Methods and compositions for feeder-free pluripotent stem cell media containing human serum

• US 8,623,650 B2
• Filed: 10/19/2007
• Issued: 01/07/2014
• Est. Priority Date: 10/19/2007
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Robins, Allan J., Schulz, Thomas C.
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): Moloye, Titilayo
• Application Number:
  US11/875,057
• Publication Number:
  US 20090104696A1
• Time in Patent Office: 2,272 Days
• Field of Search: None
• US Class Current: 435/392
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2506/02 from embryonic 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 5/068 Stem cells; Progenitors

Abstract

The present invention provides compositions and methods for the culture and maintenance of pluripotent stem cells. More particularly, the present invention provides for compositions and methods for culturing, maintaining, growing and stabilizing primate pluripotent stem cells in a feeder-free defined media further comprising human serum, or a soluble attachment component of the human serum, for promoting cell attachment.

Claims

1. A method of culturing primate pluripotent stem cells comprising:

culturing the primate pluripotent stem cells in a tissue culture vessel containing a defined culture medium under conditions essentially free of feeder cells such that said stem cells grow in an undifferentiated state, wherein the medium comprises sufficient human serum to promote attachment of the primate pluripotent stem cells to said tissue culture vessel.

2. The method of claim 1, wherein the culture medium comprises about 0.5% to about 10% human serum.

3. The method of claim 2, wherein the culture medium further comprises a fraction of serum comprising a soluble attachment activity, wherein the soluble attachment activity is a fraction selected from the group consisting of fractions having a molecular weight greater than 100 kDa, 150 kDa, 200 kDa, 250 kDa, and 300 kDa enriched by fractionation.

4. The method of claim 3, wherein the fraction of serum comprising the soluble attachment activity is prepared by fractionating the human serum using a size-exclusion device.

5. The method of claim 4, wherein the size-exclusion device has a 100 kDa molecular weight cut-off.

6. The method of claim 4, wherein the primate pluripotent stem cells are human embryonic stem cells (hESCs).

19. The method of claim 1, wherein the culturing step is conducted for over one month with the pluripotent stem cells proliferating in culture while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the stem cells.

7. A method of culturing primate pluripotent stem cells comprising:

culturing the primate pluripotent stem cells in a matrix free tissue culture vessel containing a defined culture medium under conditions essentially free of feeder cells such that the stem cells grow in an undifferentiated state, wherein the medium further comprising about 10% human serum, sufficient to promote attachment of the primate pluripotent stem cells to the said tissue culture vessel.

8. The method of claim 7, wherein the culture medium further comprises a fraction of human serum comprising a soluble attachment activity is prepared by fractionating human serum using a size-exclusion device.

9. The method of claim 8, wherein the size-exclusion device has a 100 kDa molecular weight cut-off.

20. The method of claim 7, wherein the primate pluripotent stem cells are human embryonic stem cells (hESCs).

10. A method of culturing primate pluripotent stem cells comprising:

culturing the primate pluripotent stem cells in a matrix free tissue culture vessel containing a defined culture medium under conditions essentially free of feeder cells such that said stem cells grow in an undifferentiated state, and wherein the medium comprises a fraction of human serum comprising a soluble attachment activity to promote attachment of the primate pluripotent stem cell to a tissue culture vessel.

11. The method of claim 10, wherein the fraction of serum comprising the soluble attachment activity is a fraction selected from the group consisting of fractions having a molecular weight greater than 100 kDa, 150 kDa, 200 kDa, 250 kDa, and 300kDa enriched by fractionation.

12. The method of claim 11, wherein the fraction of serum comprising the soluble attachment activity is prepared by fractionating the human serum using a size-exclusion device.

13. The method of claim 12, wherein the size-exclusion device has a 100 kDa molecular weight cut-off.

14. The method of claim 10, wherein the primate pluripotent stem cells are human embryonic stem cells (hESCs).

15. A method of culturing primate pluripotent stem cells comprising:

culturing the primate pluripotent stem cells in a matrix free tissue culture vessel containing a defined culture medium under conditions essentially free of feeder cells such that said stem cells grow in an undifferentiated state and wherein the culture medium further comprises about 0.5% to about 10% human serum wherein a fraction of the human serum comprises a soluble attachment activity, the culturing step being conducted for over one month with the pluripotent stem cells proliferating in culture while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the stem cells.

16. The method of claim 15, wherein the fraction of human serum comprising the soluble attachment activity is prepared by fractionating human serum using a size-exclusion device.

17. The method of claim 16, wherein the size-exclusion device has a 100 kDa molecular weight cut-off.

18. The method of claim 15, wherein the primate pluripotent stem cells are human embryonic stem cells (hESCs).