Method of preparing three-dimensional cell spheroid including adipose-derived stem cells and hepatocytes

Patent metadata

Field	Value
Publication number	US-12138283-B2
Application number	US-201916413278-A
Country	US
Kind code	B2
Filing date	May 15, 2019
Priority date	May 17, 2018
Publication date	Nov 12, 2024
Grant date	Nov 12, 2024

Abstract

Provided is a method of preparing a three-dimensional cell spheroid, the method including forming the cell spheroid by co-culturing adipose-derived stem cells or mesenchymal stem cells with hepatocytes. According to the cell spheroid prepared by the method, the secretome secreted by the adipose-derived stem cells affects hepatocyte maturation, and therefore, hepatic functions of the finally formed three-dimensional cell spheroid, i.e., organoid, may be enhanced. Further, a composition including a culture medium of the adipose-derived stem cells may prevent or treat liver diseases including hepatitis, hepatotoxicity, cholestasis, fatty liver, etc., and may enhance hepatic functions.

First claim

What is claimed is:

1. A method of preparing a three-dimensional cell co-spheroid, the method comprising:
introducing Foxa3 factor and hepatocyte nuclear factor 4 alpha (HNFα) to hepatic progenitors, and culturing the hepatic progenitors in a hepatocyte maturation medium for 7 days to 28 days, thereby differentiating the hepatic progenitors into hepatic differentiated cells and thereby increasing expression of hepatocyte-specific gene HNFα, alpha-fetoprotein (AFP), albumin (ALB), asialoglycoprotein Receptor 1 (ASGR1), and Cytochromes P450 (CYP450);
adherent-culturing adipose-derived mesenchymal stem cells in a culture plate having a hydrophobic surface, wherein the culture plate is coated with maltose-binding protein-fibroblast growth factor 2 (MBP-FGF2) and poly-(N-p-vinylbenzyl-O-β-D-galactopyranosyl-[1-4]-D-gluconamide (PVLA) and the culture plate is two-dimensional or the hydrophobic surface is flat; and
seeding the hepatic differentiated cells into the culture plate having a hydrophobic surface, thereby co-culturing the adherent-culturing adipose-derived mesenchymal stem cells with the hepatic differentiated cells; and
forming the three-dimensional cell co-spheroid by detaching the adipose-derived mesenchymal stem cells and the hepatic differentiated cells from the culture plate.

2. The method of claim 1 , wherein the cell co-spheroid has a diameter of 300 μm and 2000 μm.

3. The method of claim 1 , wherein the adipose-derived mesenchymal stem cells and the hepatic differentiated cells are mixed at a ratio of 1:5 to 5:1.

4. The method of claim 1 , wherein the co-culture is performed for 20 hours to 60 hours.

5. The method of claim 1 , wherein the adipose-derived mesenchymal stem cells are human adipose-derived mesenchymal stem cells.

6. A three-dimensional cell co-spheroid prepared by the method of claim 1 .

7. A method of preventing or treating liver disease, the method comprising administering to a subject a cellular therapeutic agent comprising three-dimensional cell co-spheroids prepared by the method of claim 1 .

8. The method of claim 7 , wherein the cellular therapeutic agent comprises a culture medium comprising the cell co-spheroids.

9. The method of claim 8 , wherein the culture medium is mixed at a ratio of 100 μl to 500 μl per 1 unit of the cell co-spheroids.

10. The method of claim 8 , wherein the culture medium is obtained from a medium where the cell co-spheroids are three-dimensionally cultured.

11. The method of claim 8 , wherein the culture medium comprises a factor promoting hepatocyte division and maturation.

12. The method of claim 7 , wherein the liver disease is selected from the group consisting of hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, liver ischemia, alcoholic liver disease, hepatic abscess, hepatic coma, hepatatrophia, and hepatic cancer.

Assignees

• Korea Inst Sci & Tech

Inventors

• Kim Sang-Heon
• Hong Seokheon
• Oh Seung Ja
• Park Kwi Deok

Classifications

• C12N2501/165

  Vascular endothelial growth factor [VEGF] · CPC title

• C12N2501/135

  Platelet-derived growth factor [PDGF] · CPC title

• C12N2501/115

  Basic fibroblast growth factor (bFGF, FGF-2) · CPC title

• C12N2501/113

  Acidic fibroblast growth factor (aFGF, FGF-1) · CPC title

• C12N2501/12

  Hepatocyte growth factor [HGF] · CPC title