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.
Claims56
A method for expanding hepatocytes in vitro, wherein the method comprises culturing hepatocytes in a hepatocyte culture medium, wherein the hepatocyte culture medium comprises:
- i. 50 to 500 ng/ml of a Fibroblast Growth Factor (FGF), optionally selected from the group consisting of FGF7, FGF10 and a combination thereof;
- ii. a Wnt agonist comprising an R-spondin 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.
The method of any one of the preceding claims, wherein the GSK3 inhibitor is at a concentration of 1 to 10 μM, 0.5 to 5 μM, 1 to 5 μM, 2 to 4 μM, or preferably about 3 μM.
The method of any one of the preceding claims, wherein the hepatocyte culture medium comprises FGF7 at a concentration of 50 to 400 ng/ml, 50 to 300 ng/ml, 75 to 200 ng/ml, 80 to 150 ng/ml or preferably about 100 ng/ml.
The method of any one of the preceding claims, wherein the EGF is at a concentration of 10 ng/ml to 100 ng/ml, 20 ng/ml to 100 ng/ml, 20 ng/ml to 80 ng/ml, 40 ng/ml to 80 ng/ml, or preferably about 50 ng/ml.
The method of any one of the preceding claims, wherein the HGF is at a concentration of 10 ng/ml to 100 ng/ml, 20 ng/ml to 100 ng/ml, 20 ng/ml to 80 ng/ml, 40 ng/ml to 80 ng/ml, or preferably about 50 ng/ml.
The method of any one of the preceding claims, wherein the TGF-§ inhibitor is at a concentration of 1 to 10 μM, 0.5 to 5 μM, 1 to 4 μM, 1 to 3 μM, or preferably about 2 μM.
The method of any one of the preceding claims, wherein the hepatocyte culture medium further comprises nicotinamide, optionally at a concentration of 1 to 100 mM.
The method of any one of the preceding claims, wherein the hepatocyte culture medium further comprises gastrin, optionally at a concentration of 1 to 100 nM.
The method of any one of the preceding claims, wherein the hepatocyte culture medium further comprises transforming growth factor alpha (TGF-α).
The method of any one of the preceding claims, wherein the hepatocyte culture medium further comprises a Rho-associated protein kinase (ROCK) inhibitor;
- optionally wherein the ROCK inhibitor is selected from the group consisting of:
- Y-27632, GSK429286A, Fasudil, Thiazovivin, Rho Kinase Inhibitor IV and a combination thereof;
- optionally wherein the ROCK inhibitor is Y-27632.
The method of any one of the preceding claims, wherein the hepatocyte culture medium further comprises N-acetylcysteine.
The method of any one of the preceding claims, wherein the hepatocyte culture medium further comprises B27, optionally wherein the B27 does not comprise vitamin A.
The method of any one of the preceding claims, wherein the hepatocyte culture medium further comprises a cell growth medium, optionally wherein the cell growth medium comprises antibiotics, optionally penicillin and streptomycin;
- wherein the cell growth medium is optionally AdDMEM/F12, optionally comprising Hepes and/or Glutamax.
The method of any one of the preceding claims, wherein the hepatocyte culture medium does not comprise a cAMP pathway activator.
The method of any one of the preceding claims, wherein the hepatocytes are primary hepatocytes, preferably isolated cells from liver tissue.
The method of any one of the preceding claims, wherein the hepatocytes are cultured in contact with a three dimensional extracellular matrix (ECM) in the presence of the hepatocyte culture medium.
The method of any one of the preceding claims, wherein the hepatocytes comprise cells selected from the group consisting of:
- fetal hepatocytes, adult hepatocytes and a combination thereof.
The method of any one of the preceding claims, wherein at least part of the expanded cells form one or more hepatocyte organoids.
Dependencies are inferred from the claim text.