Media for culturing stem cells

• US 9,404,079 B2
• Filed: 06/04/2013
• Issued: 08/02/2016
• Est. Priority Date: 08/29/2005
• Status: Active Grant

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Inventors: Amit, Michal, Itskovitz-Eldor, Joseph
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US13/909,128
• Publication Number:
  US 20130252329A1
• Time in Patent Office: 1,155 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced 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 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0037 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/0043 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 5/0056 Xeno-free medium
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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

Abstract

Well-defined, xeno-free culture media which comprise a TGF-beta isoform or the chimera formed between IL6 and the soluble IL6 receptor (IL6RIL6), which are capable of mainataining stem cells, and particularly, human embryonic stem cells, in an undifferentiated state are provided. Also provided are cell cultures comprising the culture media and the stem cells and methods of expanding and deriving embryonic stem cells in such well-defined, xeno-free culture media. In addition, the present invention provides methods of differentiating ESCs or EBs formed therefrom for the generation of lineage specific cells.

Claims

1. A method of expanding and maintaining human pluripotent stem cells in an undifferentiated state, the method comprising culturing the human pluripotent stem cells in a culture medium comprising a transforming growth factor beta 3 (TGFβ

3) isoform— and

factors that maintain and expand human pluripotent stem cells in an undifferentiated state, thereby expanding and maintaining the human pluripotent stem cells in the undifferentiated state.

2. The method of claim 1, wherein said human pluripotent stem cells are human embryonic stem cells.

3. The method of claim 1, wherein the human pluripotent stem cells are cultured in suspension.

4. The method of claim 1, wherein the human pluripotent stem cells are cultured on a feeder-layer free matrix.

5. The method of claim 4, wherein said feeder-layer free matrix is a fibronectin matrix.

6. The method of claim 1, wherein the human pluripotent stem cells are cultured on feeder cells.

7. The method of claim 1, wherein said TGFβ

3 isoform is provided at a concentration of at least 0.5 ng/ml.

8. The method of claim 1, wherein said TGFβ

3 isoform is provided at a concentration of at least 2 ng/ml.

9. The method of claim 1, wherein said culture medium further comprises basic fibroblast growth factor (bFGF).

10. The method of claim 9, wherein said bFGF is provided at a concentration of at least 2 ng/ml.

11. The method of claim 1, wherein said culture medium is serum-free.

12. The method of claim 1, wherein said culture medium is devoid of animal contaminant.

13. The method of claim 3, wherein said suspension is protein carrier-free.