Simplified basic media for human pluripotent cell culture

• US 9,279,103 B2
• Filed: 08/05/2011
• Issued: 03/08/2016
• Est. Priority Date: 08/05/2010
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Chen, Guokai, Thomson, James A.
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US13/204,354
• Publication Number:
  US 20120202291A1
• Time in Patent Office: 1,677 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/92 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• 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 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Fully defined media that support pluripotent cell viability, proliferation, cloning, and derivation, as well as methods and compositions including these media are described. Methods for deriving iPS cells from adult individuals under defined, xeno-free conditions are also described.

Claims

1. A defined, albumin-free medium free of any component obtained from a non-human animal, the medium comprising water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β

and NODAL, each in an amount sufficient to support human pluripotent stem cell proliferation.

13. The defined, albumin-free medium of claim 1, consisting essentially of:

water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and

NODAL.

14. The defined, albumin-free medium of claim 1, wherein the medium comprises TGF-β

.

15. The defined, albumin-free medium of claim 1, wherein the medium comprises NODAL.

16. The defined, albumin-free medium of claim 1, further comprising a ROCK inhibitor.

17. The defined, albumin-free medium of claim 16, wherein the ROCK inhibitor is selected from the group consisting of HA100 and Y27632.

18. The defined, albumin-free medium of claim 1, further comprising blebbistatin.

19. The defined, albumin-free medium of claim 1, consisting of:

water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and

NODAL.

2. A method for culturing human pluripotent stem cells, the method comprising the steps of:

placing pluripotent stem cells on a matrix; and

contacting the cells with a defined, albumin-free medium free of any component obtained from a non-human animal, the medium comprising water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and

NODAL, each in an amount sufficient to support human pluripotent stem cell proliferation.

3. The method of claim 2, wherein the matrix comprises laminin.

4. The method of claim 2, wherein the matrix comprises vitronectin.

5. The method of claim 2, wherein the cells are contacted with the medium under hypoxic conditions.

6. The method of claim 2, wherein the pluripotent stem cells are embryonic stem cells.

7. The method of claim 2, wherein the pluripotent stem cells are induced pluripotent stem cells.

8. A method for cloning a human pluripotent stem cell, the method comprising the step of:

plating human pluripotent stem cells at cloning density in a defined albumin-free medium free of any component obtained from a non-human animal, the medium comprising water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and

NODAL, each in an amount sufficient to support human pluripotent stem cell cloning.

9. The method of claim 8, wherein the medium further comprises a ROCK inhibitor.

10. The method of claim 9, wherein the ROCK inhibitor is selected from the group consisting of HA100 and Y27632.

11. The method of claim 8, wherein the medium further comprises blebbistatin.

12. A method of cryopreserving human pluripotent stem cells, the method comprising the step of:

freezing human pluripotent stem cells in a defined, albumin-free medium free of any component obtained from a non-human animal, the medium comprising water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and

NODAL, each in an amount sufficient to support human pluripotent stem cell proliferation.