Granted Patent B2

US 12,723,235 · App. 17/281,161 · Granted Sep 1, 2026

Inventors: Lijian Hui (Shanghai, CN); Ludi Zhang (Shanghai, CN); Kun Zhang (Shanghai, CN)

Assignee: Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences

C12N5/067A61K35/407A61P1/16

Abstract

Claims (55)

1 . 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 [Leu 15 ]-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 [Leu 15 ]-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.

2 . The method according to claim 1 , wherein said basal medium is selected from the group consisting of DMEM, MEM, RPMI, Neuronal basal and Fischers.

3 . The method according to claim 1 , wherein the method further comprises inducing the expanded hepatocytes into mature hepatocytes, including adding forskolin, dexamethasone, and oncostatin M to the cell culture medium, wherein concentrations of forskolin, dexamethasone, and oncostatin M in the cell culture medium are as follows

forskolin 3 to 7 μM;

dexamethasone 5 to 20 μM; and

oncostatin M 10 to 30 ng/ml.

4 . The method according to claim 1 , wherein the method further comprises sub-culturing the expanded hepatocytes under hypoxic conditions, wherein the hypoxic conditions contain 1-10% oxygen by volume, and wherein the sub-culture is a three-dimensional culture to form organoids.

5 . The method according to claim 1 , wherein the expanded hepatocytes are between mature hepatocytes and liver progenitor cells, and characterized by expressing liver progenitor cell-associated markers SOX9, CK19 and CK7, presenting lower expression levels of ALB, HNF4A, TTR or CYP3A4 compared to hepatocyte not cultured in the medium of claim 1 hepatocytes, and presenting similar or higher expression levels of CYP1A2, CAR, C3 or UGT1A1 compared to hepatocyte not cultured in the medium of claim 1 hepatocytes.

6 . 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 [Leu 15 ]-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 [Leu 15 ]-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.

Assignments (1)

Priority Claims (1)

Continuity (1)

References Cited (26)