Methods of freezing stem cells

Patent metadata

Field	Value
Publication number	US-9402388-B2
Application number	US-201314069557-A
Country	US
Kind code	B2
Filing date	Nov 1, 2013
Priority date	Nov 1, 2012
Publication date	Aug 2, 2016
Grant date	Aug 2, 2016

Abstract

The invention is directed to a method of freezing stem cells comprising introducing the HSCs into a cell culture medium that has been conditioned with Wharton's Jelly mesenchymal stem cells (WJSCs), thereby producing a stem cell culture, and slowly freezing the stem cell culture. In other aspects, the invention is directed to compositions comprising stem cells produced by the methods provided herein. In yet other aspects, the invention is directed to pharmaceutical compositions comprising the stem cells produced by the methods provided herein.

First claim

What is claimed:

1. A method of freezing CD34+ stem cells, comprising:
a) introducing the stem cells into a cell culture medium that has been conditioned with Wharton's Jelly mesenchymal stem cells (WJSCs), thereby producing a stem cell culture; and b) slowly freezing the stem cell culture at a rate that maintains stem cell viability, thereby freezing the stem cells.

2. The method of claim 1 wherein the cell culture medium has been conditioned with WJSCs for about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours or 96 hours.

3. The method of claim 1 wherein the cell culture medium is about 5% v/v, 10% v/v, 15% v/v, 20% v/v, 25% v/v, 30% v/v, 35% v/v, 40% v/v, 45% v/v, 50% v/v, 55% v/v, 60% v/v, 65% v/v, 70% v/v, 75% v/v, 80% v/v, 85% v/v, 90% v/v, 95% v/v or 100% v/v.

4. The method of claim 1 wherein the cell culture medium further comprises one or more cryoprotectants, cell culture supplements, amino acids, antibiotics, antimyotics or combinations thereof.

5. The method of claim 4 wherein the one or more cryoprotectants comprises dimethyl sulfoxide (DMSO), glycerol, ethylene glycol or combinations thereof.

6. The method of claim 4 wherein the one or more cell culture supplements comprises serum, serum replacement, albumin or combinations thereof.

7. The method of claim 6 wherein the serum is bovine serum, fetal bovine serum, human serum, serum replacement, human serum albumin or combinations thereof.

8. The method of claim 4 wherein the one or more amino acids comprise alanine, arginine, asparagines, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine or combinations thereof.

9. The method of claim 4 wherein the one or more antibiotics comprises penicillin, streptomycin, gentamycin or combinations thereof.

10. The method of claim 4 wherein the one or more antimyotics comprises amphotericin B.

11. The method of claim 1 wherein the stem cells are slowly frozen to about −100° C., −110° C., −120° C., −130° C., −140° C. or −150° C.

12. The method of claim 1 wherein the stem cells are slowly frozen using a freezing rate of about −0.5° C. per minute, −1° C. per minute, −1.5° C. per minute, −2° C. per minute, −2.5° C. per minute, −3° C. per minute, −3.5° C. per minute, −4° C. per minute, −4.5° C. per minute or −5° C. per minute.

13. The method of claim 1 further comprising storing the stem cells.

14. The method of claim 13 wherein the stem cells are stored in a liquid phase of nitrogen or in a vapor phase of nitrogen.

15. The method of claim 1 further comprising thawing the stem cells.

16. The method of claim 15 wherein the stem cells are thawed in a 37° C. water bath.

17. The method of claim 15 wherein the stem cells are thawed and cultured in a cell culture medium that has been conditioned with Wharton's Jelly mesenchymal stem cells (WJSCs).

18. The method of claim 17 wherein the stem cells are cultured in the cell culture medium for about 6 hours, 12 hours, 24 hours, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days 28 days or 30 days.

19. The method of claim 1 wherein the stem cells are obtained from bone marrow, cord blood, peripheral blood, blood from placenta or a combination thereof.

20. The method of claim 1 wherein the stem cells and the WJSCs are obtained from different individuals.

21. The method of claim 1 wherein the stem cells and WJSCs are obtained from the same individual.

22. The method of claim 1 wherein the cell culture medium is conditioned with the WJSCs that are obtained from the same source as the stem cells.

23. The method of claim 1 wherein the stem cells are human stem cells.

24. The method of claim 23 wherein the cell culture medium comprises human serum.

25. The method of claim 23 wherein the cell culture medium comprises human serum albumin.

26. The method of claim 25 wherein the human serum albumin is recombinant human serum albumin or fraction V.

27. The method of claim 23 wherein the cell culture medium comprises human serum replacement.

28. The method of claim 1 wherein the WJSCs are human WJSCs.

29. The method of claim 1 wherein the method further comprises expanding the stem cells.

30. The method of claim 29 wherein the stem cells are expanded prior to freezing of the stem cells.

31. The method of claim 29 wherein the stem cells are expanded after freezing and thawing of the stem cells.

32. The method of Claim 29 wherein the stem cells are expanded in a cell culture medium comprising Wharton's Jelly mesenchymal stem cells (WJSCs), a cell culture medium that has been conditioned with WJSCs, or a combination thereof.

33. A method of freezing CD34+ stem cells, comprising:
a) introducing the stem cells and a cell culture medium that has been conditioned with Wharton's Jelly mesenchymal stem cells (WJSCs) into a cryovial, thereby producing a stem cell culture; and
b) freezing the stem cell culture at a rate of about −0.5° C. per minute, −1 ° C. per minute, −1.5° C. per minute, −2° C. per minute, −2.5° C. per minute, −3° C. per minute, −3.5° C. per minute, −4° C. per minute, −4.5° C. per minute or −5° C. per minute, thereby freezing the stem cells.

34. Frozen, isolated CD34+stem cells in a culture medium that has been conditioned with Wharton's Jelly mesenchymal stem cells (WJSCs) wherein the culture medium further comprises a cryoprotectant selected from dimethyl sulfoxide (DMSO), glycerol, ethylene glycol or combinations thereof.

Assignees

• Nat Univ Singapore

Inventors

• Fong Chui Yee
• Bongso Tuan Ariffeen
• Lin Daniel Hao

Classifications

• A01N1/126

  Physiologically active agents, e.g. antioxidants or nutrients · CPC title

• C12N5/0647

  Haematopoietic stem cells; Uncommitted or multipotent progenitors · CPC title

• A01N1/125 Primary

  Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators · CPC title

• A01N1/0226

  Human Necessities · mapped topic

• A01N1/0221 Primary

  Human Necessities · mapped topic