Hepatocyte expansion methods
AssigneeKoninklijke Nederlandse Akademie van Wetenschappen
InventorsJohannes Carolus Clevers, Huili Hu
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Abstract
This invention relates to culture methods and media for in vitro expansion of hepatocytes, particularly primary hepatocytes; hepatocyte cultures and organoids obtainable and obtained by said methods; and uses of said hepatocyte cultures and organoids.
Claims11
A method for expanding hepatocytes in vitro to form a hepatocyte organoid, wherein the method comprises culturing isolated primary hepatocytes in a hepatocyte culture medium, wherein the hepatocyte culture medium comprises:
- i) 50 to 500 ng/ml of a Fibroblast Growth Factor (FGF) selected from FGF7, FGF10, and a combination thereof;
- ii) a Wnt agonist comprising an R-spondin selected from R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4;
- and at least one glycogen synthase kinase 3 (GSK3) inhibitor;
- iii) 5 to 100 ng/ml of epidermal growth factor (EGF);
- iv) 5 to 100 ng/ml of hepatocyte growth factor (HGF);
- and
- v) a transforming growth factor beta (TGF-β) inhibitor, comprising an inhibitor of activin receptor-like kinase ALK4, ALK5 and/or ALK7 signalling pathway;
- wherein the hepatocyte organoid is formed, wherein the hepatocyte organoid does not express KRT7 and KRT19, and wherein the hepatocyte organoid expresses albumin.
A method for expanding hepatocytes in vitro to form a hepatocyte organoid, wherein the method comprises culturing isolated primary hepatocytes in a hepatocyte culture medium,
- wherein the hepatocyte culture medium comprises:
- about 100 ng/ml FGF7;
- about 100 ng/ml FGF10;
- about 20 ng/ml TGFa;
- about 50 ng/ml EGF;
- about 50 ng/ml HGF;
- about 2 μM A83-01;
- about 10 μM Y-27632;
- about 3 μM CHIR99021;
- about 10 mM nicotinamide;
- about 10 nM gastrin;
- about 1.25 mM N-acetylcysteine;
- and about 15% R-spondin conditioned medium;
- wherein the hepatocyte organoid is formed,
- wherein the hepatocyte organoid does not express KRT7 and KRT19, and
- wherein the hepatocyte organoid expresses albumin.
Dependencies are inferred from the claim text.