Some content of this application is unavailable at the moment.
If this situation persist, please contact us atFeedback&Contact
1. (WO2017132358) METHODS FOR ISOLATION AND EXPANSION OF UMBILICAL CORD MESENCHYMAL STEM CELLS
Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

A method of producing isolated umbilical cord mesenchymal stem cells

(a) incubating umbilical cord tissue with a digestive solution to yield digested umbilical cord tissue;

(b) dissociating said digested umbilical cord tissue to yield a dissociated umbilical cord tissue solution comprising umbilical cord mesenchymal stem cells dispersed in tissue debris;

(c) separating said umbilical cord mesenchymal stem cells from said dissociated umbilical cord tissue solution to yield separated umbilical cord mesenchymal stem cells;

(d) suspending said separated umbilical cord mesenchymal stem cells in a suspension comprising a quantity of xenogen-free culture medium and red blood cell lysing solution, said xenogen-free culture medium comprising human platelet lysate; and

(e) isolating said umbilical cord mesenchymal stem cells from said culture medium and said red blood cell lysing solution, to yield said isolated umbilical cord mesenchymal stem cells.

2. The method of claim 1, wherein said quantity of xenogen-free culture medium comprises from about 2% to about 20% by volume of human platelet lysate.

3. The method of claim 1, wherein blood vessels are not removed from said umbilical cord tissue prior to said incubating (a).

4. The method of claim 1, wherein said incubating (a) occurs at a temperature of from about 30°C to about 45°C.

5. The method of claim 1, wherein said incubating (a) occurs for less than 10 hours.

6. The method of claim 1, wherein said umbilical cord tissue is treated with an antiseptic solution prior to said incubating (a).

7. The method of claim 1, wherein said separating (c) comprises filtering said dissociated umbilical cord tissue solution to remove said tissue debris, and pelleting said umbilical cord mesenchymal stem cells by centrifugation.

8. The method of claim 1, wherein said incubating (a) and said dissociating (b) are carried out in the same container.

9. The method of claim 1, wherein said xenogen-free culture medium is essentially free of fetal bovine serum.

10. The method of claim 1, further comprising expanding said isolated umbilical cord mesenchymal stem cells by:

(f) adding said isolated umbilical cord mesenchymal stem cells to a tissue culture plate or seeding said isolated umbilical cord mesenchymal stem cells on a microcarrier, said tissue culture plate or said microcarrier comprising a second quantity of xenogen-free culture medium comprising human platelet lysate;

(g) incubating said isolated umbilical cord mesenchymal stem cells in said tissue culture plate or on said microcarrier to yield cultured umbilical cord mesenchymal stem cells; and

(h) transferring said cultured umbilical cord mesenchymal stem cells to a second tissue culture plate or to a second microcarrier, to yield expanded umbilical cord mesenchymal stem cells.

11. The method of claim 10, wherein said second quantity of xenogen-free culture medium comprises from about 2% to about 20% by volume of human platelet lysate.

12. The method of claim 10, wherein said transferring (h) occurs after said cultured umbilical cord mesenchymal stem cells achieve at least about 70% confluency.

13. The method of claim 10, wherein said isolated umbilical cord mesenchymal stem cells are added to said tissue culture plate at a level of about 5,000 to about 20,000 live cells per cm2.

14. The method of claim 10, wherein said microcarrier comprises a modified polystyrene or cross-linked polystyrene surface.

15. The method of claim 14, wherein said surface comprises a cationic charge, or is coated with high silica glass or recombinant RGD-containing protein.

16. The method of claim 15, wherein the surface is cross-linked polystyrene with a cationic surface charge.

17. The method of claim 1, further comprising cryopreserving said isolated umbilical cord mesenchymal stem cells.

18. The method of claim 17, wherein said isolated umbilical cord mesenchymal stem cells are crysopreserved in a cryopreservative medium with human platelet lysate.

19. The method of claim 1, wherein said digestive solution is a digestive enzyme solution.

20. A xenogen-free culture medium comprising glucose, glutamine, human platelet lysate, and heparin.

21. The culture medium of claim 20, further comprising an antibiotic-antimycotic component.

22. The culture medium of claim 20 consisting essentially of said glucose, said glutamine, said human platelet lysate, and said heparin.