Method for expanding hepatocyte in vitro and application
AssigneeCenter for Excellence In Molecular Cell Science, Chinese Academy of Sciences
InventorsLijian Hui, Ludi Zhang, Kun Zhang
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Abstract
Provided are a method for expanding a hepatocyte in vitro and an application thereof. A culture system is provided for reprogramming a human hepatocyte into a proliferating intermediate-state cell between a mature hepatocyte and a liver progenitor cell. The liver repopulation ability of the system was verified in animals. The method does not require the introduction of an exogenous gene into a hepatocyte, and the expansion of the hepatocyte can be realized by conventional culture. The obtained hepatocyte can be passaged, and can be cultured to maturation to obtain a functional mature human hepatocyte.
Claims6
A method for in vitro hepatocyte expansion, comprising culturing hepatocytes in a cell culture medium to expand the hepatocytes in vitro, wherein said cell culture medium consists of a basal medium and a Wnt signaling pathway agonist, N-acetylcysteine, nicotinamide, recombinant human FGF10, recombinant human EGF, recombinant human HGF, human [Leu15]-gastrin I, A 83-01, Y-27632, serum, N2 supplement and neuronal culture supplement;
- wherein said Wnt signaling pathway agonist is selected from Wnt3a protein or CHIR;
- wherein the concentration of each component is
- Wnt3a protein
- 30 to 70 ng/ml, or
- CHIR
- 0.05 to 2 μM;
- N-acetylcysteine
- 0.5 to 2 mM;
- nicotinamide
- 5 to 20 mM;
- recombinant human FGF10
- 1 to 4 ng/ml;
- recombinant human EGF
- 30 to 70 ng/ml;
- recombinant human HGF
- 15 to 40 ng/ml;
- human [Leu15]-gastrin I
- 5 to 20 mM;
- A 83-01
- 3 to 7 μM ;
- Y-27632
- 5 to 20 μM;
- and
- serum
- 0.5 to 2%, and
- wherein the hepatocytes are human hepatocytes.
A method for in vitro hepatocyte expansion, comprising culturing hepatocytes in a cell culture medium to expand the hepatocytes in vitro, wherein said cell culture medium consists of a basal medium and a Wnt signaling pathway agonist, N-acetylcysteine, nicotinamide, recombinant human FGF10, recombinant human EGF, recombinant human HGF, human [Leu15]-gastrin I, A 83-01, Y-27632, serum, N2 supplement and neuronal culture supplement;
- wherein said Wnt signaling pathway agonist is selected from Wnt3a protein or CHIR;
- wherein the concentration of each component is
- Wnt3a protein
- 30 to 70 ng/ml, or
- CHIR
- 0.05 to 2 μM;
- N-acetylcysteine
- 0.5 to 2 mM;
- nicotinamide
- 5 to 20 mM;
- recombinant human FGF10
- 1 to 4 ng/ml;
- recombinant human EGF
- 30 to 70 ng/ml;
- recombinant human HGF
- 15 to 40 ng/ml;
- human [Leu15]-gastrin I
- 5 to 20 mM;
- A 83-01
- 3 to 7 μM ;
- Y-27632
- 5 to 20 μM;
- and
- serum
- 0.5 to 2%, and
- wherein the hepatocytes are human hepatocytes.
Dependencies are inferred from the claim text.