Cell culture method and application thereof

• US 20100015103A1
• Filed: 01/15/2009
• Published: 01/21/2010
• Est. Priority Date: 07/16/2008
• Status: Active Grant

• Current Assignee: National Taiwan University (Government of The Republic of China)
• Original Assignee: National Taiwan University (Government of The Republic of China)
• Inventors: Chao, Kuo-Fang, Chao, Kuo-Ching, Liu, Shing-Hwa
• Granted Patent: US 9,017,657 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61K 38/27 Growth hormone [GH], i.e. somatotropin

Abstract

The invention relates to a cell culture method, particularly to a co-culture method for human mesenchymal stem cells and target animal cells, in order to solve the problem that animal cells are not easy to survive alone upon culturing. The invention also provides a method for using a stem cell conditioned medium to culture animal cells. The invention also provides a method to induce the transformation of human fetal islet-like cell clusters from human stem cells and its application thereof.

Claims

1. A cell culture method, comprising:

preparing a suspended medium containing human stem cells;

adding target animal cells to be cultured; and

co-culturing the target cells at a suitable condition.

2. The method according to claim 1, wherein the human stem cells are human mesenchymal stem cells.

3. The method according to claim 1, wherein the target animal cells are mammalian cells.

4. The method according to claim 3, wherein the mammalian cells are human cells.

5. The method according to claim 4, wherein the human cells are islet-like cell clusters.

6. The method according to claim 1, wherein the human stem cells and the target animal cells are inoculated to culture plates sequentially.

7. The method according to claim 1, wherein after the human stem cells and the target animal cells are inoculated for certain time, the culture medium is used to wash off the non-adherent cells from culture plates.

21. A method of using the islet-like cell clusters prepared according to claim 1 to inhibit type I diabetes, comprising transplanting the human islet-like cell clusters to the liver of a subject in need thereof.

8. A cell culture method, comprising:

preparing a stem cell conditioned medium;

adding target animal cells to be cultured; and

culturing the target cells at a suitable condition;

wherein the stem cell conditioned medium is composed of basic culture medium and a active component.

9. The method according to claim 8, wherein one or more active components are selected from the group consisting of interleukin-6 (IL-6), tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue inhibitor of metalloproteinase-2 (TIMP-2), monocyte chemoattractant protein-1 (MCP-1), growth related oncogene (GRO), hepatocyte growth factor (HGF), insulin-like growth factor binding protein 4 (IGFBP-4) and interleukin-8 (IL-8).

10. The method according to claim 8, wherein the target animal cells are mammalian cells.

11. The method according to claim 10, wherein the mammalian cells are human cells.

12. The method according to claim 11, wherein the human cells are islet-like cell clusters.

13. A method of inducing human stem cells differentiate into human islet-like cell clusters, comprising:

separating human stem cells;

culturing the human stem cells in a neuronal conditioned medium; and

culturing the human stem cells in a first conditioned medium and a second conditioned medium sequentially.

14. The method according to claim 13, wherein the neuronal conditioned medium is the cell medium for culturing mammalian brain cells.

15. The method according to claim 13, wherein the first conditioned medium comprises DMEM/F12 medium, glucose, insulin, nicotinamide, and B27.

16. The method according to claim 15, wherein the concentration of glucose is 20 mM to 30 mM.

17. The method according to claim 16, wherein the concentration of glucose is 25 mM.

18. The method according to claim 15, wherein the first conditioned medium further comprises 2% fetal bovine serum.

19. The method according to claim 13, wherein the second conditioned medium is prepared by adding the stem cell conditioned medium into the first conditioned medium.

20. The method according to claim 13, wherein the human stem cells are human mesenchymal stem cells.