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

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.