SINGLE PLURIPOTENT STEM CELL CULTURE

• US 20090325294A1
• Filed: 06/30/2008
• Published: 12/31/2009
• Est. Priority Date: 07/01/2007
• Status: Active Grant

• Current Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Nelson, Shelley
• Granted Patent: US 9,080,145 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0608 Germ cells production of embryos, nuclear transfer A01K67/027
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention relates to the field of pluripotent stem cell culture and methods facilitate pluripotent stem cell culture at industrial levels.

Claims

1. A method for differentiating pluripotent stem cells, comprising the steps of:

a. Culturing the pluripotent stem cells as clusters,

b. Releasing the pluripotent stem cells as single cells,

c. Plating the single pluripotent stem cells on a tissue culture substrate, and

d. Differentiating the single pluripotent stem cells.

2. The method of claim 1, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme selected from the group consisting of trypsin, TrypLE™

SELECT, TrypLE™

EXPRESS, and trypsin/EDTA.

3. The method of claim 2, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS.

4. The method of claim 2, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme at a concentration from about 0.5 g/l to about 2.5 g/l.

5. The method of claim 2, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme at a concentration of about 2.5 g/l.

6. The method of claim 2, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme for about two to about five minutes.

7. The method of claim 2, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme for about five minutes.

8. The method of claim 3, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS at a concentration from about 1×

to about 0.001×

.

9. The method of claim 3, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS at a concentration of 1×

.

10. The method of claim 3, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS for about two to about five minutes.

11. The method of claim 3, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS for about five minutes.

12. The method of claim 1, wherein the tissue culture substrate is selected from the group consisting of MATRIGEL™

, fibronectin, laminin, human serum and collagen.

13. The method of claim 12, wherein the tissue culture substrate is MATRIGEL™

.

14. The method of claim 13, wherein the MATRIGEL™

is used at a dilution from about 1;

30 to about 1;

10.

15. The method of claim 14, wherein the MATRIGEL™

is used at a dilution of 1;

10.

16. The method of claim 1, wherein the pluripotent stem cells are embryonic stem cells.

17. The method of claim 16, wherein the embryonic stem cells are human.

18. The method of claim 1, wherein the single pluripotent stem cells are subsequently differentiated.

19. A method for maintaining pluripotent stem cells, comprising the steps of:

a. Obtaining clusters of pluripotent stem cells,

b. Releasing the pluripotent stem cells as single cells, and

c. Plating the single pluripotent stem cells on a tissue culture substrate.

20. The method of claim 19, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme selected from the group consisting of trypsin, TrypLE™

SELECT, TrypLE™

EXPRESS, and trypsin/EDTA.

21. The method of claim 20, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS.

22. The method of claim 20, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme at a concentration from about 0.5 g/l to about 2.5 g/l.

23. The method of claim 20, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme at a concentration of about 2.5 g/l.

24. The method of claim 20, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme for about two to about five minutes.

25. The method of claim 20, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme for about five minutes.

26. The method of claim 21, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS at a concentration from about 1×

to about 0.001×

.

27. The method of claim 21, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS at a concentration of 1×

.

28. The method of claim 21, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS for about two to about five minutes.

29. The method of claim 21, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS for about five minutes.

30. The method of claim 19, wherein the tissue culture substrate is selected from the group consisting of MATRIGEL™

, growth factor-reduced MATRIGEL™

fibronectin, laminin, human serum and collagen.

31. The method of claim 30, wherein the tissue culture substrate is growth factor-reduced MATRIGEL™

.

32. The method of claim 31, wherein the growth factor-reduced MATRIGEL™

is used at a dilution from about 1;

30 to about 1;

10.

33. The method of claim 32, wherein the growth factor-reduced MATRIGEL™

is used at a dilution of 1;

30.

34. The method of claim 19, wherein the pluripotent stem cells are embryonic stem cells.

35. The method of claim 34, wherein the embryonic stem cells are human.

36. The method of claim 19, wherein the single pluripotent stem cells are subsequently differentiated.

37. A method for passaging pluripotent stem cells, comprising the steps of:

a. Obtaining clusters of pluripotent stem cells,

b. Releasing the pluripotent stem cells as single cells,

c. Plating the single pluripotent stem cells on a tissue culture substrate,

d. Allowing the single pluripotent stem cells to expand,

e. Releasing the single pluripotent stem cells, and

f. Plating the single pluripotent stem cells on a new tissue culture substrate.

38. The method of claim 37, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme selected from the group consisting of trypsin, TrypLE™

SELECT, TrypLE™

EXPRESS, and trypsin/EDTA.

39. The method of claim 38, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS.

40. The method of claim 38, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme at a concentration from about 0.5 g/l to about 2.5 g/l.

41. The method of claim 38, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme at a concentration of 2.5 g/l.

42. The method of claim 38, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme for about two to about five minutes.

43. The method of claim 38, wherein the pluripotent stem cells are released as single cells by treatment with an enzyme for about five minutes.

44. The method of claim 39, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS at a concentration from about 1×

to about 0.001×

.

45. The method of claim 39, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS at a concentration of 1×

.

46. The method of claim 39, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS for about two to about five minutes.

47. The method of claim 39, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS for about five minutes.

48. The method of claim 37, wherein the tissue culture substrate is selected from the group consisting of MATRIGEL™

, growth factor-reduced MATRIGEL™

fibronectin, laminin, human serum and collagen.

49. The method of claim 48, wherein the tissue culture substrate is growth factor-reduced MATRIGEL™

.

50. The method of claim 49, wherein the growth factor-reduced MATRIGEL™

is used at a dilution from about 1;

30 to about 1;

10.

51. The method of claim 50, wherein the growth factor-reduced MATRIGEL™

is used at a dilution of 1;

30.

52. The method of claim 37, wherein the pluripotent stem cells are embryonic stem cells.

53. The method of claim 52, wherein the embryonic stem cells are human.

54. The method of claim 37, wherein the single pluripotent stem cells on a new tissue culture substrate are passaged onto another tissue culture substrate by releasing the cells and plating the released cells onto another tissue culture substrate.

55. The method of claim 37, wherein the single pluripotent stem cells are subsequently differentiated.