and 61. [Figure 9]FIG. 2 is a series of photomicrographs showing immunohistochemistry of explants on day 61 containing unexpanded control PHHs and PHHs grown for 10 days in the presence of growth medium as described in FIG. 1. H&E, hOTC, and Ck18 staining are shown. [Figure 10] A series of photomicrographs showing bright field images of proliferating cells at different days of PHHs cultured in collagen I and laminin 521 ECM at P0 and P1 in low oxygen culture conditions (5% O2) and in the presence of modified growth medium referred to as Expand 3.0 formulation. The modified growth medium contained modified Dulbecco's modified Eagle's medium / Ham's nutrient mixture F-12 supplemented with Wnt signaling activators R-spondin1 and Wnt3a; the receptor tyrosine kinase ligands recombinant epidermal growth factor, recombinant transforming growth factor, recombinant human fibroblast growth factor 7, recombinant human fibroblast growth factor 10, and recombinant human hepatocyte growth factor; the epithelial phenotype stabilizer A83-01; N-acetylcysteine, nicotinamide; 5-10% KNOCKOUT™ serum replacement (KOSR); non-essential amino acids (NEAA); vitamin A-free B27 supplement, and N2 supplement; 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), GLUTAMAX™, penicillin, and streptomycin. [Figure 11] 11 is a graph comparing the cumulative fold expansion (P0+P1) of PHHs cultured in modified growth medium described in FIG. 10 under hypoxic culture conditions (5% O2), in collagen I and laminin 521 ECM. [Figure 12]Heat map of RT-qPCR analysis using TaqMan probes covering different categories with mature and progenitor states of hepatocytes and epithelial and mesenchymal markers in control PHH cells plated overnight in the absence of growth medium and in PHH cultured in the presence of Expand 3.0 formulation in collagen I and laminin 521 ECM at P0 and P1 in hypoxic culture conditions (5% O2). Values ​​were normalized to sample #1 of control hepatocytes plated overnight in collagen I matrix. LGR5 data is presented as a separate bar graph for proliferating cells only, since LGR5 is not detected in control hepatocytes plated overnight. [Figure 13] FIG. 13 is a graph showing the levels of secreted albumin by control PHH cells plated overnight in the absence of growth medium compared to PHH cells cultured in the presence of Expand 3.0 formulation in collagen I and laminin 521 ECM at P0 and P1 in low oxygen culture conditions (5% O2). [Figure 14] 1 is a series of photomicrographs showing immunostaining of markers for hepatic phenotype in PHHs cultured in laminin 521 ECM in the presence of Expand 3.0 formulation in hypoxic culture conditions for 24 days (P0 13 days + P1 11 days). At day 11 of P1 culture, PHHs express HNF4a and albumin. [Figure 15] 1 shows a table and graphs showing the percentage of cells expressing HNF4a and albumin on day 11 of P1 culture from two independent wells of a 6-well plate, as measured by Imaris software. [Figure 16] Figure 1 depicts a timeline of PHH proliferation and maturation along with KOSR percentage (%) during passage (p) at various days (d). Phase contrast microscopy, secreted urea, secreted albumin, and RT-qPCR of maturation markers were used for readout of cell maturation. [Figure 17]FIG. 1 is a graph showing secreted urea (μg / million cells / day) for primary hepatocytes (Primary Hepatocytes), expanded hepatocytes (Expanded Hepatocytes Control), and mature hepatocytes expanded in either Lonza HCM medium (Lonza HCM plus maturation supplement) or William E medium (William E plus maturation supplement). Primary hepatocytes normalized to plated cells and normalized to total cells seeded are shown. Error bars=SEM, n=2 for maturation conditions and n=3 for primary hepatocytes and expanded hepatocytes control. [Figure 18] Graph showing secreted albumin (ng / million cells / day) for primary hepatocytes (Primary Hepatocytes), expanded hepatocytes (Expanded Hepatocytes Control), and mature hepatocytes expanded in either Lonza HCM medium (Lonza HCM plus maturation supplement) or William E medium (William E plus maturation supplement). Primary hepatocytes normalized to plated cells and normalized to total cells seeded are shown. Error bars=SEM, n=2 for maturation conditions and n=3 for primary hepatocytes and expanded hepatocytes control. [Figure 19] 1 is a heat map showing the fold change in transcript levels as measured using RT-PCR of mature hepatocyte markers and progenitor / bile duct cell markers for primary hepatocytes (Primary Hepatocyte Control), expanded hepatocytes (Expanded Hepatocyte Control), and mature hepatocytes expanded in either Lonza HCM medium (Lonza HCM plus Mature Supplement) or William E medium (William E plus Mature Supplement). [Figure 20] A collection of phase contrast images showing hepatocytes before maturation, during maturation, and after completion of maturation. Expanded hepatocytes are shown at 10x magnification on day 24, before maturation begins. Expanded mature hepatocytes (Lonza HCM plus maturation supplement) are shown at 4x and 10x magnification on days 1, 3, and 7 of maturation. Scale bar = 100 micrometers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] definition As used herein, unless the context clearly indicates otherwise, the terms "a," "an," and "the" include plural references. Thus, for example, reference to "a ligand" optionally includes a combination of two or more such ligands, and so forth.

[0067] As used herein, the term "basal medium" refers to any cell culture medium suitable for culturing human cells, including, but not limited to, Dulbecco's Modified Eagle's Medium / Ham's Nutrient Mixture F-12 (DMEM / F-12), LONZA™ HCM™, William E, HepatoZYME-SFM, and any modifications thereof. Modifications to cell culture media include the addition of various reagents, including, but not limited to, stabilized forms of L-glutamine, buffers such as -2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and antibiotic solutions such as penicillin and streptomycin.

[0068] As used herein, "comprise," "comprising," "comprises," and "comprised of" are synonymous with "include," "including," "includes," or "contain," "containing," or "contains," and are inclusive or open-ended terms specifying the presence of what follows, e.g., components, and do not exclude or preclude the presence of additional, unrecited components, features, elements, members, or steps that are known in the art or disclosed therein.

[0069] As used herein, the term "control cells" refers to a population of hepatocytes cultured in a control medium comprising any suitable basal medium for human cells (e.g., Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) supplemented with mammalian serum (e.g., fetal bovine serum) and any other supplier-provided media supplements suitable for hepatocytes.

[0070] As used herein, the term "culturing step" refers to the process of growing and passage cells. This process encompasses the cell culture phase, where cell numbers increase by cell division. Once the cells reach, for example, 80-90% confluence, they can be passaged and seeded onto additional cell culture surfaces. For example, one passage of primary human hepatocytes (PHH) may require at least 3 days to reach confluence. Cells can be passaged and cultured continuously for 120 days. Alternatively, cells can be passaged until transformed or until they lose the hepatic phenotype.

[0071] The terms "decrease," "reduced," "reduction," or "inhibit" are all used herein to mean a decrease by a statistically significant amount. In some embodiments, "decrease," "reduce," "decrease," or "inhibit" typically refers to a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" can encompass complete inhibition or a reduction compared to a reference level.

[0072] As used herein, the term "epithelial phenotype stabilizer" refers to any compound, natural or synthetic, that can promote the maintenance of the epithelial phenotype. Epithelial phenotype stabilizers can also prevent the development of a mesenchymal phenotype. Such compounds include, but are not limited to, inhibitors of the TGFβ signaling pathway and corticosteroids.

[0073] As used herein, "expand," "expands," "expanding," and "expansion" refer to a subsequent increase, e.g., in the number of a population of PHHs. An "expansion step" refers to a phase of cell culture in which the number of PHHs increases by cell division. When used herein in the context of multiple agents that together or collectively "expand" a population of PHHs, this describes an example where each agent individually may or may not achieve the indicated function, but when the agents are combined, the indicated expansion is achieved.

[0074] As used herein, the term "express" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing), (3) translation of the RNA into a polypeptide or protein, and (4) post-translational modification of the polypeptide or protein. In the context of a gene that encodes a protein product, the terms "gene expression" and the like are used interchangeably with the terms "protein expression" and the like. Expression of a gene or protein of interest in a patient can be determined, for example, by detecting, in a sample obtained from the patient, an increase in the amount or concentration of mRNA encoding the corresponding protein (e.g., assessed using RNA detection procedures described herein or known in the art, such as reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RNA seq techniques), an increase in the amount or concentration of the corresponding protein (e.g., assessed using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assay (ELISA), among others), and / or an increase in the activity of the corresponding protein (e.g., in the case of an enzyme, assessed using enzyme activity assays described herein or known in the art). As used herein, a cell is considered to "express" a gene or protein of interest if one or more, or all, of the above events are detectable within the cell or in the medium in which the cell resides.For example, a gene or protein of interest is considered to be "expressed" by a cell, or population of cells, if it is possible to detect: (i) production of a corresponding RNA transcript, such as an mRNA template, by the cell, or population of cells (e.g., using the RNA detection procedures described herein); (ii) processing of the RNA transcript (e.g., splicing, editing, 5' capping, and / or 3' end processing, using the RNA detection procedures described herein); (iii) translation of the RNA template into a protein product (e.g., using the protein detection procedures described herein); and / or (iv) post-translational modification of the protein product (e.g., using the protein detection procedures described herein).

[0075] The terms "expression level" or "level of expression" are generally used interchangeably and generally refer to the amount of a marker in a biological sample. "Expression" generally refers to the process by which information (e.g., genetic coding information and / or epigenetic information) is converted into structures present and functional in a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modification of a polypeptide) are also considered expressed, whether they are derived from transcripts or degraded transcripts generated by alternative splicing, or from post-translational processing of a polypeptide, e.g., by proteolysis. "Expressed genes" include genes that are transcribed into polynucleotides as mRNA and then translated into a polypeptide, and also include genes that are transcribed into RNA but not translated into a polypeptide (e.g., transfer RNA and ribosomal RNA).

[0076] As used herein, the term "hypoxic conditions" refers to conditions in which the oxygen concentration in the atmosphere is below 20.9%. The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase of a statistically significant amount. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level. In the context of a marker (e.g., albumin), "increase" is a statistically significant increase in such level.

[0077] As used herein, the term "inhibitor" refers to any compound, natural or synthetic, capable of reducing the activity of a target protein or signaling pathway. An inhibitor can either directly or indirectly attenuate or prevent the activity of a target protein. Direct inhibition can be obtained, for example, by binding to the protein and preventing the protein from interacting with endogenous molecules, such as enzymes, substrates, or other binding partners, thereby reducing the activity of the protein. For example, an inhibitor can bind to an enzyme active site and sterically exclude the binding of endogenous substrates at this location, thereby reducing the enzymatic activity of the protein. Alternatively, indirect inhibition can be obtained by binding to a protein that promotes the activity of the target protein, for example, by inducing a conformational change in the target protein or catalyzing a chemical modification of the target protein. For example, indirect inhibition of a target protein can be achieved by binding to and inactivating a kinase that catalyzes the phosphorylation of the target protein and thus activating the target protein.

[0078] As used herein, the term "level" refers to the level of a protein compared to a reference. The reference can be any useful reference as defined herein. A "decreased level" and an "increased level" of a protein refer to a decrease or increase in protein level compared to a reference (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more decrease or increase; a decrease or increase of about 10%, about 15%, about 20%, about 30%, about 40%, about 50% or more compared to a reference). By "protein level" is meant a decrease or increase of more than about 20%, about 50%, about 75%, about 100%, or about 200%; a decrease of less than about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold or less or an increase of 15-fold; or an increase of more than about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 1000-fold or more. Protein levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, or ng / mL) or as a percentage of the total protein in the sample.

[0079] As used herein, the term "marker" is used interchangeably herein to refer to a DNA, RNA, protein, carbohydrate, or glycolipid-based molecular marker, the expression or presence of which in a cell sample can be detected by standard methods (or the methods disclosed herein). Expression of such markers can be determined to be higher or lower in a population of expanded and / or mature PHHs using the disclosed compositions and / or according to the disclosed methods, compared to a population of unexpanded and / or immature PHHs not treated with the compositions or methods.

[0080] As used herein, the term "maturation" refers to the process of developing cells into a population distinct from a progenitor cell population. For example, in some cases, a population of hepatocytes that has undergone a maturation step disclosed herein has a mature hepatocyte phenotype (e.g., as assessed by gene expression analysis (e.g., upregulation of transcripts associated with mature hepatocytes and downregulation of transcripts associated with progenitor / bile duct cells), hepatocyte function assays (e.g., urea secretion, CYP3A4 activity)).

[0081] As used herein, the term "one or more" or "at least one," e.g., one or more or at least one member of a group of members, is itself explicit by way of further illustration, and the term specifically encompasses reference to any one of said members, or any two or more of said members, e.g., three or more, four or more, five or more, six or more, or seven or more of said members, and so forth, as well as all or less of said members.

[0082] As used herein, the term "pharmaceutical formulation" refers to a composition containing a population of primary human hepatocytes (PHHs) that have been grown and / or matured according to the methods described herein, formulated with a pharma- ceutical acceptable excipient, and manufactured or sold under the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a subject.

[0083] "Primary cells" and "primary cultures" are used interchangeably herein to refer to cells and cell cultures that are harvested from a human liver and immediately cryopreserved without any in vitro culture for any time. Cells can be harvested from an individual by any convenient method, such as biopsy or isolation from a donated whole liver. An appropriate solution can be used to disperse or suspend the harvested cells. The cells can be used immediately or they can be stored, frozen, thawed and reused for an extended period of time. In such cases, the cells are typically frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other solution commonly used in the art to preserve cells at such freezing temperatures, and thawed by methods commonly known in the art to thaw frozen cultured cells.

[0084] As used herein, the term "primary human hepatocytes" (or "PHH") refers to the major parenchymal cells in the liver. Specifically, these cells are of human origin and have the ability to replicate and increase cell numbers in response to liver injury. Such cells are known in the art to express one or more genes selected from HNF4α, albumin, and members of the CYP gene family. It is also known that such cells do not express the AFP gene. It is further known in the art that such cells express proteins including, but not limited to, HNF4α, albumin, A1AT, transferrin, and urea.

[0085] As used herein, the term "receptor tyrosine kinase ligand" refers to any compound, natural or recombinant, capable of binding to a receptor tyrosine kinase. Such ligands include, but are not limited to, natural and recombinant epidermal growth factors (EGFs), natural and recombinant fibroblast growth factors (FGFs), natural and recombinant hepatocyte growth factors (HGFs), and natural and recombinant transforming growth factors (TGFs).

[0086] As used herein, the term "recipient" refers to any organism to which the composition according to the present invention may be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). The subject may be a subject seeking or needing treatment, a subject in need of treatment, a subject undergoing treatment, a subject to be treated in the future, or a human or animal under the care of a trained professional for a particular disease or condition. In a preferred embodiment, the subject is a human.

[0087] As used herein, the term "serum replacement components" refers to components present in serum-free media. As used herein, transforming growth factor beta "(TGFβ) inhibitor" refers to a substance (e.g., a small molecule, protein, interfering RNA, or other natural or synthetic compound) that can attenuate or prevent the transcription of one or more genes transcribed by the activity of SMAD transcriptional coactivator proteins. TGFβ inhibitors can disrupt the signal transduction cascade that leads to SMAD-induced gene transcription at one or more points in the pathway. For example, TGFβ inhibitors can prevent or prevent TGFβ or TGFβ superfamily ligands, such as activin, inhibin, nodal, lefty, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), or Müllerian inhibitory factors (MIFs), from binding to their endogenous receptors, thereby inhibiting phosphorylation and activation of receptor-associated SMAD proteins. TGFβ pathway inhibitors can function, for example, by binding to SMAD proteins and preventing or preventing the interaction of SMAD proteins with nucleoporins, thereby preventing the translocation of one or more SMAD proteins to the nucleus. TGFβ signaling pathway inhibitors can stabilize the interaction of one or more SMAD proteins with SMAD anchor for receptor activation (SARA), thereby sequestering the SMAD proteins in the cytoplasm and preventing the SMAD proteins from translocating to the nucleus. Other examples of TGFβ signaling pathway inhibitors include substances such as neurogenin that bind to SMAD proteins and sequester them from DNA-binding transcription factors, thereby preventing the transcription of target genes. Alternative inhibitors of the TGFβ signaling pathway include substances that promote the ubiquitination of one or more SMAD proteins, thereby marking the proteins for degradation by the proteasome and preventing the transcription of target genes. Exemplary assays that can be used to measure the inhibitory activity of TGFβ signaling pathway inhibitors include, but are not limited to, electrophoretic mobility shift assays, antibody supershift assays, as well as TGFβ-induced gene reporter assays, among others.

[0088] As used herein, "Wnt signaling activator" refers to an agonist of the canonical Wnt signaling pathway. Agonists of this pathway further include Wnt proteins or other compounds that directly bind to Frizzled and LRP56 co-receptor proteins in a manner that promotes increased concentrations of β-catenin in the nucleus of mammalian cells. Wnt signaling activators include, but are not limited to, Wnt-3a (R&D systems). Alternatively, Wnt pathway agonists can function by inhibiting one or more secreted Frizzled-related proteins (SFRPs) or Wnt inhibitory proteins (WIFs), which bind to and sequester Wnt proteins from endogenous Wnt co-receptors. These Wnt signaling activators can include molecules that activate non-canonical Wnt signaling pathways by stimulating Wnt signaling independent of Frizzled. Non-canonical Wnt signaling activators preferably stimulate the Wnt pathway via the LGR5 cell surface receptor. Well-known LGR5 agonists include, but are not limited to, roof plate specific spondin (R-spondin) proteins.

[0089] Detailed Description The present disclosure provides compositions and methods that can be used for 2-dimensional expansion and 3-dimensional aggregation of primary human hepatocytes (PHH). According to the compositions and methods described herein, a recipient (e.g., a human) can be transplanted with a population of expanded PHH. The disclosed methods for expanding PHH result in robust and broadly applicable culture expansion. Furthermore, these methods are not limited by restrictive age restrictions on donor PHH, thereby expanding the pool of eligible PHH donors and hopefully alleviating the shortage of donor PHH.

[0090] The present invention is based, at least in part, on the discovery of fully defined culture conditions, including a basal medium for human cells, one or more activators of Wnt signaling, one or more receptor tyrosine kinase ligands, one or more epithelial phenotype stabilizers, and optionally one or more cell survival or proliferation agents, that allow PHHs to be cultured for a greater number of passages compared to existing cell culture medium formulations. The defined culture conditions can include the presence of serum replacement components (e.g., KNOCKOUT™ Serum Replacement (KOSR)) and / or culture under hypoxic conditions. The fully defined, xeno-free culture conditions also result in large-scale, robust, two-dimensional expansion of PHHs, advantageously allowing more cells to be obtained from a single starting cell or from a collection of starting cells than was possible using previous methods. These advantages allow for increased availability of donor cells for therapeutic purposes (e.g., to replenish or restore natural liver function).

[0091] The present disclosure also provides compositions and methods that can be used for maturing PHH. According to the compositions and methods described herein, a population of mature hepatocytes can be transplanted into a recipient (e.g., human). The disclosed methods of maturing PHH result in robust and widely applicable cell maturation.

[0092] The present invention is based, at least in part, on the discovery of a basal medium for human cells that includes one or more hepatocyte maturation supplements that mature hepatocytes into a distinct population, and fully defined culture conditions for maturing PHHs, including, optionally, removing one or more supplements and preventing cell proliferation and / or progenitor cell phenotype.

[0093] In some embodiments, the methods include culturing one or more PHHs in contact with an extracellular matrix (ECM) in the presence of a growth medium comprising a basal medium for human cells supplemented with one or more activators of Wnt signaling, one or more receptor tyrosine kinase ligands, and one or more epithelial stabilizing agents.

[0094] In some embodiments, the growth medium comprises a basal medium for human cells supplemented with one or more activators of Wnt signaling, one or more receptor tyrosine kinase ligands, one or more epithelial stabilizing agents, and one or more cell survival agents.

[0095] In some embodiments, the growth medium comprises a basal medium for human cells supplemented with one or more activators of Wnt signaling, one or more receptor tyrosine kinase ligands, one or more epithelial stabilizing agents, and one or more cell proliferation agents.

[0096] In some embodiments, the growth medium comprises one or more activators of Wnt signaling, one or more receptor tyrosine kinase ligands, one or more epithelial stabilizing agents, one or more cell survival agents, and a basal medium for human cells supplemented with one or more cell survival agents.

[0097] In some embodiments, the maturation medium comprises a basal medium for human cells supplemented with one or more maturation supplements. In some embodiments, the maturation medium does not include one or more supplements that promote cell growth. For example, compared to the growth medium, the maturation medium disclosed herein may lack one or more supplements that promote cell growth.

[0098] In some embodiments, the maturation medium does not include one or more supplements that promote progenitor cell phenotype.For example, compared to the proliferation medium, the maturation medium disclosed herein may lack one or more supplements that promote progenitor cell phenotype.

[0099] liver cells In some aspects of the compositions and methods described herein, the hepatocytes are PHHs. In some embodiments, culturing PHHs with the expansion methods disclosed herein allows the cells to expand while maintaining hepatic phenotype. In some embodiments, the expanded population of PHHs formed using the expansion methods comprises hepatic stem cell or progenitor-like cells. In some aspects, the method further comprises maturing the expanded population of PHHs.

[0100] In some embodiments, the PHH is obtained from mature tissue. In some embodiments, the PHH is not derived from, for example, a hepatocyte cell line that has been differentiated in vitro. In some embodiments, the PHH is or is derived from a primary hepatocyte.

[0101] PHH can be obtained by any suitable method. In some embodiments, cells are isolated by collagenase digestion, for example as described in the Examples and in Dorell et al., 2008 (Hepatology. 2008 48:1282-91). In some embodiments, collagenase digestion is performed on tissue biopsies. In some embodiments, collagenase and actase digestion are used to obtain PHH.

[0102] PHHs are present in the liver. In some embodiments, the method comprises culturing a tissue fragment comprising liver epithelium. In some embodiments, the PHHs are isolated from the tissue fragment. For example, in the context of the liver, the tissue fragment can comprise liver bile duct or bile duct tissue. Liver PHHs can be isolated from EpCAM using FACS-based sorting. + By excluding progenitor cells, PHH isolated from normal liver tissue can contain EpCAM+ progenitor cells, in some embodiments.

[0103] In another embodiment, the cells of the present invention can be isolated by immunoaffinity purification, a separation method well known in the art. Simply by way of example, the cells of the present invention can be isolated by immunoaffinity purification directed against c-kit. As will be apparent to those skilled in the art, this method relies on the immobilization of an antibody on a purification column. The cell sample is then loaded onto the column, allowing the appropriate cells to be bound by the antibody and thus to the column. After a washing step, the cells are eluted from the column using a competitor that preferentially binds to the immobilized anti-c-kit antibody, allowing the cells to be released from the column.

[0104] In some embodiments, the cells can be cultured for at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 days after isolation. In some embodiments, the cells can be cultured for at least about 5 days after isolation. In some embodiments, the cells can be cultured for at least about 10 days after isolation. In some embodiments, the cells can be cultured for at least about 15 days after isolation. In some embodiments, the cells can be cultured for at least about 20 days after isolation. In some embodiments, the cells can be cultured for at least about 25 days after isolation. In some embodiments, the cells can be cultured for at least about 25 days after isolation.

[0105] In certain aspects, the cells are expanded in culture for an extended period of time to improve the homogeneity of the cell phenotype in the cell population or to stabilize the cellular state of the expanded cells. In some embodiments, a population of cells can be used as a starting point, for example a population of cells contained in a liver fragment as described above, and thus the methods of the invention are not limited to using a single cell as a starting point.

[0106] In some embodiments, methods are provided for obtaining a population of expanded PHHs comprising culturing PHHs in a growth medium using the methods described herein. In some embodiments, the method comprises culturing the PHH or obtaining a population of expanded PHH from a single cell. Advantageously, this allows for the formation of a homogenous population of cells. In some embodiments, the method comprises culturing the PHH in a growth medium of the invention for 3-120 days (e.g., 4-119 days, 5-118 days, 10-117 days, 15-116 days, 20-115 days, 30-100 days, 40-90 days, 50-80 days, 60-70 days, or 65 days), thereafter dissociating the cells to a single cell density, seeding one or more cells at a ratio of one cell per vessel (e.g., per well), and expanding the cells using a growth medium of the invention.

[0107] For example, in some embodiments, the method comprises culturing the PHH in the growth medium of the present invention for 3 to 120 days. In some embodiments, the method comprises culturing the PHH in the growth medium of the present invention for 4 to 119 days. In some embodiments, the method comprises culturing the PHH with the growth method of the present invention for 5 to 118 days. In some embodiments, the method comprises culturing the PHH with the growth method of the present invention for 10 to 117 days. In some embodiments, the method comprises culturing the PHH with the growth method of the present invention for 15 to 116 days. In some embodiments, the method comprises culturing the PHH with the growth method of the present invention for 20 to 115 days. In some embodiments, the method comprises culturing the PHH with the growth method of the present invention for 30 to 100 days. In some embodiments, the method comprises culturing the PHH with the growth method of the present invention for 40 to 90 days. In some embodiments, the method comprises culturing the PHH with the growth method of the present invention for 50 to 80 days. In some embodiments, the method comprises culturing the PHH with the growth method of the present invention for 60 to 70 days. In some embodiments, the method comprises culturing the PHH in a growth method of the invention for 65 days.

[0108] In some embodiments, the culturing step includes expanding the plated cells (step P0) and passaging the expanded cells for the first time (step P1). In some embodiments, the P0 step has a duration of 7 to 16 days (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days). In some embodiments, the P0 step has a duration of 11 days. In some embodiments, the P0 step has a duration of 13 days.

[0109] In some embodiments, the P1 step has a duration of 7 to 20 days (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P1 step has a duration of 11 days. In some embodiments, the P1 step has a duration of 13 days.

[0110] In some embodiments, the P0 step comprises administering hepatocytes at a concentration of 200 to 13,333 cells / cm. 2 , e.g., 200 cells / cm 2 ~1,000 cells / cm 2 (e.g., 200 cells / cm 2 , 300 cells / cm 2 , 400 cells / cm 2 , 500 cells / cm 2 , 600 cells / cm 2 , 700 cells / cm 2 , 800 cells / cm 2 , 900 cells / cm 2 , or 1,000 cells / cm 2 ), 1,000 cells / cm 2 ~10,000 cells / cm 2 (e.g., 1,000 cells / cm 2 , 2,000 cells / cm 2 , 3,000 cells / cm 2 , 4,000 cells / cm 2 , 5,000 cells / cm 2 , 6,000 cells / cm 2 , 7,000 cells / cm 2 , 8,000 cells / cm2 , 9,000 cells / cm 2 , or 10,000 cells / cm 2 ), or 10,000 cells / cm 2 ~13,333 cells / cm 2 (e.g., 10,000 cells / cm 2 , 11,000 cells / cm 2 , 12,000 cells / cm 2 , 13,000 cells / cm 2 , or 13,333 cells / cm 2 In some embodiments, the P0 step comprises seeding at a density of 667 cells / cm. 2 In some embodiments, the P1 step includes seeding hepatocytes at a density of 333 to 13,333 cells / cm. 2 In some embodiments, the P1 step comprises seeding hepatocytes at a density of 1,333 cells / cm. 2 The method includes seeding hepatocytes at a density of 100-200 μg / ml.

[0111] In some embodiments, the PHH are cultured in the presence of about 5% carbon dioxide. In some embodiments, the PHH are cultured at a temperature of about 37° C. In some embodiments, the PHH are cultured under low oxygen conditions.

[0112] Advantageously, the culture method provided by the present invention results in a cell population in which hepatocytes are formed that remain non-tumorigenic when the cells are cultured for an extended period of time, as measured by molecular characterization of the expanded cells. Thus, in some embodiments, a population of PHHs of the present invention remains non-tumorigenic as measured by molecular characterization of expanded cells after 4 months or more (e.g., 5, 6, 7, or 8 months) of culture in a growth medium of the present invention. For example, in some embodiments, a population of PHHs of the present invention remains non-tumorigenic as measured by molecular characterization of expanded cells after 5 months of culture in a growth medium of the present invention. In some embodiments, a population of PHHs of the present invention remains non-tumorigenic as measured by molecular characterization of expanded cells after 6 months of culture in a growth medium of the present invention. In some embodiments, a population of PHHs of the present invention remains non-tumorigenic as measured by molecular characterization of expanded cells after 7 months of culture in a growth medium of the present invention. In some embodiments, populations of PHHs of the invention remain non-tumorigenic after 8 months of culture in a growth medium of the invention, as measured by molecular characterization of the expanded cells.

[0113] After culturing, the method may further include obtaining and / or isolating one or more PHHs. For example, after culturing the PHHs, it may be useful to remove one or more PHHs cultured in the growth medium from the culture medium for use in a subsequent application. For example, it may be useful to isolate a single cell for culturing using the growth medium of the present invention. Alternatively, it may be useful to obtain a population of cells for culturing using the growth medium of the present invention.

[0114] A population of expanded PHHs of the invention preferably comprises at least 50% (e.g., at least 60%, 70%, 80%, or 90%) viable cells. For example, in some embodiments, a population of expanded PHHs of the invention preferably comprises at least 60% viable cells. In some embodiments, a population of expanded PHHs of the invention preferably comprises at least 70% viable cells. In some embodiments, a population of expanded PHHs of the invention preferably comprises at least 80% viable cells. In some embodiments, a population of expanded PHHs of the invention preferably comprises at least 90% viable cells. Cell viability can be assessed using Hoechst or propidium iodide staining on a FACS.

[0115] In some embodiments, one or more frozen populations of PHH of the present invention are provided. Also provided is a method of preparing a population of expanded PHH for freezing, which comprises dissociating the expanded population of PHH culture, mixing them with a freezing medium such as harvested cell culture freezing medium (Gibco) or CryoStor (Biolife Solutions), and freezing according to standard procedures. Also provided is a method of thawing frozen PHH, which comprises thawing the frozen PHH, embedding the thawed PHH in ECM (e.g., collagen I, or laminin 521), and culturing the PHH in the growth medium of the present invention.

[0116] In some examples, the medium can be first thawed and then supplemented with Y-27632, for example, about 10 μM Y-27632. In some embodiments, the culture medium is supplemented with Y-27632 for the first 1, 2, 3, 4, 5 days or less after thawing, preferably the first 3 or 4 days. In some embodiments, Y-27632 is not present in the culture medium after the first 3, 4, 5, 6 days or more, preferably the first 3 or 4 days. This thawing method can be used to grow the PHHs of the present invention. In other examples, a ROCK inhibitor such as Y-27632 may not be present.

[0117] The cells produced by the methods described herein can be used immediately. Alternatively, the cells can be frozen at liquid nitrogen temperature, stored for a long period of time, thawed, and reused. For example, the cells can be frozen in 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution commonly used in the art to preserve cells at such freezing temperatures, and thawed by methods commonly known in the art to thaw frozen cultured cells.

[0118] In some embodiments, the cells grow at a population doubling rate of greater than 2 (e.g., 3, 4, 5, 10, 20, 30) per week. For example, in some embodiments, the cells grow at a population doubling rate of greater than 3 per week. In some embodiments, the cells grow at a population doubling rate of greater than 4 per week. In some embodiments, the cells grow at a population doubling rate of greater than 5 per week. In some embodiments, the cells grow at a population doubling rate of greater than 10 per week. In some embodiments, the cells grow at a population doubling rate of greater than 20 per week. In some embodiments, the cells grow at a population doubling rate of greater than 30 per week.

[0119] In some embodiments, the method can include replacing the medium with fresh medium during the culture process, as the components of the medium are used up during the culture. The frequency with which the medium needs to be replaced with fresh medium will be clear to those skilled in the art. In some embodiments, the medium is replaced every two days, but it is also contemplated that it can be replaced every day, every three days, or as needed.

[0120] The growth medium preferably induces or promotes cell survival and / or proliferation for at least 5 (e.g., at least 5, 10, 25, 50, or 100) days in culture. For example, in some embodiments, the growth medium induces or promotes cell survival and / or proliferation for at least 10 days (e.g., at least 25 days). In some embodiments, the growth medium induces or promotes cell survival and / or proliferation for at least 25 days. In some embodiments, the growth medium induces or promotes cell survival and / or proliferation for at least 50 days. In some embodiments, the growth medium induces or promotes cell survival and / or proliferation for at least 100 days.

[0121] Proliferation can be assessed using techniques well known in the art, such as BrdU staining, Edu staining, Ki67 staining, and proliferation curve assays can be used. For example, 1000 cells / cm of frozen PHHs were cultured in a cell culture vessel suitable for 2D cell culture. 2 After thawing and plating at 200-1000 cells per cm, at passage 0 (P0), at least a 10-fold (e.g., at least 15-fold, 20-fold, or 30-fold) cell expansion is achieved, and the cell yield after expansion is 2 Winning 2 x 10 3 ~10×10 3 Once confluence is reached, the expanded PHH are then passaged and cultured at 1000× until approximately at least 500-fold (e.g., at least 1000-fold) expansion of the PHH is achieved. 2 It is possible to seed at a density of 200-1000 cells per mouse, or even higher. In some embodiments, once confluence is reached, the expanded PHHs can then be passaged at a ratio of 1:3 until approximately 100-fold expansion of PHHs is achieved. This is important for the industry, as the availability of PHHs for transplantation poses a serious problem. For mouse transplantation, for example, a minimum of 10 5 In some cases, 1 × 10 cells are needed for human transplantation to be successful. 7 Cells may be required.

[0122] In other words, the growth medium used in accordance with the present invention is capable of expanding a population of PHHs to form an expanded population of PHHs that, under appropriate conditions, maintains a hepatic phenotype for at least six passages or achieves at least a 500-fold expansion (e.g., at least a 1000-fold expansion).

[0123] In some embodiments, after the culturing step, the expression profile of the PHH comprises expression of Ki67 (i.e., protein) by at least 15% of the PHH (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more). In some embodiments, after the culturing step, the expression profile of the PHH comprises expression of Ki67 by up to 15% of the PHH (e.g., up to 10%, 5%, 4%, 3%, 2%, or 1%).

[0124] In some embodiments, after the culturing step, the expression profile of the PHH comprises expression of Ki67 (i.e., gene) by at least 15% of the PHH (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more). In some embodiments, after the culturing step, the expression profile of the PHH comprises expression of Ki67 by up to 15% of the PHH (e.g., up to 10%, 5%, 4%, 3%, 2%, or 1%).

[0125] In some embodiments, after the culturing step, the hepatocyte yield increases at least 2-fold (e.g., at least 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 30-fold) within 14-28 days (e.g., within 15-27 days, within 16-26 days, within 17-25 days, within 18-24 days, within 19-23 days, within 20-21 days, or within 22 days) of culture.

[0126] In some embodiments, after the culturing step, the hepatocyte yield is increased by at least 500-fold within 30 days (e.g., within 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, or 14 days) of culture. In some embodiments, after the culturing step, the hepatocyte yield is increased by at least 500-fold within 24 days of culture.

[0127] In some embodiments, after the culturing step, the hepatocyte yield is increased by 500-fold to 2000-fold (e.g., 1000-fold to 2000-fold, e.g., 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 1,100-fold, 1,200-fold, 1,300-fold, 1,400-fold, 1,500-fold, 1,600-fold, 1,700-fold, 1,800-fold, 1,900-fold, or 2,000-fold) within 30 days (e.g., 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, or 14 days) of culture. In some embodiments, after the culturing step, the hepatocyte yield is increased by 500-fold to 2000-fold (e.g., 1000-fold to 2000-fold, e.g., 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 1,100-fold, 1,200-fold, 1,300-fold, 1,400-fold, 1,500-fold, 1,600-fold, 1,700-fold, 1,800-fold, 1,900-fold, or 2,000-fold) within 24 days of culture.

[0128] In some embodiments, following hepatocyte expansion, the expanded hepatocyte population can undergo maturation in the presence of a maturation medium comprising a basal medium for human cells to which one or more hepatocyte maturation supplements are added. In some embodiments, the maturation step occurs immediately after hepatocyte expansion. In some embodiments, the maturation step does not immediately follow hepatocyte expansion. In some embodiments, the maturation step has a duration of 3 to 12 days (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days). In some embodiments, the maturation step is 7 days.

[0129] Maturation can be assessed using techniques well known in the art, such as phase contrast microscopy to visualize cell morphology, detection of secreted urea and albumin, and RT-qPCR of hepatocyte maturation markers.

[0130] Activator of Wnt signaling The growth medium of the present invention includes one or more Wnt agonists. The Wnt signaling pathway is usually defined by a series of events that occur when the cell surface Wnt receptor complex, which includes the Frizzled receptor, LRP, and LGR, which are extracellular signaling molecules such as members of the Wnt family, is activated. This leads to the activation of the Disheveled family proteins, which inhibit a complex of proteins including Axin, GSK-3, and the protein APC from degrading intracellular β-catenin. The resulting concentrated nuclear β-catenin enhances transcription by TCF / LEF family transcription factors. A Wnt agonist is an agent that activates TCF / LEF-mediated transcription in cells. Thus, the Wnt agonist is selected from Wnt agonists that bind to and activate the Wnt receptor complex, which includes all of the Wnt family proteins, such as inhibitors of intracellular β-catenin degradation, GSK inhibitors (such as CHIR9901), and activators of TCF / LEF.

[0131] In some embodiments, the Wnt agonist is a secreted glycoprotein selected from the following list: Wnt-I / Int-1, Wnt-2 / Irp (InM-related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a (R&D systems), Wnt-4, Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi H et al., 2001 Biochem Biophys Res Com 283 798-805), Wnt-7a (R&D systems), Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, WnMI, and Wnt-16. An overview of human Wnt proteins is provided in "THE WNT FAMILY OF SECRETED PROTEINS," R&D Systems Catalog, 2004.

[0132] The Wnt agonist in the growth medium is preferably any agonist that can stimulate the Wnt pathway by LGR5 cell surface receptor, for example, in one embodiment, the Wnt agonist in the growth medium is an LGR5 agonist. Well-known LGR5 agonists include R-spondin, its fragments and derivatives, and anti-LGR5 antibodies (see, for example, WO2012 / 140274, and De Lau, W. et al. Nature, 2011 July 4; 476 (7360): 293-7). The preferred LGR5 agonist is R-spondin. Any suitable R-spondin can be used, for example, any selected from the list including R-spondin1, R-spondin2, R-spondin3, R-spondin4, or their derivatives. For example, any of R-spondin1 (NU206, Nuvelo, San Carlos, Calif.), R-spondin2 (R&D systems), R-spondin3, and R-spondin4 can be used.

[0133] The Wnt agonist is preferably added to the culture medium in an amount effective to stimulate Wnt activity in cells. As is well known to those skilled in the art, Wnt activity can be determined by measuring the transcriptional activity of Wnt, for example, with pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (Korinek et al., 1997. Science 275:1784-1787).

[0134] In some embodiments, the secreted glycoprotein Wnt agonist is Wnt3a. The growth medium of the present invention can contain at least 10 ng / mL Wnt3a (e.g., at least 50 ng / mL, 100 ng / mL, 1 μg / mL, or 10 μg / mL). For example, in some embodiments, the medium contains at least 50 ng / mL Wnt3a. In some embodiments, the medium contains at least 100 ng / mL Wnt3a. In some embodiments, the medium contains at least 1 μg / mL Wnt3a. In some embodiments, the medium contains at least 10 μg / mL Wnt3a.

[0135] In some embodiments, the LGR5 agonist is selected from R-spondin1, R-spondin2, R-spondin3, or R-spondin4. In some embodiments, the growth medium comprises at least 10 ng / mL (e.g., at least 50 ng / mL, 100 ng / mL, 1 μg / mL, or 5 μg / mL) of R-spondin. For example, in some embodiments, the growth medium comprises at least 50 ng / mL of R-spondin. In some embodiments, the growth medium comprises at least 100 ng / mL of R-spondin. In some embodiments, the growth medium comprises at least 1 μg / mL of R-spondin. In some embodiments, the growth medium comprises at least 5 μg / mL of R-spondin.

[0136] In some embodiments, during the culture of PHH, one or more Wnt agonists can be added to culture medium when necessary, for example, every day or once every two days.Wnt agonist is preferably added to culture medium once every two days.In some embodiments, Wnt agonist can be mixed with culture medium before adding medium to cells.

[0137] Receptor tyrosine kinase ligands Receptor tyrosine kinase ligands described herein include epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and transforming growth factor (TGF), preferably all present in the growth medium. Many receptor tyrosine kinase ligands are mitogenic growth factors.

[0138] EGF is a protein that stimulates cell proliferation and differentiation by binding to its receptor, the epidermal growth factor receptor (EGFR). FGF family members have broad mitogenic and cell survival activities and are involved in a variety of biological processes, including embryonic development, cell proliferation, morphogenesis, tissue repair, tumor growth, and invasion. FGFs stimulate cells by interacting with cell surface fibroblast growth factor receptors (FGFRs). Four closely related receptors (FGFR1-FGFR4) have been identified. Most FGFs bind to more than one receptor (Ornitz J. Biol. Chem. 1998 Feb. 27;273(9):5349-57). However, fibroblast growth factor 10 (FGF-10) and fibroblast growth factor 7 (FGF-7) are unique among FGFs in that they interact only with a specific isoform of FGFR2 (designated FGFR2b), which is expressed exclusively by epithelial cells (Igarashi, J. Biol. Chem. 1998 273(21):13230-5).

[0139] Hepatocyte growth factor / scatter factor (HGF / SF) is a morphogenetic factor that regulates cell proliferation, cell motility, and morphogenesis by activating a tyrosine kinase signaling cascade after binding to the proto-oncogene c-Met receptor.

[0140] Transforming growth factors (TGFs) are polypeptide growth factors. One type of TGF, transforming growth factor alpha (TGFα), binds to the EGFR and induces the development of an epithelial phenotype.

[0141] In some embodiments, the one or more receptor tyrosine kinase ligands in the growth medium are selected from the group consisting of EGF, FGF, HGF, and TGF, with the FGF preferably being FGF-7 or FGF10.

[0142] In some embodiments, the growth medium comprises one or more receptor tyrosine kinase ligands. In some embodiments, only one receptor tyrosine kinase ligand is included in the growth medium, which may be selected from FGF, HGF, EGF, and TGF. In some embodiments, the growth medium comprises two receptor tyrosine kinase ligands. In some embodiments, the two receptor tyrosine kinase ligands in the growth medium are EGF and FGF. In some embodiments, the two receptor tyrosine kinase ligands in the growth medium are EGF and HGF. In some embodiments, the two receptor tyrosine kinase ligands in the growth medium are HGF and FGF. In some embodiments, the two receptor tyrosine kinase ligands in the growth medium are TGF and FGF. In some embodiments, the two receptor tyrosine kinase ligands in the growth medium are TGF and HGF. In some embodiments, the two receptor tyrosine kinase ligands in the growth medium are EGF and TGF. In some embodiments, the growth medium comprises three receptor tyrosine kinase ligands. In some embodiments, the three receptor tyrosine kinase ligands in the growth medium are EGF, FGF, and TGF. In some embodiments, the three receptor tyrosine kinase ligands in the growth medium are EGF, FGF, and HGF. In some embodiments, the three receptor tyrosine kinase ligands in the growth medium are TGF, HGF, and EGF. In some embodiments, the three receptor tyrosine kinase ligands in the growth medium are TGF, HGF, and FGF. In some embodiments, the growth medium comprises four receptor tyrosine kinase ligands. In some embodiments, the four receptor tyrosine kinase ligands are EGF, FGF, HGF, and TGF.

[0143] In some embodiments, any suitable EGF can be used. In some embodiments, the EGF comprises human EGF. In some embodiments, the EGF comprises recombinant human EGF.

[0144] In some embodiments, EGF is added to the growth medium at a concentration of 5-500 ng / mL. For example, in some embodiments, the growth medium comprises 10 ng / mL EGF. In some embodiments, the growth medium comprises 50 ng / mL EGF. In some embodiments, the growth medium comprises 100 ng / mL EGF. In some embodiments, the growth medium comprises 1 μg / mL EGF. In some embodiments, the growth medium comprises 5 μg / mL EGF. In some embodiments, the growth medium comprises 10 μg / mL EGF. In some embodiments, the EGF is replaced with an alternative compound that activates EGFR. For example, it is envisioned that EGF may be replaced with insulin-like growth factor (IGF).

[0145] The FGF used in the growth medium is preferably an FGF capable of binding to fibroblast growth factor 2 (FGFR2), preferably FGF-7 or FGF-10. In some embodiments, not more than one FGF is used. In some embodiments, the FGF is FGF-7. In some embodiments, the FGF is FGF-10. In other embodiments, more than one FGF is used, for example, two, three, or more FGFs. In some embodiments, two FGFs are used. In some embodiments, the two FGFs are FGF-7 and FGF-10. In some embodiments, the FGF is replaced with a compound that activates the FGFR2 pathway ("FGF pathway activator"). In some embodiments, the growth medium comprises 20-500 ng / mL FGF. For example, in some embodiments, the growth medium comprises 50 ng / mL FGF. In some embodiments, the growth medium comprises 100 ng / mL FGF. In some embodiments, the growth medium comprises 250 ng / mL FGF.

[0146] In some embodiments, the HGF is human HGF. In some embodiments, the HGF is recombinant human HGF. In some embodiments, the growth medium comprises 1-50 ng / mL HGF. For example, in some embodiments, the growth medium comprises 1 ng / mL HGF. In some embodiments, the growth medium comprises 5 ng / mL HGF. In some embodiments, the growth medium comprises 10 ng / mL HGF. In some embodiments, the growth medium comprises 25 ng / mL HGF. In some embodiments, the growth medium comprises 50 ng / mL HGF. In some embodiments, the HGF is replaced with a compound that activates the HGF receptor, such as Dihexa.

[0147] In some embodiments, the FGF is human FGF. In some embodiments, the FGF is recombinant human FGF-7 and recombinant human FGF-10. In some embodiments, the growth medium comprises at least 5 ng / mL FGF. For example, in some embodiments, the growth medium comprises 10 ng / mL FGF. In some embodiments, the growth medium comprises 50 ng / mL FGF. In some embodiments, the growth medium comprises 100 ng / mL FGF. In some embodiments, the growth medium comprises 250 ng / mL FGF.

[0148] In some embodiments, the TGF is human TGFα. In some embodiments, the TGF is recombinant human TGFα. In some embodiments, the growth medium comprises at least 2 ng / mL of TGF. For example, in some embodiments, the growth medium comprises 2.5 ng / mL of TGF. In some embodiments, the growth medium comprises 5 ng / mL of TGF. In some embodiments, the growth medium comprises 10 ng / mL of TGF. In some embodiments, the growth medium comprises 50 ng / mL of TGF. In some embodiments, the growth medium comprises 100 ng / mL of TGF. In some embodiments, the growth medium comprises 250 ng / mL of TGF.

[0149] In some embodiments, during the culture of PHH, one or more receptor tyrosine kinase ligands (e.g., EGF, FGF-7, FGF-10, HGF, and TGFα) are added to the culture medium when needed, for example, every day or once every two days. These can be added alone or in combination. They are preferably added once every two days. In some embodiments, one or more receptor tyrosine kinase ligands can be added directly to the cell culture medium before addition to the cells.

[0150] Epithelial phenotype stabilizers The growth medium described herein may include one or more epithelial phenotype stabilizers. In some embodiments, the one or more epithelial phenotype stabilizers are transforming growth factor beta (TGFβ) inhibitors. The presence of TGFβ inhibitors in the growth medium is advantageous because it prevents PHHs from differentiating. In other words, TGFβ inhibitors reduce or inhibit the activity of the TGFβ signaling pathway, thereby preventing the development of a mesenchymal phenotype. TGFβ signaling is involved in many cellular functions, including cell proliferation, cell fate, and apoptosis. Signaling typically begins with the binding of TGFβ superfamily ligands to type II receptors, which recruit and phosphorylate type I receptors. Type I receptors then phosphorylate SMADs, which act as transcription factors in the nucleus and control target gene expression.

[0151] TGFβ superfamily ligands include bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-Mullerian hormone (AMH), activin, nodal, and TGFβ. Generally, Smad2 and Smad3 are phosphorylated by ALK4, 5, and 7 receptors in the TGFβ / activin pathway. In contrast, Smad1, Smad5, and Smad8 are phosphorylated as part of the bone morphogenetic protein (BMP) pathway. Although there is some overlap between the pathways, in the context of the present invention, a "TGFβ inhibitor" or an "inhibitor of TGFβ signaling" is preferably an inhibitor of the TGFβ pathway that acts via Smad2 and Smad3. Thus, in some embodiments, the TGFβ inhibitor is not a BMP inhibitor, e.g., the TGFβ inhibitor is not Noggin. In some embodiments, in addition to the TGFβ inhibitor, a BMP inhibitor is added to the culture medium (see below).

[0152] Thus, a TGFβ inhibitor can be any agent that reduces the activity of the TGFβ signaling pathway. There are many ways to disrupt the TGFβ signaling pathway that are well known in the art and can be used with the present invention. For example, TGFβ signaling can be disrupted by inhibition of TGFβ expression by small interfering RNA methods; inhibition of Furin (a TGFβ-activating protease); inhibition of the pathway by physiological inhibitors; neutralization of TGFβ by monoclonal antibodies; inhibition of TGFβ receptor kinase 1 (also known as ALK5) or other TGFβ-related receptor kinases by small molecule inhibitors; inhibition of Smad2 and Smad3 signaling, for example by overexpression of its physiological inhibitor Smad7, or by using thioredoxin as a Smad anchor that disables Smad activation (Fuchs, O. Inhibition of TGF-Signaling for the Treatment of Tumor Metastasis and Fibrotic Diseases. Current Signal Transduction Therapy, Volume 6, Number 1, January 2011, pp. 29-43(15)).

[0153] Various methods are known to determine whether a substance is a TGFβ inhibitor and can be used with the present invention. For example, a cellular assay can be used in which cells are stably transfected with a reporter construct containing the human PAI-1 promoter or Smad binding sites to drive a luciferase reporter gene. Inhibition of luciferase activity compared to a control group can be used as an indicator of the activity of a compound (De Gouville et al., Br J Pharmacol. 2005 May; 145(2): 166-177).

[0154] The TGFβ inhibitor according to the present invention can be a protein, a peptide, a small molecule, a small interfering RNA, an antisense oligonucleotide, an aptamer, or an antibody. The inhibitor can be naturally occurring or synthetic. In one embodiment, the TGFβ inhibitor is an inhibitor of ALK5. For example, the TGFβ inhibitor can bind to and directly inhibit ALK5. A preferred small molecule TGFβ inhibitor that can be used in the context of the present invention includes A83-01. In some embodiments, the TGFβ inhibitor is a small molecule inhibitor. In some embodiments, the TGFβ inhibitor is A83-01.

[0155] In some embodiments, not more than one TGFβ inhibitor is present in the growth medium.In other embodiments, two or more (e.g., 2, 3, or 4 or more) TGFβ inhibitors are present in the growth medium.Those skilled in the art will understand that many other small molecule inhibitors, which are primarily designed to target other kinases but can also inhibit TGFβ signaling pathways at high concentrations, can also be used in the context of the present invention.

[0156] In some embodiments, the TGFβ inhibitor is an ALK5 inhibitor. In some embodiments, the ALK5 inhibitor is A83-01. In some embodiments, the growth medium comprises at least 10 nM (e.g., at least 50 nM, 100 nM, 1 μM, 5 μM, or 10 μM). For example, in some embodiments, the growth medium comprises at least 50 nM A83-01. In some embodiments, the growth medium comprises at least 100 nM A83-01. In some embodiments, the growth medium comprises at least 1 μM A83-01. In some embodiments, the growth medium comprises at least 5 μM A83-01. In some embodiments, the growth medium comprises at least 10 μM A83-01.

[0157] In some embodiments, the epithelial phenotype stabilizer can further include a corticosteroid. In some embodiments, the corticosteroid includes hydrocortisone. In some embodiments, the growth medium includes at least 10 ng / mL (e.g., at least 25 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1 μg / mL, 5 μg / mL, or 10 μg / mL) of hydrocortisone. For example, in some embodiments, the growth medium includes at least 25 ng / mL of hydrocortisone. In some embodiments, the growth medium includes at least 50 ng / mL of hydrocortisone. In some embodiments, the growth medium includes at least 100 ng / mL of hydrocortisone. In some embodiments, the growth medium includes at least 250 ng / mL of hydrocortisone. In some embodiments, the growth medium includes at least 500 ng / mL of hydrocortisone. In some embodiments, the growth medium includes at least 1 μg / mL of hydrocortisone. In some embodiments, the growth medium comprises at least 5 μg / mL of hydrocortisone, hi some embodiments, the growth medium comprises at least 10 ng / mL.

[0158] In some embodiments, during the culture of PHH, one or more epithelial phenotype stabilizers are added to the culture medium as needed, for example, every day or once every two days. It is preferable to add once every two days. In some embodiments, one or more epithelial phenotype stabilizers can be added directly to the cell culture medium before adding to the PHH.

[0159] Promoting cell survival and proliferation The growth media described herein optionally include one or more agents that promote cell survival and / or proliferation selected from the group consisting of N-acetylcysteine, B27, N2, nicotinamide, and Rho kinase inhibitors, which are believed to control cell survival, cell proliferation, and aid in DNA stability.

[0160] In some embodiments, the growth medium is supplemented with at least 0.25 mM (e.g., at least 0.5 mM, 1 mM, or 5 mM) N-acetylcysteine. For example, in some embodiments, the growth medium is supplemented with at least 0.5 mM N-acetylcysteine. In some embodiments, the growth medium is supplemented with at least 1 mM N-acetylcysteine. In some embodiments, the growth medium is supplemented with at least 5 mM N-acetylcysteine.

[0161] In some embodiments, B27 optionally does not contain vitamin A. B27 supplement can be used to formulate media containing biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin. B27 supplement excluding vitamin A has been shown to work particularly well in liver growth media. B27 supplement, which contains biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin, among other ingredients, is provided as a 50X concentrate. Among these components, at least linoleic acid, retinol, retinyl acetate, and triiodothyronine (T3) are nuclear hormone receptor agonists. B27 supplement can be added to the culture medium as a concentrate, or can be diluted before being added to the culture medium as a concentrate, or can be diluted before being added to the culture medium. B27 supplement can be used at a final concentration of 1X, or at other final concentrations. Using B27 supplement is a convenient way to incorporate biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin into the culture medium of the present invention. It is also contemplated that instead of using B27 supplement, some or all of these components can be added separately to the growth medium. Thus, the growth medium can include some or all of these components.

[0162] In some embodiments, the amount of B27 in the growth medium can be from about 0.1X to about 100X (e.g., from about 0.1X to about 90X, from about 0.5X to about 80X, from about 1X to about 70X, from about 5X to about 60X, and from about 10X to about 50X).

[0163] For example, in some embodiments, the concentration of B27 in the growth medium is about 0.1X. In some embodiments, the concentration of B27 in the growth medium is about 0.5X. In some embodiments, the concentration of B27 in the growth medium is about 1X. In some embodiments, the concentration of B27 in the growth medium is about 5X. In some embodiments, the concentration of B27 in the growth medium is about 10X. In some embodiments, the concentration of B27 in the growth medium is about 50X. In some embodiments, the concentration of B27 in the growth medium is about 70X. In some embodiments, the concentration of B27 in the growth medium is about 80X. In some embodiments, the concentration of B27 in the growth medium is about 90X. In some embodiments, the concentration of B27 in the growth medium is about 100X.

[0164] The N2 supplement is provided as a 100X liquid concentrate containing human transferrin, bovine insulin, progesterone, putrescine, and sodium selenite. The N2 supplement can be added to the culture medium as a concentrate or can be diluted before adding to the culture medium. The N2 supplement can be used at a final concentration of 1X or other final concentrations. Using the N2 supplement is a convenient way to incorporate transferrin, insulin, progesterone, putrescine, and sodium selenite into the culture medium of the present invention. Of course, instead of using the N2 supplement, it is also contemplated to add some or all of these components separately to the growth medium. Thus, in some embodiments, the growth medium can include some or all of these components.

[0165] In some embodiments, the amount of N2 in the growth medium can be from about 0.1X to about 100X (e.g., from about 0.1X to about 90X, from about 0.5X to about 80X, from about 1X to about 70X, from about 5X to about 60X, and from about 10X to about 50X).

[0166] For example, in some embodiments, the concentration of N2 in the growth medium is about 0.1X. In some embodiments, the concentration of N2 in the growth medium is about 0.5X. In some embodiments, the concentration of N2 in the growth medium is about 1X. In some embodiments, the concentration of N2 in the growth medium is about 5X. In some embodiments, the concentration of N2 in the growth medium is about 10X. In some embodiments, the concentration of N2 in the growth medium is about 50X. In some embodiments, the concentration of N2 in the growth medium is about 70X. In some embodiments, the concentration of N2 in the growth medium is about 80X. In some embodiments, the concentration of N2 in the growth medium is about 90X. In some embodiments, the concentration of N2 in the growth medium is about 100X.

[0167] In some embodiments where the medium includes B27, the medium does not include N2. Embodiments of the invention can therefore be adapted to exclude N2, if desired, when B27 is present.

[0168] In some embodiments, N2 is absent from the growth medium. In some embodiments where the medium includes N2, the medium does not include B27. Embodiments of the invention can therefore be adapted to exclude B27, if desired, when N27 is present.

[0169] In some embodiments, B27 is absent from the growth medium. In some embodiments, the growth medium is supplemented with B27 and / or N2. In some embodiments, the growth medium comprises at least 1 mM (e.g., at least 5 mM, 10 mM, or 50 mM) nicotinamide. For example, in some embodiments, the growth medium comprises at least 5 mM nicotinamide. In some embodiments, the growth medium comprises at least 10 mM nicotinamide. In some embodiments, the growth medium comprises at least 50 mM nicotinamide.

[0170] In some embodiments, the Rho kinase inhibitor is Y-27632. In some embodiments, the growth medium comprises at least 1 μM (e.g., at least 5 μM, 10 μM, or 50 μM) Y-27632. For example, in some embodiments, the growth medium comprises at least 5 μM Y-27632. In some embodiments, the growth medium comprises at least 10 μM Y-27632. In some embodiments, the growth medium comprises at least 50 μM Y-27632. In some embodiments, the Rho kinase inhibitor is not included in the growth medium.

[0171] In some embodiments, during the culture of the PHH, one or more agents that promote cell survival and / or proliferation selected from the group consisting of N-acetylcysteine, B27, N2, nicotinamide, and a Rho kinase inhibitor are added to the culture medium as needed, for example, every day or once every two days, preferably once every two days. In some embodiments, one or more agents that promote cell survival and / or proliferation can be added directly to the cell culture medium before adding it to the PHH.

[0172] In some embodiments, the maturation medium disclosed herein does not include one or more agents that promote cell survival and / or proliferation, such as any of the agents that promote cell survival and / or proliferation disclosed herein.

[0173] Accelerated maturation Hepatocytes as described herein can be matured according to the methods described herein. In some embodiments, hepatocytes have previously been expanded according to the methods described herein. In some embodiments, hepatocytes are expanded according to the methods described herein and then matured. The maturation medium as described herein can include a basal medium for human cells. In some embodiments, the maturation basal medium is LONZA™ HCM™, William E, or HepatoZYME-SFM. LONZA™ HCM™ is a hepatocyte culture medium that includes HBM™ basal medium or is obtained by combining HBM™ basal medium with transferrin, ascorbic acid, human epidermal growth factor (HEGF), insulin, hydrocortisone, fatty acid-free bovine serum albumin, and gentamicin sulfate-amphotericin (GA-1000). William E medium is a reduced serum supplement medium for expanding primary hepatocytes. HepatoZYME-SFM is a serum-free medium for long-term maintenance of hepatocytes.

[0174] The maturation media described herein optionally include one or more agents that promote hepatocyte maturation selected from the group consisting of antibiotics, HEPES, GLUTAMAX™, ITS, Notch inhibitors, EGFR inhibitors, oncostatin M, antioxidants, glucocorticoids, pregnane X receptor (PXR) activators, bile acids, cAMP or cAMP analogs, cholesterol, thyroid hormones, serum replacement components, or any combination of the foregoing.

[0175] In some embodiments of the aforementioned aspects, the Notch inhibitor is Compound E, gamma secretase inhibitor XX, or a combination thereof. In some embodiments of the aforementioned aspects, the antibiotic is penicillin, streptomycin, or a combination thereof. In some embodiments of the aforementioned aspects, the EGFR inhibitor is erlotinib hydrochloride. In some embodiments of the aforementioned aspects, the antioxidant is vitamin C. In some embodiments of the aforementioned aspects, the glucocorticoid is dexamethasone, hydrocortisone, or a combination thereof. In some embodiments of the aforementioned aspects, the PXR activator is vitamin K2. In some embodiments of the aforementioned aspects, the bile acid is lithocholic acid, ursodeoxycholic acid, or a combination thereof. In some embodiments of the aforementioned aspects, the cAMP analog is 8-bromo-cAMP, forskolin, or a combination thereof. In some embodiments of the aforementioned aspects, the thyroid hormone is T3. In some embodiments of the aforementioned aspects, the serum replacement component is ITS, KOSR, Trace Elements A, Trace Elements B, or a combination thereof.

[0176] The maturation medium described herein, for example, is subjected to removal of supplements to prevent cell proliferation or progenitor cell phenotype, compared with the proliferation medium disclosed herein.In some embodiments of any of the above aspects, the maturation medium lacks one or more of R-spondin1 / Wnt3a, epidermal growth factor (EGF), transforming growth factor alpha (TGFα), N-acetylcysteine, nicotinamide, B27 supplement, N2 supplement, fibroblast growth factor 7 (FGF7), and fibroblast growth factor 10 (FGF10).

[0177] Serum replacement ingredient The basal or maturation media described herein may further comprise serum replacement components.

[0178] Any suitable serum replacement component can be used. In some embodiments, the serum replacement component is KNOCKOUT™ Serum Replacement (KOSR), human platelet lysate, human serum, ITS, Trace Elements A, or Trace Elements B. KOSR contains albumin or an albumin supplement or an albumin or albumin supplement supplemented with glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and trace element portions of Ag. + , Al 3+ , B.A. 2+ , Cd 2+ , Co 2+ , Cr3+, Ge 4+ , Se 4+ , Br - , I - , Mn 2+ , F - , Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ , and Zr 4+ and one or more components selected from the group consisting of compounds containing the formula:

[0179] In some embodiments, the culturing step includes expanding the plated cells (step P0) and first passaging the expanded cells (step P1). In some embodiments, the P0 step has a duration of 7 to 16 days (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days). In some embodiments, the P0 step has a duration of 11 days. In some embodiments, the P0 step has a duration of 13 days.

[0180] In some embodiments, the P1 step has a duration of 7 to 20 days (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P1 step has a duration of 11 days. In some embodiments, the P1 step has a duration of 13 days.

[0181] In some embodiments, the P0 step comprises administering hepatocytes at a concentration of 200 to 13,333 cells / cm. 2 , e.g., 200 cells / cm 2 ~1,000 cells / cm 2 (e.g., 200 cells / cm 2 , 300 cells / cm 2 , 400 cells / cm 2 , 500 cells / cm 2 , 600 cells / cm 2 , 700 cells / cm 2 , 800 cells / cm 2 , 900 cells / cm 2 , or 1,000 cells / cm 2 ), 1,000 cells / cm 2 ~10,000 cells / cm 2 (e.g., 1,000 cells / cm 2 , 2,000 cells / cm 2 , 3,000 cells / cm 2 , 4,000 cells / cm 2 , 5,000 cells / cm 2 , 6,000 cells / cm 2 , 7,000 cells / cm 2 , 8,000 cells / cm 2, 9,000 cells / cm 2 , or 10,000 cells / cm 2 ), or 10,000 cells / cm 2 ~13,333 cells / cm 2 (e.g., 10,000 cells / cm 2 , 11,000 cells / cm 2 , 12,000 cells / cm 2 , 13,000 cells / cm 2 , or 13,333 cells / cm 2 In some embodiments, the P0 step comprises seeding at a density of 667 cells / cm. 2 In some embodiments, the P1 step includes seeding hepatocytes at a density of 333 to 13,333 cells / cm. 2 In some embodiments, the P1 step comprises seeding hepatocytes at a density of 1,333 cells / cm. 2 The method includes seeding hepatocytes at a density of 100-200 μg / ml.

[0182] In some embodiments, the growth medium comprises a serum replacement component, and the concentration of the serum replacement component is varied over the duration of the culture step. In some embodiments, the concentration of the serum replacement component is 1% (v / v) on day 0 of the P0 step.

[0183] In some embodiments, the concentration of serum replacement is increased to 5% (v / v) (i) when the cell density reaches 15%-30% (e.g., 15%, 20%, 25%, or 30%) confluency, or (ii) between days 3-7 (e.g., days 3, 4, 5, 6, or 7) of the P0 step. In some embodiments, the concentration of serum replacement is increased to 5% (v / v) on day 5 of the P0 step. In some embodiments, the concentration of serum replacement component is 5% (v / v) on day 0 of the P0 step. In some embodiments, the concentration of serum replacement component is 5% (v / v) on day 0 of P0 and remains at 5% (v / v) until the concentration of serum replacement component is increased. In some embodiments, the concentration of serum replacement is increased to 10% (v / v) (i) when the cell density reaches 40%-60% (e.g., 40%, 45%, 50%, 55%, or 60%) confluency, or (ii) between days 7 and 13 (e.g., days 7, 8, 9, 10, 11, 12, or 13) of the P0 step. In some embodiments, the concentration of serum replacement is increased to 10% (v / v) on day 9 of the P0 step.

[0184] In some embodiments, the concentration of serum replacement components is 1% (v / v) on day 0 of the P1 step. In some embodiments, the concentration of serum replacement is increased to 5% (v / v) (i) when the cell density reaches 15%-30% (e.g., 15%, 20%, 25%, or 30%) confluency, or (ii) between days 3-7 (e.g., days 3, 4, 5, 6, or 7) of the P1 step. In some embodiments, the concentration of serum replacement is increased to 5% (v / v) on day 5 of the P1 step.

[0185] In some embodiments, the concentration of the serum replacement component is 5% (v / v) on day 0 of the P1 step. In some embodiments, the concentration of the serum replacement component is 5% (v / v) on day 0 of the P1 step and remains at 5% (v / v) until the concentration of the serum replacement component is increased.

[0186] In some embodiments, the concentration of serum replacement is increased to 10% (v / v) (i) when the cell density reaches 40%-60% (e.g., 40%, 45%, 50%, 55%, or 60%) confluency, or (ii) between days 5 and 13 (e.g., days 5, 6, 7, 8, 9, 10, 11, 12, or 13) of the P1 step. In some embodiments, the concentration of serum replacement is increased to 10% (v / v) on day 9 of the P1 step. In some embodiments, the concentration of serum replacement is increased to 10% (v / v) on day 7 of the P1 step.

[0187] Additional Components In some instances, the basal media described herein may additionally comprise a buffering agent.

[0188] In some embodiments, the buffering agent is -2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). In some embodiments, the growth medium comprises at least 1 mM (e.g., 5 mM, 10 mM, or 50 mM) HEPES. For example, in some embodiments, the growth medium comprises 5 mM HEPES. In some embodiments, the growth medium comprises 10 mM HEPES. In some embodiments, the growth medium comprises 50 mM HEPES.

[0189] In some embodiments, the basal medium comprises L-glutamine or a derivative thereof. In some embodiments, the L-glutamine is an L-glutamine supplement, e.g., L-alanyl-L-glutamine dipeptide, e.g., 0.200 mM L-alanyl-L-glutamine dipeptide (e.g., GLUTAMAX™) in 0.85% NaCl. In some embodiments, the growth medium comprises at least 0.1% (e.g., at least 0.5%, 1%, or 5%) GLUTAMAX™. For example, in some embodiments, the growth medium comprises at least 0.5% GLUTAMAX™. In some embodiments, the growth medium comprises at least 1% GLUTAMAX™. In some embodiments, the growth medium comprises at least 5% GLUTAMAX™. In some embodiments, the growth medium comprises 0.1% to 10%, e.g., 0.1% to 1% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 1% to 10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) GLUTAMAX™.

[0190] In some embodiments, the basal medium further comprises an antibiotic. In some embodiments, antibiotics include, but are not limited to, a solution of penicillin and streptomycin. In some embodiments, the growth medium or maturation medium comprises at least 0.1% (e.g., at least 0.5%, 1%, or 5%) of a solution of penicillin and streptomycin. For example, in some embodiments, the growth medium or maturation medium comprises at least 0.5% of a solution of penicillin and streptomycin. In some embodiments, the growth medium or maturation medium comprises at least 1% of a solution of penicillin and streptomycin. In some embodiments, the growth medium or maturation medium comprises at least 5% of a solution of penicillin and streptomycin. In some embodiments, the growth or maturation medium comprises 0.1% to 10%, e.g., 0.1% to 1% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 1% to 10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) of a solution of penicillin and streptomycin.

[0191] In some embodiments, the growth medium further comprises an amino acid supplement. In some embodiments, the amino acid supplement is a non-essential amino acid (NEAA) supplement. In some embodiments, the NEAA supplement comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. In some embodiments, the growth medium may contain, for example, 1 μM to 100 mM, for example, 1 μM to 10 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM), 10 μM to 100 μM (e.g., 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or 100 μM), 100 μM to 1 mm (e.g., 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 90 In some embodiments, the growth medium comprises a non-essential amino acid supplement containing 100 μM each of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, at 1 mM to 10 mM (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM), or 10 mM to 100 mM (e.g., 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM). In some embodiments, the growth medium comprises 100 μM each of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

[0192] In some embodiments, the growth medium does not include a NEAA supplement. In some embodiments, serum is added to the growth medium. In some embodiments, serum includes, but is not limited to, fetal bovine serum. In some embodiments, the growth medium comprises at least 1% (e.g., at least 5%, 10%, or 15%) fetal bovine serum. For example, in some embodiments, the growth medium comprises at least 5% fetal bovine serum. In some embodiments, the growth medium comprises at least 10% fetal bovine serum. In some embodiments, the growth medium comprises at least 15% fetal bovine serum. In some embodiments, serum is absent from the growth medium.

[0193] In some embodiments, during the cultivation of PHH, the above-mentioned agents are optionally added to the culture medium when necessary, for example, every day or once every two days. They are preferably added once every two days. In some embodiments, the above-mentioned agents can be added directly to the cell culture medium before being added to the PHH.

[0194] In some embodiments, a Notch inhibitor is not present in the growth medium. In some embodiments, a Notch agonist is not present in the growth medium. In some embodiments, neither a Notch inhibitor nor a Notch agonist is present in the growth medium.

[0195] Hypoxic conditions The culture method described herein can include culturing PHH under hypoxic conditions or in the presence of hypoxia mimics.The hypoxic conditions described herein include any conditions in which oxygen is present at a concentration below normal oxygen concentration (normoxic conditions).The hypoxia mimics mimic hypoxia by inducing the accumulation of hypoxia-inducible factor 1α (HiF1α), a protein subunit of a transcription factor that responds to a decrease in available oxygen.

[0196] In some embodiments, culturing comprises culturing the cells under hypoxic conditions. Hypoxic conditions can include, for example, an oxygen concentration of less than 20%. In some embodiments, culturing under hypoxic conditions comprises culturing the cells at an oxygen concentration of 1% to 19% (e.g., 1% to 10%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some embodiments, culturing under hypoxic conditions comprises culturing the cells at an oxygen concentration of 1% to 19%. In some embodiments, culturing comprises culturing the cells at an oxygen concentration of 1% to 10%. In some embodiments, culturing comprises culturing the cells at an oxygen concentration of 5%. In some embodiments, culturing comprises culturing the cells under normoxic conditions.

[0197] In some embodiments, the culturing includes culturing the cells in the presence of a hypoxia mimetic (e.g., a HIF-1α stabilizer, or a PHD inhibitor). Exemplary hypoxia mimetics include iron chelators (e.g., deferoxamine mesylate (DFO), Compound A, deferasirox, and 2,2'-dipyridyl (DP)), iron competitors (e.g., cobalt chloride (CoCl2), or Ni 2+ , Mn2+, Co 2+ , or Zn 2+Examples of hypoxia mimetics include, but are not limited to, divalent metal ions such as 2-oxoglutarate (20G) analogs (e.g., dihydroxybenzoic acid (DHB), N-oxalylglycine, dimethyloxalylglycine (DMOG)), PHD inhibitors (e.g., FG-4497, GSK360A, TM6008, or folic acid), or HIF-1α stabilizers (e.g., miR-335, isoflurane, N-acetylcysteine, MG-132, BSc2118, and tilorone). Hypoxia mimetics are also described, for example, in Davis et al. Front. Cell Dev. Biol. 6:175, 2018. In some embodiments, culturing in the presence of a hypoxia mimetic comprises culturing the cells in the presence of cobalt chloride. In some embodiments, culturing in the presence of a hypoxia mimetic comprises culturing the cells in the presence of desferrioxamine (e.g., DFO). In some embodiments, culturing in the presence of a hypoxia mimetic comprises culturing the cells in the presence of DMOG, hi some embodiments, culturing comprises culturing the cells in the absence of a hypoxia mimetic.

[0198] Culture surface In some examples of the methods described herein, PHHs are cultured on a two-dimensional surface. The cell culture surface can be made of any material suitable for culturing mammalian cells. For example, the surface can be a material that can be easily sterilized, such as plastic or other artificial polymeric materials, as long as the material is biocompatible. In some embodiments, the cell culture surface can contain plastic or glass. In some embodiments, the surface includes any material that allows for cell attachment. In some embodiments, the cells are grown on a single plate.

[0199] Any number of materials can be used to form the surface, including, but not limited to, polyamides, polyesters, polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinyl chloride), polycarbonates, polytetrafluoroethylene (PTFE), nitrocellulose, cotton, polyglycolic acid (PGA), cellulose, dextran, gelatin, glass, fluoropolymers, fluorinated ethylene propylene, polyvinylidene, polydimethylsiloxane, and silicon substrates (fused silica, polysilicon, or single silicon crystals). Metals (e.g., gold, silver, titanium films) can also be used.

[0200] In some embodiments, the surface can be modified to promote cell adhesion (e.g., coated with an adhesive material). For example, a glass surface can be treated with a protein (i.e., a peptide of at least two amino acids) such as collagen or fibronectin to support tissue cells to adhere to the substrate. In some embodiments, a single protein adheres to the surface. In some embodiments, two or more proteins adhere to the surface. Proteins suitable for use in modifying substrates to promote adhesion include proteins to which specific cell types adhere under cell culture conditions. In some embodiments, the surface is coated with an ECM to promote cell adhesion.

[0201] In some embodiments, the PHHs are cultured on a two-dimensional surface, the two-dimensional surface being 9.5 cm 2 ~10,000cm 2 In some embodiments, the PHHs are cultured on a two-dimensional surface, the two-dimensional surface comprising a surface area of ​​500 cm 2 ~10,000cm 2 For example, in some embodiments, the surface area is 9.5 cm 2 ~500cm 2 (For example, 9.5 cm 2 , 100cm 2 , 200cm 2 , 300cm 2 , 400cm2 , or 500cm 2 ), 500cm 2 ~1,000cm 2 (For example, 500 cm 2 , 600cm 2 , 700cm 2 , 800cm 2 , 900cm 2 , or 1,000 cm 2 ) or 1,000 cm 2 ~10,000cm 2 (For example, 1,000 cm 2 , 2,000cm 2 , 3,000cm 2 , 4,000cm 2 , 5,000cm 2 , 6,000cm 2 , 7,000cm 2 , 8,000cm 2 , 9,000cm 2 , or 10,000 cm 2 In some embodiments, the PHHs are cultured on a two-dimensional surface, and the two-dimensional surface has a diameter of 636 cm 2 ~6360cm 2 Includes a surface area of

[0202] ECM As described herein, the method of culturing PHHs can include culturing one or more PHHs in contact with ECM. In some embodiments, PHHs are contacted with ECM by physical, mechanical, or chemical means, or any combination thereof. Any suitable ECM can be used. Isolated PHHs are preferably cultured in a microenvironment that at least partially mimics the cellular niche in which the PHHs naturally reside. The cellular niche is determined in part by the PHHs and surrounding cells, as well as the ECM produced by cells within the niche. This cellular niche can be mimicked by culturing the PHHs in the presence of a biomaterial, such as an ECM that provides key regulatory signals that control hepatocyte fate.

[0203] In some embodiments, the PHH adheres to the ECM, hi some embodiments, the surface of the cell culture is coated with an ECM. The ECM comprises various polysaccharides, water, elastin, and glycoproteins, including collagen, entactin (nidogen), fibronectin, and laminin. The ECM is secreted by connective tissue cells. Various types of ECM are known, including various compositions containing various types of glycoproteins and / or various combinations of glycoproteins. The ECM can be provided by removing these cells and culturing ECM-producing cells, such as fibroblasts, in a container before adding isolated tissue fragments or isolated PHH. Examples of ECM-producing cells are chondrocytes, which produce mainly collagen and proteoglycans, fibroblasts, which produce mainly type IV collagen, laminin, interstitial procollagen, and fibronectin, and colonic myofibroblasts, which produce mainly collagen (types I, III, and V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tenascin-C.

[0204] Alternatively, the ECM is commercially available. An example of a commercially available ECM is ECM Proteins (Invitrogen). Synthetic ECM materials can be used. If desired, a mixture of ECM materials can be used. In some embodiments, the ECM does not include a hydrogel (e.g., MATRIGEL™). In some embodiments, the ECM includes a hydrogel (e.g., MATRIGEL™). In some embodiments, the hydrogel is MATRIGEL™.

[0205] In some embodiments, the ECM comprises collagen. In some embodiments, the collagen is collagen I or collagen IV. In some embodiments, the collagen is collagen I. In some embodiments, the collagen is collagen IV.

[0206] In some embodiments, the ECM comprises laminin. In some embodiments, the laminin is laminin 111, laminin 211, laminin 221, laminin 332, laminin 411, laminin 421, laminin 511, or laminin 521. In some embodiments, the laminin is laminin 111. In some embodiments, the laminin is laminin 511. In some embodiments, the laminin is laminin 521.

[0207] In some embodiments, the culture medium is disposed above the ECM. The culture medium can then be removed and replenished as and when necessary. In some embodiments, the culture medium is replenished every day. In some embodiments, the culture medium is replenished every two days. In some embodiments, the culture medium is replenished every three days.

[0208] In some embodiments, when a component is "added" to or "removed" from a medium, this can mean that the medium itself is removed from the ECM and then new medium containing the "added" component or excluding the "removed" component is placed on top of the ECM.

[0209] In some embodiments, the culture medium of the present invention contacts the ECM or a 3D matrix that mimics the ECM by interacting with cell membrane proteins such as integrins.

[0210] Cell signatures Albumin is a protein produced in the liver that prevents fluid from leaking out of the bloodstream. It is a marker for hepatoblasts and terminally differentiated hepatocytes. In some embodiments, after culture, PHH secrete albumin. Proliferated PHH secrete lower levels of albumin compared to control PHH plated overnight. In some embodiments, after maturation, mature hepatocytes secrete albumin.

[0211] In some embodiments, the expanded PHH secrete at least 1 ug / million cells / day (e.g., at least 2 μg / million cells / day, 5 μg / million cells / day, 10 μg / million cells / day, 25 μg / million cells / day, or 50 μg / million cells / day) of albumin. For example, in some embodiments, the expanded PHH secrete at least 2 μg / million cells / day of albumin. In some embodiments, the expanded PHH secrete at least 5 μg / million cells / day of albumin. In some embodiments, the expanded PHH secrete at least 10 μg / million cells / day of albumin. In some embodiments, the expanded PHH secrete at least 25 μg / million cells / day of albumin. In some embodiments, the expanded PHH secrete at least 50 μg albumin per million cells per day.

[0212] Self-renewing cell populations have been discovered that are capable of expressing LGR5 on their surface. LGR5-positive cells proliferate by dividing to form clones that further divide into clones, thus expanding the size of the cell population without the need for external intervention to evolve into cells with a more restricted differentiation potential.

[0213] In some embodiments, a population of PHHs cultured in a growth medium of the present invention expresses the LGR5 cell surface marker. In some embodiments, LGR5 expression is induced in expanded PHH populations, but LGR5 expression is not seen in control PHHs plated overnight.

[0214] The liver breaks down proteins and produces nitrogen-containing ammonia. Nitrogen then combines with other elements, such as carbon, hydrogen, and oxygen, to form urea. In some embodiments, after culturing and / or growing PHH, the cells downregulate the transcription levels of urea cycle enzymes and secreted urea is not detected, suggesting that an additional maturation step is required to enable the grown PHH to secrete urea. In some embodiments, after maturation, mature hepatocytes secrete urea.

[0215] Adult mature PHHs are also known to express one or more proteins selected from the cytochrome p450 (CYP) protein family, including Cyp3a4, Cyp1a2; the nuclear receptor NR1I2; apical and basolateral polar membrane proteins, such as ABCG2, ABCC2, ABCB11, SR-B1, SLC10A1, etc. In some embodiments, the expanded PHHs downregulate one or more of these proteins characteristic of a mature cell state, compared to unexpanded overnight plated control PHHs. In some embodiments, the expanded PHHs upregulate one or more of fetal / liver progenitor / cholangiocyte markers, such as AFP, Cyp3a7, EPCAM, LGR5, KRT7, KRT19, AQP1, compared to unexpanded overnight plated control PHHs. In some embodiments, the expanded PHHs require an additional maturation step to upregulate urea cycle enzymes, proteins characteristic of the mature cell state, and to downregulate fetal / hepatic progenitor / cholangiocyte markers.

[0216] The presence and / or expression levels / amounts of the various markers described herein in a sample may be analyzed by a number of methodologies, many of which are known in the art and understood by those of skill in the art, including immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofiltration assays (ELIFA), fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, quantitative blood-based assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, fluorescence in situ hybridization (FIH), and the like. These include, but are not limited to, any one of a wide variety of assays that can be performed by PCR, including quantitative real-time PCR (qRT-PCR) and other amplified detection methods, such as NANOSTRING®, branched DNA, SISBA, TMA, RNA-Seq (bulk and single cell), microarray analysis, gene expression profiling, and / or serial analysis of gene expression ("SAGE"), and protein, gene, and / or tissue array analysis. Exemplary protocols for assessing the status of genes and gene products can be found, for example, in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting), and 18 (PCR Analysis). Multiplexed immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD"), may also be used.

[0217] In some embodiments of any of the methods described herein, DNA from the PHH can be isolated and then sequenced using next-generation sequencing methods, such as the targeted gene pull-down and sequencing method described in Frampton et al. (Nature Biotechnology. 31:1023-1033, 2013), which is incorporated by reference in its entirety.

[0218] In any of the preceding methods, the presence and / or expression level / amount of a marker (e.g., LGR5) is measured by determining the protein expression level of the marker. In some embodiments, the method includes contacting a biological sample with an antibody that specifically binds to the marker (e.g., an anti-LGR5 antibody) under conditions that allow binding of the marker, and detecting whether a complex is formed between the antibody and the marker. Such a method may be an in vitro method or an in vivo method. Any method known in the art or described herein that measures protein expression levels may be used. For example, in some embodiments, the protein expression level of the marker is determined using a method selected from the group consisting of flow cytometry (e.g., fluorescence-activated cell sorting (FACS™)), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry (IHC), immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC.

[0219] In some embodiments, the presence and / or expression level / amount of a marker (e.g., LGR5) is measured by determining the mRNA expression level of the marker. In some embodiments, the expression level of a gene is determined using a method comprising: (a) performing gene expression profiling, PCR (such as RT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY techniques, or FISH on a sample (such as a liver disease sample from a subject); and b) determining the presence and / or expression level / amount of the marker in the sample. In one embodiment, the PCR method is qRT-PCR. In one embodiment, the PCR method is multiplex PCR. In some embodiments, gene expression is measured by microarray. In some embodiments, gene expression is measured by qRT-PCR. In some embodiments, expression is measured by multiplex PCR.

[0220] Methods for evaluating mRNA in cells are well known, and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization, Northern blots, and related techniques using labeled riboprobes specific to one or more genes), and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific to one or more genes, and other amplification-based detection methods, such as branched DNA, SISBA, TMA, etc.). Samples from mammals can be conveniently assayed for mRNA using Northern, dot blot, or PCR analysis. In addition, such methods can include one or more steps that allow the level of target mRNA to be determined in biological samples (for example, by simultaneously testing the level of a control mRNA sequence of a "housekeeping" gene, such as an actin family member).

[0221] In addition to or as an alternative to mRNA expression analysis, other markers such as protein expression can be quantified according to the methods described above. For example, the methods of the present invention include testing a sample for a genomic marker (e.g., the presence of AFP) and further testing the sample for a protein marker (e.g., the protein transcript of AFP).

[0222] In some embodiments of any of the methods, DNA sequence can serve as marker. DNA can be quantified according to any method known in the art, including but not limited to PCR, exome-seq (e.g., whole exome sequencing), DNA microarray analysis, NANOSTRING®, or whole genome sequencing.

[0223] In some cases, the expression level of a gene in a sample is the average (e.g., average expression or median expression) of the gene, and the reference expression level of a gene is the average (e.g., average expression or median expression) of the genes in the reference, and the average of the gene in the sample is compared to the average of the genes in the reference.

[0224] In some embodiments, the presence and / or expression level / amount of the marker in the first sample is increased or elevated compared to its presence / absence and / or expression level / amount in the second sample. In some embodiments, the presence / absence and / or expression level / amount of the marker in the first sample is decreased or decreased compared to its presence and / or expression level / amount in the second sample. In some embodiments, the second sample is a reference sample (e.g., PHH cultured in the absence of the disclosed and defined culture medium), a reference cell, a reference tissue, a control sample, a control cell, or a control tissue.

[0225] Containers and incubators The present disclosure also relates to tissue cultures and incubators that can be used with the media and methods described herein. For example, the culture methods described herein can include culturing PHHs in a container or incubator.

[0226] In some embodiments, the tissue culture vessel can include a growth medium as described herein. In some embodiments, the incubator can include a tissue culture vessel. In some embodiments, the incubator can include a tissue culture vessel and maintain the tissue culture vessel under hypoxic conditions. Hypoxic conditions can include, for example, an oxygen concentration of less than 20%. In some embodiments, the incubator maintains an oxygen concentration of 1%-19% (e.g., 1%-10%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some embodiments, the oxygen concentration is 1%-10%. In some embodiments, the oxygen level is 5%. In some embodiments, the incubator can include a tissue culture vessel and maintain the tissue culture vessel under normoxic conditions.

[0227] Hepatocyte aggregates The grown and / or mature PHHs of the present invention can aggregate. The aggregates described herein include a population of PHHs. In some embodiments, the PHHs are mixed under conditions that allow the cell population to form aggregates. In some embodiments, the PHHs are mixed using tissue preparation techniques. In some embodiments, the PHHs are cultured in hanging drops; microwell molds; non-adherent surfaces; spheroid suspension culture using spinner flasks, vertical wheel bioreactors, horizontal wheel bioreactors, or microfluidic spheroid systems. Additional methods include using sonication and using positively charged surfaces on plates. In some embodiments, the PHHs are mixed in the presence of stromal cells (e.g., normal human dermal fibroblasts (NHDFs)). In some embodiments, the PHHs are mixed in the presence of NHDFs. In some embodiments, the PHHs are mixed in the absence of stromal cells (e.g., NHDFs). In some embodiments, the PHHs are mixed in the absence of NHDFs.

[0228] In other aspects, the compositions provided herein can contain additional components including, but not limited to, growth factors, ligands, cytokines, drugs, etc. In some embodiments, the cell mixture can include molecules that induce additional microenvironmental cues, such as small molecules or growth factors, that stimulate or enhance the proliferation and expansion of the cell population.

[0229] In certain embodiments, the aggregates disclosed herein include one or more adhesive materials that promote the maintenance of a desired phenotype of the transplanted cells in vivo. The materials can include, but are not limited to, antibodies, proteins, peptides, nucleic acids, peptide aptamers, nucleic acid aptamers, sugars, proteoglycans, or cell receptors. The type of adhesive material (e.g., ECM materials, sugars, proteoglycans, etc.) is determined, in part, by the cell type (e.g., PHH) being cultured.

[0230] In some embodiments, organization of cells and materials into spatial arrangements, such as aggregates, can be achieved by physically constraining the arrangement of cells / materials using wells or grooves, or by injecting cells into microfluidic channels or oriented voids / pores. In certain embodiments, cells can be organized by physically positioning the cells with electric fields, magnetic tweezers, optical tweezers, ultrasound, pressure waves, or micromanipulators.

[0231] The cells produced by the methods described herein can be used immediately in the production of aggregates. Alternatively, the cells can be frozen at liquid nitrogen temperature, stored for a long period of time, thawed, and reused. For example, the cells can be frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution commonly used in the art to preserve cells at such freezing temperatures, and thawed by methods commonly known in the art to thaw frozen cultured cells.

[0232] cell transplantation As described herein, the methods disclosed herein can include introducing the expanded and / or matured population of PHHs, or their progeny, into a recipient. In some embodiments, the expanded and / or matured population of PHHs, or their progeny, is introduced into the recipient in the form of hepatocyte aggregates. In some embodiments, the recipient is a human. In some embodiments, the recipient is a human patient suffering from liver disease. The expanded and / or matured population of PHHs, or their progeny, can be incorporated into a recombinant tissue construct, e.g., for transplantation into a subject. The recombinant tissue construct can include a biocompatible hydrogel scaffold (e.g., containing fibrin). The biocompatible scaffold can contain an encapsulated population of aggregated PHHs.

[0233] In some embodiments, the invention also provides for the use of PHHs cultured, expanded, and / or matured as described herein. For example, in some embodiments, the invention also provides for the use of populations of expanded and / or matured PHHs of the invention, or multiple frozen PHHs derived from the populations of expanded PHHs, in discovery screens; toxicity assays; gene expression studies, including recombinant gene expression; studies of mechanisms involved in tissue injury and repair; studies of inflammatory and infectious diseases; studies of pathogenic mechanisms; or studies of mechanisms of cell transformation and pathogenesis of liver disease.

[0234] In some embodiments, the invention also provides cells derived from the expanded and / or mature PHH populations of the invention for use in medicine. In some embodiments, the invention also provides cells derived from the expanded and / or mature PHH populations of the invention for use in treating a disorder, condition, or disease. In some embodiments, the invention also provides expanded PHH populations of the invention or cells derived from the expanded PHH populations for use, for example, in regenerative medicine, where the use involves transplanting the expanded cell population or cells derived from the expanded PHH population into a patient. In some embodiments, the invention also provides mature PHH populations of the invention or cells derived from the mature PHH population for use, for example, in regenerative medicine, where the use involves transplanting the mature cell population or cells derived from the mature PHH population into a patient.

[0235] Pharmaceutical preparations The present invention also provides pharmaceutical formulations comprising one or more populations of grown and / or mature PHHs and a pharma- ceutically acceptable diluent and / or excipient. In some embodiments, the pharmaceutical formulation comprises a population of PHHs and one or more excipients. In some embodiments, the excipient is selected from water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0236] In some embodiments, the growth or maturation medium can be mixed with pharma- ceutically acceptable active and / or inactive substances to prepare a pharmaceutical composition or formulation. The composition and method for pharmaceutical formulation depends on several criteria, including but not limited to the route of administration, the extent of the disease, or the dose to be administered.

[0237] In some embodiments, the pharmaceutical composition comprising the population of PHHs includes any pharma- ceutically acceptable salt of the inhibitor, an ester of the inhibitor, or a salt of such an ester. In some embodiments, the pharmaceutical composition comprising the population of PHHs can result (directly or indirectly) in a biologically active metabolite or residue thereof upon administration to a subject (e.g., a human). Thus, for example, the present disclosure is directed to pharma- ceutically acceptable salts of the inhibitors, prodrugs, pharma- ceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharma- ceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the prodrugs include one or more conjugate groups attached to the growth medium, where the conjugate groups are cleaved by endogenous enzymes in the body.

[0238] In some embodiments, the pharmaceutical formulation includes a co-solvent system. Certain such co-solvent systems include, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In some embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of 3% w / v benzyl alcohol, 8% w / v of the non-polar surfactant Polysorbate 80™, and 65% w / v of polyethylene glycol 300 in absolute ethanol. The proportions of such a co-solvent system may vary significantly without significantly altering its solubility and toxicity characteristics. Additionally, the identity of the co-solvent components may be changed, for example, other surfactants may be used in place of Polysorbate 80™, the fractional amount of polyethylene glycol may be changed, other biocompatible polymers, for example, polyvinylpyrrolidone, may replace polyethylene glycol, and dextrose may be replaced with other sugars or polysaccharides.

[0239] In some embodiments, the pharmaceutical formulation is prepared for administration by injection (e.g., intraocular (e.g., intravitreal), intravenous, subcutaneous, intramuscular, intrathecal, intracerebroventricular, etc.). In some embodiments, the pharmaceutical formulation includes a carrier and is formulated in an aqueous solution, e.g., water or a physiologically compatible buffer, e.g., Hank's solution, Ringer's solution, or saline buffer, etc. In some embodiments, other ingredients (e.g., ingredients that aid in solubility or act as preservatives) are included. In some embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, etc. Certain injectable pharmaceutical formulations are provided in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical formulations for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending agents, stabilizing agents, and / or dispersing agents. Certain solvents suitable for use in injectable pharmaceutical formulations include, but are not limited to, lipophilic solvents and fatty oils such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

[0240] kit The compositions described herein can be provided in kits for use in the propagation of freshly extracted or previously frozen PHHs or in the maturation of PHHs. In some embodiments, the kits can include one or more components of cell culture media as described herein. In some embodiments, the kits can include a package insert instructing a user of the kit, e.g., a laboratory scientist, to perform any one of the methods described herein. In some embodiments, the kits can include frozen PHHs previously propagated according to the methods described herein. In some embodiments, the kits can include frozen PHHs previously matured according to the methods described herein. In some embodiments, the kits can optionally include a device for administering the pharmaceutical formulation.

[0241] Working Example The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein may be used and evaluated, are intended to be purely illustrative of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0242] Example 1. Evaluation of cell culture methods using growth media A frozen vial of primary human hepatocytes (PHH) was thawed according to the instructions provided by the supplier. 2 Winning 5 x 10 3 ~6×10 3 PHHs were plated at a density of 1000 μg / ml on tissue culture vessels coated with rat tail collagen I or recombinant human laminin 521 (Table 1, Table 2). After 4 hours, the plating medium was replaced with the disclosed growth medium, which was replaced every 48 hours. Figure 1 shows bright field images of PHH cells cultured with growth medium after 6 days (P0) in a T-75 culture flask. PHHs were harvested at different time points and immunofluorescence was performed by fixing PHHs in 4% formaldehyde, permeabilizing PHHs in 0.4% Triton X-100, blocking with bovine serum albumin, incubating overnight with primary antibodies selected from albumin, HNF4α, and LGR5, and incubating with the appropriate secondary antibodies for 1 hour. Images were taken with a fluorescent microscope. After 13 days of growth on collagen I-coated, 2D plastic surfaces, PHHs expressed albumin, HNF4α, and LGR5 (Figure 2). After 13 days of growth on 2D plastic surfaces coated with laminin 521, PHH expressed albumin, HNF4α, and LGR5 (Figure 3). Qualitative comparisons between immunostained cell panels show that collagen I and laminin 521 yielded similar staining results. The use of ECM to culture PHH improved their long-term survival and the continued presence of a proliferative hepatic phenotype.

[0243] [Table 1]

[0244] [Table 2]

[0245] Example 2. Assessment of PHH proliferation PHHs were passaged and grown on laminin 521-coated 2D surfaces in the presence of growth medium for 120 days. Medium was changed every 48 hours. During this period, the cells maintained their morphology and epithelial phenotype as seen in (Figure 4). On day 68, immunostaining of the cells revealed that the cells at the tip of the colony were proliferative hepatocyte phenotype cells and expressed HNF4α, LGR5, and Ki67 (Figure 5).

[0246] The panels in Figure 6 represent PHHs at days 2, 7, 9, and 17, respectively, cultured in the growth medium described in Table 2. By day 2, approximately 20-25% of the plated cells appear to have attached to the cell culture surface. By day 7, the cells are actively dividing and have increased in size. At day 9, the cells are fully confluent and display a cobblestone pattern, characteristic of an epithelial phenotype. At day 17, the cells are densely packed, displaying a well-defined hepatocyte morphology. They appear to have decreased in size compared to earlier time points.

[0247] PHH (per well, 50 x 10 3 cells) were seeded onto the surface of a 6-well tissue culture plate and cultured in the presence of growth medium (Figure 7, day 1). By day 17, the cells had expanded 3.1-fold, with one well of the 6-well plate containing 15.5 x 10 cells per well. 4 cells (Figure 7, day 17) (Figure 8).

[0248] Example 3. Expanded cells transplanted into mice PHH(4.5×10 5 cells) in 7 mg / mL bovine fibrinogen at 9 × 10 5One cell graft was prepared by aggregating with 10 normal human dermal fibroblasts (NHDFs). Lot D of PHHs listed in Table 3 was used in this experiment. Grafts for transplantation were prepared using unexpanded PHHs and PHHs grown for 10 days in collagen I matrix using the growth medium shown in Figure 1. Three NOD-scid IL2Rγ null NSG™ mice (5-8 weeks old on arrival) each received one graft of 10-day-old expanded PHH aggregates, and six NSG™ mice each received one graft of control unexpanded PHH aggregates. NSG™ mice are immunodeficient mice. Long-term clinical observations were performed mainly by taking blood samples three times every two weeks for two months, and secreted human albumin levels were detected and quantified by ELISA (Figure 8). The detection of human albumin in mice transplanted with these aggregates indicated that the PHHs in the aggregates were alive. Furthermore, the expanded PHHs were confirmed to be healthy because they were able to continue to synthesize and secrete human albumin even two months after transplantation. Terminal analysis of the expanded grafts was also performed by immunohistochemistry to confirm the presence of hepatocytes in the grafts. Immunohistochemistry confirmed the presence of healthy liver aggregates by H&E, hOTC, and CK18 staining (Figure 9).

[0249] [Table 3]

[0250] Example 4. Evaluation of the growth limits of PHH PHHs were passaged and grown on 2D surfaces coated with ECM containing collagen I and laminin 511 in the presence of growth medium. Growth medium contained the Wnt signaling activators Wnt3a and roof plate specific spondin protein 4, the receptor tyrosine kinase ligands epidermal growth factor and transforming growth factor α, the epithelial phenotype stabilizer A83-01, and the cell survival agents nicotinamide and B27. Medium was changed every 2 days and PHHs were passaged until confluence was reached. PHHs were seeded on new surfaces at 1:10 dilution. Samples of PHHs were collected after 6 months of culture. Immunofluorescence was performed to evaluate PHH expression profile. Quantification by immunofluorescence showed that up to 15% (e.g., up to 10%, 5%, or 1%) of the PHH expressed Ki67, at least 10% (e.g., at least 11%, 12%, 13%, 14%, or 15%) of the cells expressed Cyp3a4, and at least 80% (e.g., at least 85%, 90%, or 95%) of the cells expressed one or more proteins selected from hepatocyte nuclear factor 4α, leucine-rich repeat-containing G protein-coupled receptor 5, keratin 18, and albumin.

[0251] Example 5. Growth of PHHs using modified growth medium - EXPAND 3.0 cocktail PHHs were grown in a similar manner as described in Example 1 using a modified growth medium, EXPAND 3.0 cocktail. The modified growth medium lacked ROCK inhibitor and contained KNOCKOUT™ serum replacement (KOSR) and non-essential amino acids (NEAA) (Table 4). KOSR was added in gradient increments of 5-10% to the EXPAND 3.0 cocktail. Growing PHHs were placed in an incubator with a low oxygen atmosphere of 5% oxygen for the EXPAND 3.0 cocktail. These changes in culture conditions resulted in robust growth of PHHs in both collagen I and laminin 521 matrices (Figure 10). With further optimization of passaging and P1 growth, Expand 3.0 supported a cumulative growth of over 1000-fold in P0+P1 (Figure 11). RT-qPCR analysis of expanded hepatocytes at P0 and P1 demonstrated a hepatic progenitor state of expanded hepatocytes compared to control non-expanded hepatocytes plated overnight with collagen I or laminin 521 (Figure 12). Lower amounts of secreted albumin indicate a less mature cellular state of expanded hepatocytes compared to control non-expanded hepatocytes (Figure 13). Immunostaining for HNF4a and albumin (Figure 14) and quantification of the percentage of positive cells (Figure 15) confirm the hepatic phenotype of expanded hepatocytes at the end of p1 expansion.

[0252] [Table 4]

[0253] Example 6. Maturation of expanded hepatocytes PHHs were grown and then matured in a similar manner as described in Examples 1 and 5 (Figure 16). Frozen vials of PHHs were thawed and plated in t75 flasks containing growth medium containing 5% KOSR (step P0). After 9 days, KOSR was increased to 10%. On day 13, cells were transferred to 6-well plates containing fresh medium containing 5% KOSR (step P1) and after 7 days, KOSR was increased to 10% and cells were grown for another 4 days. The total time of cell growth was 24 days. After growth, cells were transferred to 6-well plates containing maturation basal medium of LONZA™ HCM™, William E, or Hepatozyme-SFM with various hepatocyte maturation supplements (Table 5).

[0254] [Table 5]

[0255] After 7 days of maturation, samples were collected to measure urea, albumin, and maturation markers. Maturation of expanded hepatocytes led to induction and secretion of urea (Figure 17), while the level of secreted albumin was maintained (Figure 18). Transcripts corresponding to the mature hepatocyte phenotype were found to be upregulated upon maturation of expanded hepatocytes, while progenitor / cholangiocyte transcripts were downregulated (Figure 19). Phase contrast microscopy images showed morphological differences between expanded hepatocytes and hepatocytes after completion of maturation (Figure 20).

[0256] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0257] While the invention has been described in relation to specific embodiments, it will be understood that the invention is capable of further modifications, and this application is generally intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention, including departures therefrom which come within known or customary practice in the art to which this invention pertains and which may be applied to the essential features hereinbefore described, and which fall within the scope of the appended claims.

[0258] Other embodiments are within the scope of the claims.

Claims

1. 1. A method for culturing primary human hepatocytes, the method comprising: The method comprises culturing one or more hepatocytes in contact with an extracellular matrix (ECM) in the presence of a growth medium comprising a basal medium for human cells supplemented with one or more Wnt signaling activators, one or more receptor tyrosine kinase ligands, and one or more epithelial phenotype stabilizers.

2. the one or more activators of Wnt signalling (a) R-spondin1, R-spondin2, R-spondin3, R-spondin4, Wnt3a, or any combination of the foregoing; or (b) R-spondin1 and Wnt3a The method of claim 1 , comprising: (a) the one or more receptor tyrosine kinase ligands comprise epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor alpha (TGFα), or any combination of the foregoing; and / or (b) the epithelial phenotype stabilizer is a TGFβ inhibitor and / or comprises a corticosteroid; The method of claim 1. (a) the one or more receptor tyrosine kinase ligands comprise human EGF, FGF-7, FGF-10, HGF, TGFα, or any combination of the foregoing; and / or (b) the TGFβ inhibitor is an activin receptor-like kinase (ALK) 5 inhibitor; and / or (c) the corticosteroid is hydrocortisone; The method of claim 3. (a) the one or more receptor tyrosine kinase ligands include human EGF, FGF-7, FGF-10, HGF, and TGFα; and / or (b) the ALK5 inhibitor is A83-01; The method of claim 4. (a) the growth medium comprises N-acetylcysteine, nicotinamide, a Rho kinase inhibitor, or any combination of the foregoing; or (b) the growth medium does not contain a Rho kinase inhibitor; The method of claim 1.

7. The method of claim 6, wherein the Rho kinase inhibitor is Y-27632.

8. The method of claim 1 , wherein the ECM comprises collagen, laminin, or both collagen and laminin. (a) the collagen is collagen I or collagen IV; and / or (b) the laminin is laminin 111, laminin 211, laminin 221, laminin 332, laminin 411, laminin 421, laminin 511, or laminin 521; The method of claim 8.

10. The culturing step comprises: (a) performed on a surface, wherein the hepatocytes are adherently attached to the surface during the culturing step; and / or (b) culturing the cells under hypoxic conditions or in the presence of a hypoxia mimetic; The method of claim 1.

11. 11. The method of claim 10, wherein the surface is a two-dimensional surface, and the two-dimensional surface is coated with an ECM.

12. The growth medium comprises: (a) further comprising a B27 supplement, an N2 supplement, or a combination thereof; (b) further comprising an amino acid supplement; (c) further comprising a serum replacement component; and / or (d) does not contain a Notch inhibitor, a Notch agonist, or gastrin; The method of claim 1.

13. 13. The method of claim 12, wherein the B27 supplement does not contain vitamin A.

14. The method of claim 1 , wherein the culturing step includes expanding the plated cells (step P0) and initially passage the expanded cells (step P1). (a) the P0 step has a duration of 7 to 16 days; and / or (b) the P1 step has a duration of 7 to 20 days; 15. The method of claim 14. (a) The P0 step is 200 to 13,333 cells / cm ２ and / or seeding the hepatocytes at a density of (b) the P1 step comprises seeding the hepatocytes at a density of 333 to 13,333 cells / cm 2 ; 15. The method of claim 14.

17. 16. The method of claim 15, wherein the growth medium comprises a serum replacement component, and the concentration of the serum replacement component is varied over the duration of the culturing step. (a) the concentration of the serum replacement component is 1% (v / v) on day 0 of the P0 step and remains at 1% (v / v) until the concentration of the serum replacement component is increased; or (b) the concentration of the serum replacement component is 5% (v / v) on day 0 of the P0 step; 18. The method of claim 17.

19. (a) the concentration of the serum replacement component is 1% (v / v) on day 0 of the P0 step, and (i) when the cell density reaches 15% to 30% confluency, or (ii) between days 3 and 7 of the P0 step, the concentration of the serum replacement is increased to 5% (v / v); and / or (b) (i) when the cell density reaches 40% to 60% confluency, or (ii) between days 7 and 13 of the P0 step, the concentration of the serum replacement is increased to 10% (v / v); 20. The method of claim 18. (a) the concentration of the serum replacement component is 1% (v / v) on day 0 of the P1 step; or (b) the concentration of the serum replacement component is 5% (v / v) on day 0 of the P1 step and remains at 5% (v / v) until the concentration of the serum replacement component is increased; 20. The method of claim 18.

21. (a) the concentration of the serum replacement component is 1% (v / v) on day 0 of the P1 step, and (i) when the cell density reaches 15% to 30% confluency, or (ii) between days 3 and 7 of the P1 step, the concentration of the serum replacement is increased to 5% (v / v); and / or (b) (i) when the cell density reaches 40% to 60% confluency, or (ii) between days 5 and 13 of the P1 step, the concentration of the serum replacement is increased to 10% (v / v); 21. The method of claim 20.

22. 2. The method of claim 1, wherein the method further comprises measuring an expression profile of the hepatocytes after the culturing step, and wherein the duration of the culturing step is at least 3 days.

23. After the culturing step, the expression profile of the hepatocytes is (a) expression by at least 80% of the hepatocytes of one or more proteins selected from hepatocyte nuclear factor 4 alpha (HNF4α), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), keratin 18 (CK18), and albumin; (b) expression of one or more genes selected from HNF4α, LGR5, CK18, and ALB by at least 80% of the hepatocytes; (c) expression of Ki67 protein or Ki67 gene by up to 15% of said hepatocytes; (d) expression of Ki67 protein or Ki67 gene by at least 15% of said hepatocytes; or (e) at least 10% downregulation of one or more proteins characteristic of a mature cell state and upregulation of one or more fetal / liver progenitor / cholangiocyte markers compared to control hepatocytes plated overnight; 23. The method of claim 22, comprising:

24. After the culturing step, the expression profile of the hepatocytes comprises at least 10% downregulation of one or more proteins characteristic of a mature cell state and upregulation of one or more fetal / liver progenitor / cholangiocyte markers compared to control hepatocytes plated overnight, and optionally (a) the mature hepatocyte state protein is NR1I2, a urea cycle enzyme, Cyp3a4, Cyp1a2, ABCG2, ABCC2, ABCB11, SR-B1, or SLC10A1; and / or (b) the fetal / liver progenitor / bile duct cell marker is AFP, Cyp3a7, EPCAM, LGR5, KRT7, KRT19, or AQP1; 24. The method of claim 23.

25. After the culturing step, (a) the expression profile of the hepatocytes comprises decreased expression of urea cycle enzymes; (b) the hepatocyte yield is at least 5 x 10 3 hepatocytes per cm 2 ; (c) the yield of hepatocytes increases at least two-fold within 14 days of culture; (d) the yield of hepatocytes is increased by at least 500-fold within 24 days or within 30 days of culture; or (e) the yield of the hepatocytes increases between 500-fold and 2000-fold within 24 days or within 30 days of culture; The method of claim 1.

26. 26. The method of any one of claims 1 to 25, wherein the method further comprises maturing the hepatocytes in a maturation medium comprising a basal medium for human cells supplemented with one or more hepatocyte maturation supplements.

27. A method for maturing a population of hepatocytes, the method comprising maturing an expanded population of hepatocytes in the presence of a maturation medium comprising a basal medium for human cells to which one or more hepatocyte maturation supplements are added.

28. 28. The method of claim 27, wherein the maturation step has a duration of 3 to 12 days.

29. 28. The method of claim 27, wherein the one or more maturation supplements comprise an antibiotic, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), GLUTAMAX™, ITS, a Notch inhibitor, an EGFR inhibitor, oncostatin M, an antioxidant, a glucocorticoid, a pregnane X receptor (PXR) activator, a bile acid, cAMP or a cAMP analog, cholesterol, a thyroid hormone, a serum replacement component, or any combination of the foregoing.

30. (a) the Notch inhibitor is Compound E, gamma-secretase inhibitor XX, or a combination thereof; (b) the EGFR inhibitor is erlotinib hydrochloride; (c) the antioxidant is vitamin C; (d) the glucocorticoid is dexamethasone, hydrocortisone, or a combination thereof; (e) the PXR activator is vitamin K2; (f) the bile acid is lithocholic acid, ursodeoxycholic acid, or a combination thereof; (g) the cAMP analog is 8-bromo-cAMP, forskolin, or a combination thereof; (h) the thyroid hormone is T3, and / or (i) the serum replacement component is ITS, KOSR, Trace Elements A, Trace Elements B, or a combination thereof; 30. The method of claim 29.

31. 28. The method of claim 27, wherein the maturation medium lacks one or more of R-spondin1 / Wnt3a, epidermal growth factor (EGF), transforming growth factor alpha (TGFα), N-acetylcysteine, nicotinamide, B27 supplement, N2 supplement, fibroblast growth factor 7 (FGF7), and fibroblast growth factor 10 (FGF10).

32. 26. A kit comprising a growth medium comprising a basal medium for human cells supplemented with one or more Wnt signaling activators, one or more receptor tyrosine kinase ligands, and one or more epithelial phenotype stabilizers, the kit further comprising a package insert instructing a user of the kit to culture one or more hepatocytes according to the method of any one of claims 1 to 25.

33. A growth medium comprising a basal medium for human cells supplemented with one or more Wnt signaling activators, one or more receptor tyrosine kinase ligands, and one or more epithelial phenotype stabilizers.

34. the one or more activators of Wnt signalling (a) R-spondin1, R-spondin2, R-spondin3, R-spondin4, Wnt3a, or any combination of the foregoing; or (b) R-spondin1 and Wnt3a 34. The growth medium of claim 33, comprising:

35. (a) the receptor tyrosine kinase ligand comprises EGF, FGF, HGF, TGFα, or any combination of the foregoing; and / or (b) the epithelial phenotype stabilizer is a TGFβ inhibitor and / or comprises a corticosteroid; 34. The growth medium of claim 33.

36. (a) the receptor tyrosine kinase ligand comprises human EGF, FGF-7, FGF-10, HGF, or TGFα; (b) the TGFβ inhibitor is an ALK5 inhibitor; and / or (c) the corticosteroid is hydrocortisone; 36. The growth medium of claim 35.

37. 37. The growth medium of claim 36, wherein the ALK5 inhibitor is A83-01.

38. (a) the growth medium comprises N-acetylcysteine, nicotinamide, or a Rho kinase inhibitor; or (b) the growth medium does not contain a Rho kinase inhibitor; 34. The growth medium of claim 33.

39. The growth medium comprises: (a) further comprising a B27 supplement, an N2 supplement, or a combination thereof; and / or (b) further comprising an amino acid supplement; (c) further comprising a serum replacement component; and / or (d) does not contain a Notch inhibitor, a Notch agonist, or gastrin; 34. The growth medium of claim 33.

40. 32. A kit comprising a maturation medium comprising a basal medium for human cells supplemented with one or more maturation supplements, the kit further comprising a package insert instructing a user of the kit to mature one or more hepatocytes according to the method of any one of claims 27 to 31.

41. A maturation medium comprising a basal medium for human cells to which one or more hepatocyte maturation supplements are added.

42. 42. The maturation medium of claim 41 , wherein the one or more maturation supplements comprise an antibiotic, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), GLUTAMAX™, ITS, a Notch inhibitor, an EGFR inhibitor, oncostatin M, an antioxidant, a glucocorticoid, a pregnane X receptor (PXR) activator, a bile acid, cAMP or a cAMP analogue, cholesterol, a thyroid hormone, a serum replacement component, or any combination of the foregoing.

43. (a) the Notch inhibitor is Compound E, gamma-secretase inhibitor XX, or a combination thereof; (b) the EGFR inhibitor is erlotinib hydrochloride; (c) the antioxidant is vitamin C; (d) the glucocorticoid is dexamethasone, hydrocortisone, or a combination thereof; (e) the PXR activator is vitamin K2; (f) the bile acid is lithocholic acid, ursodeoxycholic acid, or a combination thereof; (g) the cAMP analog is 8-bromo-cAMP, forskolin, or a combination thereof; (h) the thyroid hormone is T3; or (i) the serum replacement component is ITS, KOSR, Trace Elements A, Trace Elements B, or a combination thereof; 43. The maturation medium of claim 42.

44. 44. The maturation medium of any one of claims 41 to 43, wherein the maturation medium lacks one or more of R-spondin1 / Wnt3a, epidermal growth factor (EGF), transforming growth factor alpha (TGFα), N-acetylcysteine, nicotinamide, B27 supplement, N2 supplement, fibroblast growth factor 7 (FGF7), and fibroblast growth factor 10 (FGF10).

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