Methods of culturing cells in a medium comprising transforming growth factor beta 1 and basic fibroblast growth factor

• US 8,563,311 B2
• Filed: 05/08/2012
• Issued: 10/22/2013
• Est. Priority Date: 12/16/2002
• Status: Expired due to Term

• 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): Crouch, Deborah
• Application Number:
  US13/466,161
• Publication Number:
  US 20120214232A1
• Time in Patent Office: 532 Days
• Field of Search: None
• US Class Current: 435/384
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/84 from mammals
• 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 2501/10 Growth factors
• 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 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0031 Serum-free culture media
• 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 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0611 : Primordial germ cells, e.g. embryonic germ cells [EG]
• CPC Class Codes: C12N 5/0623 : Stem cells
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention is of methods of establishing and propagating human embryonic stem cell lines using feeder cells-free, xeno-free culture systems and stem cells which are capable of being maintained in an undifferentiated, pluripotent and proliferative state in culture which is free of xeno contaminants and feeder cells.

Claims

1. A method of propagating mammalian pluripotent stem cells in an undifferentiated, pluripotent and proliferative state under culturing conditions devoid of feeder cells, the method comprising culturing the mammalian pluripotent stem cells in a tissue culture medium which comprises transforming growth factor β

₁ (TGFβ

₁) and basic fibroblast growth factor (bFGF), to thereby maintain the mammalian pluripotent stem cells in an undifferentiated, pluripotent and proliferative state.

2. The method of claim 1, further comprising culturing the mammalian pluripotent stem cells on an extracellular matrix.

3. The method of claim 2, wherein said extracellular matrix is a fibronectin matrix.

4. The method of claim 3, wherein said fibronectin is selected from the group consisting of bovine fibronectin, recombinant bovine fibronectin, human fibronectin, recombinant human fibronectin, mouse fibronectin, recombinant mouse fibronectin, and synthetic fibronectin.

5. The method of claim 2, wherein said culturing conditions are substantially free of animal contaminant and wherein said extracellular matrix is selected from the group consisting of human plasma fibronectin matrix, recombinant human plasma fibronectin matrix, human cellular fibronectin matrix, recombinant human cellular fibronectin matrix, synthetic fibronectin.

6. The method of claim 1, wherein said mammalian pluripotent stem cells are human pluripotent stem cells.

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

8. The method of claim 7, wherein said embryonic stem cells are human embryonic stem cells.

9. The method of claim 1, wherein said tissue culture medium further comprises serum.

10. The method of claim 1, wherein said tissue culture medium further comprises serum replacement.

11. The method of claim 1, wherein said TGFβ

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

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

13. The method of claim 1, further comprises leukemia inhibitor factor (LIF).

14. The method of claim 13, wherein said LIF is provided at a concentration of at least 500 u/ml.

15. A cell culture comprising mammalian pluripotent stem cells in a culture medium which comprises transforming growth factor β

₁ (TGFβ

₁) and basic fibroblast growth factor (bFGF), wherein the cell culture is substantially free of animal- and/or feeder cells contaminants and whereas said mammalian pluripotent stem cells are maintained in an undifferentiated, pluripotent and proliferative state when cultured in said culture medium.

16. The cell culture of claim 15, wherein the culture medium comprises serum replacement.

17. The cell culture of claim 16, wherein said serum replacement is provided at a concentration of at least 10%.

18. The cell culture of claim 15, wherein said TGFβ

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

19. The cell culture of claim 15, wherein said bFGF is provided at a concentration of at least 2 ng/ml.

20. The cell culture of claim 15, wherein said culture medium further comprises leukemia inhibitor factor (LIF).

21. The cell culture of claim 20, wherein said LIF is provided at a concentration of at least 500 u/ml.

22. The cell culture of claim 15, wherein said mammalian pluripotent stem cells are maintained in an undifferentiated, pluripotent and proliferative state for at least 38 passages when cultured in said culture medium.

23. The cell culture of claim 15, wherein said mammalian pluripotent stem cells are human embryonic stem cells.

24. A method of propagating cells in a culture, comprising culturing the cells in a culture medium which comprises transforming growth factor β

₁ (TGFβ

₁) and basic fibroblast growth factor (bFGF), wherein said bFGF is provided at a concentration of at least 2 ng/ml, and wherein said TGFβ

₁ is provided at a concentration range of 0.06-0.24 ng/ml, there propagating the cells in the culture.

25. A cell culture comprising cells and a culture medium, wherein said culture medium comprises transforming growth factor β

₁ (TGFβ

₁) and basic fibroblast growth factor (bFGF), wherein said bFGF is provided at a concentration of at least 2 ng/ml, and wherein said TGFβ

₁ is provided at a concentration range of 0.06-0.24 ng/ml.