# US 2021/0340494 A1 · Method for Expanding Hepatocyte in Vitro and Application > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0340494 A1 - Document ID: US20210340494A1 - Status: Published application - Kind: A1 - Application number: 17/281,161 - Filed: 2019-08-14 - Published: 2021-11-04 - Earliest claimed: 2018-09-30 - Assignees: Center for Excellence In Molecular Cell Science, Chinese Academy of Sciences - Applicants: Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences - Inventors: Lijian Hui; Ludi Zhang; Kun Zhang - Web page: https://patentgrep.com/patent/US20210340494A1 ## CPC - C12N 5/067: {Hepatocytes} - C12N 5/0671: {Three-dimensional culture, tissue culture or organ culture; Encapsulated cells} - A61K 35/407: Liver; Hepatocytes - A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics - C12N 5/0672: {Stem cells; Progenitor cells; Precursor cells; Oval cells} - C12N 2500/02: Atmosphere, e.g. low oxygen conditions - C12N 2501/11: Epidermal growth factor [EGF] - C12N 2501/119: Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10 - C12N 2501/12: Hepatocyte growth factor [HGF] - C12N 2501/15: Transforming growth factor beta (TGF-β) - C12N 2501/345: Gastrin; Cholecystokinins [CCK] - C12N 2501/415: Wnt; Frizzeled - C12N 2501/727: Kinases (EC 2.7.) - C12N 2506/14: from hepatocytes - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS ## 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. ## Claims ### Claim 1 A method for in vitro hepatocyte expansion, comprising culturing hepatocytes in a cell culture medium containing a Wnt signaling pathway agonist to expand the hepatocytes in vitro. ### Claim 2 (depends on 1) The method according to claim 1, wherein the cell culture medium also comprises a component selected from the group consisting of N-acetylcysteine, nicotinamide, FGF10, EGF, HGF, [Leu15]-Gastin I, A 83-01, and Y-27632. ### Claim 3 (depends on 1) The method according to claim 1, wherein the Wnt signaling pathway agonist includes an agonist selected from the group consisting of Wnt3a protein, Wnt3a conditioned medium, and CHIR, or a combination thereof. ### Claim 4 (depends on 1) The method according to claim 1, wherein the cell culture medium also includes a cell growth supplement selected from the group consisting of N2 supplement, and B27 supplement; or further includes serum. ### Claim 5 (depends on 1) The method according to claim 1, wherein the cell culture medium comprises a basal medium selected from the group consisting of DMEM, MEM, RPMI, Neuronal basal and Fischers. ### Claim 6 (depends on 1) The method according to claim 1, wherein the method also includes inducing the expanded hepatocytes into mature hepatocytes, including adding Forskolin, dexamethasone, and oncostatin M to the cell culture medium. ### Claim 7 (depends on 1) The method according to claim 1, wherein the concentration of the components in the cell culture medium is as follows: Wnt3a protein 30 to 70 ng/ml 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 Forskolin 3 to 7 μM Dexamethasone 5 to 20 μM Oncostatin M 10 to 30 ng/ml Serum 0.5 to 2% ### Claim 8 (depends on 1) The method according to claim 1, wherein the method further includes sub-culturing the expanded hepatocytes under hypoxic conditions, wherein the hypoxic conditions contain 0-15% oxygen by volume, wherein the culture is a three-dimensional culture to form organoids. ### Claim 9 (depends on 1) The method according to claim 1, wherein the hepatocytes are human hepatocytes. ### Claim 10 (depends on 1) A hepatocyte culture obtained by the method according to claim 1, or isolated hepatocytes or organoids from the hepatocyte culture. ### Claim 11 (depends on 10) The hepatocyte culture or the isolated hepatocytes or organoids according to claim 10, wherein the hepatocytes are intermediate-state cells between mature hepatocytes and liver progenitor cells, which are obtained by the method of culturing hepatocytes in a cell culture medium containing a Wnt signaling pathway agonist to expand the hepatocytes in vitro; or the hepatocytes or organoids are mature hepatocytes, which are obtained by the method of culturing hepatocytes in a cell culture medium containing a Wnt signaling pathway agonist to expand the hepatocytes in vitro, and adding Forskolin, dexamethasone, and oncostatin M to the cell culture medium to induce the expanded hepatocytes into mature hepatocytes. ### Claim 12 (depends on 10) A method for promoting liver regeneration and preventing, alleviating or treating liver diseases, comprising administrating an effective amount of the hepatocyte culture or the isolated hepatocytes or organoids according to claim 10 to a patient in need thereof. ### Claim 13 (canceled) ### Claim 14 (depends on 10) A pharmaceutical composition comprising the hepatocyte culture or the isolated hepatocytes or organoids according to claim 10, and a pharmaceutically acceptable carrier. ### Claim 15 (depends on 10) A kit comprising the hepatocyte culture or the isolated hepatocytes or organoids according to claim 10; or comprising the pharmaceutical composition comprising the hepatocyte culture or the isolated hepatocytes or organoids according to claim 10, and a pharmaceutically acceptable carrier. ### Claim 16 A culture medium for in vitro expansion of hepatocytes, comprising Wnt signaling pathway agonist, N-acetylcysteine, nicotinamide, FGF10, EGF, HGF, [Leu15]-gastrin I, A 83-01, and Y-27632, wherein the Wnt signaling pathway agonist is selected from the group consisting of Wnt3a protein, Wnt3a conditioned medium, and CHIR, or a combination thereof. ### Claim 17 (depends on 16) The culture medium according to claim 16, wherein the culture medium also includes a component selected from the group consisting of N2 supplement, B27 supplement and serum. ### Claim 18 (depends on 16) A culture medium for in vitro expansion of hepatocytes and induction of hepatocyte maturation, comprising the culture medium for hepatocyte expansion in vitro according to claim 16, and Forskolin, dexamethasone, and oncostatin M. ### Claim 19 (depends on 16) The culture medium according to claim 16, wherein the components of cell culture medium are present in a basal medium selected from the group consisting of DMEM, MEM, RPMI, Neuronal basal and Fischers. ### Claim 20 (depends on 16) The culture medium according to claim 16, wherein the concentration of the components in the cell culture medium is as follows: Wnt3a protein 30 to 70 ng/ml 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 Forskolin 3 to 7 μM Dexamethasone 5 to 20 μM Oncostatin M 10 to 30 ng/ml Serum 0.5 to 2% ### Claim 21 (canceled) ### Claim 22 (depends on 16) A kit for in vitro expansion of hepatocytes and induction of hepatocyte maturation, comprising the culture medium according to claim 16. ### Claim 23 (depends on 1) A method for research on liver related diseases or drugs, comprising using the hepatocyte culture obtained by the method according to claim 1 or isolated hepatocytes or organoids from the hepatocyte culture as an in vitro model.