Media for culturing stem cells

• US 10,385,312 B2
• Filed: 07/14/2016
• Issued: 08/20/2019
• Est. Priority Date: 08/29/2005
• Status: Active Grant

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Inventors: Amit, Michal, Itskovitz-Eldor, Joseph
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US15/209,776
• Publication Number:
  US 20160319241A1
• Time in Patent Office: 1,132 Days
• 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 maintaining 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 culturing primate pluripotent stem cells in a pluripotent and undifferentiated state, the method comprising culturing the primate pluripotent stem cells in a suspension culture which comprises a culture medium, said culture medium comprising basic fibroblast growth factor (bFGF) and at least one factor selected from the group consisting of transforming growth factor beta-3 (TGFβ

3) isoform, transforming growth factor beta-1 (TGFβ

1) isoform, and IL6RIL6 chimera, wherein the primate pluripotent stem cells are expanded for at least 5 passages in said suspension culture without adherence to an external substrate, wherein the primate pluripotent stem cells are maintained in said suspension culture in a pluripotent and undifferentiated state, wherein the pluripotent stem cells express OCT4, and are capable of forming teratomas containing cells of the mesoderm, ectoderm and endoderm embryonic germ layers.

2. The method of claim 1, wherein said culturing is in a bioreactor.

3. The method of claim 1, wherein the primate pluripotent stem cells comprise primate embryonic stem cells.

4. The method of claim 1, wherein said culture medium is devoid of serum.

5. The method of claim 1, wherein said culture medium is devoid of serum replacement.

6. The method of claim 1, wherein said culture medium is animal contaminant-free.

7. The method of claim 1, wherein said suspension culture is feeder cell layer-free.

8. The method of claim 1, wherein said culture medium is protein carrier-free.

9. The method of claim 1, wherein said IL6RIL6 chimera comprises amino acids 112-355 of SEQ ID NO:

32.

10. The method of claim 1, wherein said IL6RIL6 chimera is provided at a concentration of at least 25 ng/ml.

11. The method of claim 1, wherein said bFGF is provided at a concentration of at least 2 ng/ml.

12. The method of claim 1, wherein said TGFβ

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

13. The method of claim 1, wherein said TGFβ

1 is at a concentration of at least 0.06 ng/ml.

14. The method of claim 1, wherein said primate pluripotent stem cells are human pluripotent stem cells.

15. The method of claim 1, wherein said primate pluripotent stem cells are monkey pluripotent stem cells.