Proliferative liver organoid, metabolically activated liver organoid, and use thereof

1 . A method for producing a metabolically activated liver organoid, the method comprising:

culturing primary human hepatocytes or cryopreserved primary human hepatocytes in a growth medium and with contact to an extracellular matrix to obtain a proliferative liver organoid for at least 10 days and up to 14 days; and

culturing the proliferative liver organoid in a differentiation medium for 1 week to obtain a metabolically activated liver organoid,

wherein the growth medium comprises an interleukin-6 family cytokine, at least one growth factor, a Wnt agonist, a transforming growth factor-β inhibitor, a bone morphogenetic protein inhibitor, and a forskolin,

wherein the interleukin-6 family cytokine comprises at least one selected from the group consisting of interleukin-6, interleukin-11, oncostatin M, a leukemia inhibitory factor, cardiotrophin-1, and a ciliary neurotrophic factor,

wherein the differentiation medium comprises a growth factor, a transforming growth factor-β inhibitor, and a forskolin, and the differentiation medium does not substantially contain an interleukin-6 family cytokine,

wherein the extracellular matrix comprises basement membrane matrix, collagen I, or a 1:1 mixture of collagen I and basement membrane matrix, and

wherein the at least one growth factor in the growth medium is selected from the group consisting of:

epidermal growth factor;

hepatocyte growth factor;

heparin-binding EGF-like growth factor;

amphiregulin;

a combination of epidermal growth factor, fibroblast growth factor 10, and hepatocyte growth factor;

a combination of epidermal growth factor and fibroblast growth factor 10;

a combination of hepatocyte growth factor and fibroblast growth factor 10; and

a combination of epidermal growth factor and hepatocyte growth factor.

2 . The method according to claim 1 ,

wherein the growth medium does not substantially contain nicotinamide.

3 . The method according to claim 1 ,

wherein the growth factor in the growth medium further comprises at least one selected from the group consisting of an epidermal growth factor, a fibroblast growth factor, a hepatocyte growth factor, amphiregulin, and a heparin-binding EGF-like growth factor.

4 . The method according to claim 1 ,

wherein the growth medium further comprises a Rho kinase inhibitor.

5 . The method according to claim 1 ,

wherein the differentiation medium does not substantially contain nicotinamide.

6 . The method according to claim 1 ,

wherein the growth factor in the differentiation medium comprises at least one selected from the group consisting of an epidermal growth factor, a fibroblast growth factor, and a hepatocyte growth factor.

7 . The method according to claim 1 ,

wherein the differentiation medium further comprises a Wnt agonist.

8 . The method according to claim 1 ,

wherein the differentiation medium further comprises a Rho kinase inhibitor.

9 . The method according to claim 1 ,

wherein the differentiation medium further comprises a bone morphogenetic protein inhibitor.

10 . The method according to claim 1 ,

wherein the differentiation medium further comprises vitamin D.

11 . The method according to claim 1 ,

wherein the differentiation medium further comprises a Notch inhibitor.

12 . The method according to claim 1 , wherein the extracellular matrix comprises collagen I.

13 . The method according to claim 1 , wherein the extracellular matrix comprises laminin, entactin, and collagen IV.