Single pluripotent stem cell culture

• US 9,080,145 B2
• Filed: 06/30/2008
• Issued: 07/14/2015
• Est. Priority Date: 07/01/2007
• Status: Expired due to Fees

• Current Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Nelson, Shelley
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US12/164,795
• Publication Number:
  US 20090325294A1
• Time in Patent Office: 2,570 Days
• Field of Search: 435/380
• US Class Current: 1/1
• 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 maintaining pluripotent stem cells, comprising the steps of:

a) obtaining clusters of pluripotent stem cells;

b) culturing the cluster of pluripotent stem cells in MEF-conditioned media;

c) releasing the pluripotent stem cells as single cells with an enzyme; and

d) plating the single pluripotent stem cells on a tissue culture substrate comprising an extracellular matrix, and either bFGF or fibroblast conditioned media;

wherein the enzyme;

(1) is TrypLE Select™

or TrypLE Express™

;

(2) is present in a concentration from about 0.5 g/l to about 2.5 g/l;

wherein the enzyme is used to culture the cells for two minutes; and

wherein there is no gain of chromosome abnormalities after releasing the cells.

2. The method of claim 1, wherein the pluripotent stem cells are released as single cells by treatment with the enzyme at a concentration of about 2.5 g/l.

3. The method of claim 1, wherein the pluripotent stem cells released as single cells are subsequently cultured with MEF-conditioned media.

4. The method of claim 1, wherein the tissue culture substrate is selected from the group consisting of matrigel, growth factor-reduced matrigel, fibronectin, laminin, human serum and collagen.

5. The method of claim 4, wherein the tissue culture substrate is growth factor-reduced matrigel.

6. The method of claim 5, wherein the growth factor-reduced matrigel is used at a dilution from about 1:

30 to about 1;

10.

7. The method of claim 6, wherein the growth factor-reduced matrigel is used at a dilution of 1:

30.

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

9. The method of claim 8, wherein the embryonic stem cells are human.

22. The method of claim 1, wherein cell viability is at least 98% after releasing the cells.

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

a) obtaining clusters of pluripotent stem cells;

b) culturing the cluster of pluripotent stem cells in MEF-conditioned media;

c) releasing the pluripotent stem cells as single cells with an enzyme;

d) plating the single pluripotent stem cells on a tissue culture substrate;

e) allowing the single pluripotent stem cells to expand;

f) releasing the single pluripotent stem cells; and

g) plating the single pluripotent stem cells on a new tissue culture substrate comprising an extracellular matrix, and either bFGF or fibroblast conditioned media;

wherein the enzyme;

(1) is TrypLE Select™

or TrypLE Express™

;

(2) is present in a concentration from about 0.5 g/l to about 2.5 g/l;

wherein the enzyme is used to culture the cells for two minutes; and

wherein there is no gain of chromosome abnormalities after releasing the cells.

11. The method of claim 10, wherein the pluripotent stem cells are released as single cells by treatment with the enzyme at a concentration of 2.5 g/l.

12. The method of claim 10, wherein the pluripotent stem cells released as single cells are subsequently cultured in MEF-conditioned media.

13. The method of claim 10, wherein the tissue culture substrate is selected from the group consisting of matrigel, growth factor-reduced matrigel, fibronectin, laminin, human serum and collagen.

14. The method of claim 13, wherein the tissue culture substrate is growth factor-reduced matrigel.

15. The method of claim 14, wherein the growth factor-reduced matrigel is used at a dilution from about 1:

30 to about 1;

10.

16. The method of claim 15, wherein the growth factor-reduced matrigel is used at a dilution of 1:

30.

17. The method of claim 10, wherein the pluripotent stem cells are embryonic stem cells.

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

19. The method of claim 10, 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.

23. The method of claim 10, wherein cell viability is at least 98% after releasing the cells.

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

a) obtaining clusters of pluripotent stem cells;

b) culturing the cluster of pluripotent stem cells in MEF conditioned media;

c) releasing the pluripotent stem cells as single cells with an enzyme; and

d) plating the single pluripotent stem cells on a tissue culture substrate comprising an extracellular matrix, and MEF-conditioned media;

wherein the enzyme;

(1) is TrypLE Select™

or TrypLE Express™

;

(2) is present in a concentration from about 0.5 g/l to about 2.5 g/l;

wherein the enzyme is used to culture the cells from two to five minutes; and

wherein there is no gain of chromosome abnormalities after releasing the cells.

24. The method of claim 20, wherein cell viability is at least 98% after releasing the cells.

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

a) obtaining clusters of pluripotent stem cells;

b) culturing the cluster of pluripotent stem cells in MEF conditioned media;

c) releasing the pluripotent stem cells as single cells with an enzyme; and

d) plating the single pluripotent stem cells on a tissue culture substrate;

e) allowing the single pluripotent stem cells to expand;

f) releasing the single pluripotent stem cells; and

g) plating the single pluripotent stem cells on a new tissue culture substrate comprising an extracellular matrix, and MEF-conditioned media;

wherein the enzyme;

(1) is TrypLE Select™

or TrypLE Express™

;

(2) is present in a concentration from about 0.5 g/l to about 2.5 g/l;

wherein the enzyme is used to culture the cells from two to five minutes; and

wherein there is no gain of chromosome abnormalities after releasing the cells.

25. The method of claim 21, wherein cell viability is at least 98% after releasing the cells.