Medium comprising transforming growth factor β;and basic fibroblast growth factor

• US 8,945,925 B2
• Filed: 10/21/2013
• Issued: 02/03/2015
• Est. Priority Date: 12/16/2002
• Status: Expired due to Term

• 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): Crouch, Deborah
• Application Number:
  US14/058,347
• Publication Number:
  US 20140045266A1
• Time in Patent Office: 470 Days
• US Class Current: 435/405
• 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 together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• 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 culture medium which comprises transforming growth factor β

₁ (TGFβ

₁), and basic fibroblast growth factor (bFGF), wherein the culture medium is free of animal- and/or feeder cells contaminants, and said culture medium is capable of maintaining pluripotent stem cell in an undifferentiated state for at least 5 passages.

2. The culture medium of claim 1, further comprises serum replacement.

3. The culture medium of claim 2, wherein said serum replacement is provided at a concentration of at least 10%.

4. The culture medium of claim 2, wherein said serum replacement is provided at a concentration of 15%.

5. The culture medium of claim 1, further comprises human serum.

6. The culture medium of claim 5, wherein said human serum is provided at a concentration of at least 10%.

7. The culture medium of claim 5, wherein said human serum is provided at a concentration of 15%.

8. The culture medium of claim 1, wherein said TGFβ

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

9. The culture medium of claim 1, wherein said TGFβ

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

10. The culture medium of claim 1, wherein said bFGF is provided at a concentration of at least 2 ng/ml.

11. The culture medium of claim 1, wherein said bFGF is provided at a concentration rage of 2-8 ng/ml.

12. The culture medium of claim 1, further comprising leukemia inhibitor factor (LIF).

13. The culture medium of claim 12, wherein said LIF is provided at a concentration of at least 500 units/ml.

14. The culture medium of claim 12, wherein said LIF is provided at a concentration range of 500-2000 units/ml.

15. A culture medium comprising transforming growth factor β

₁ (TGFβ

₁), and basic fibroblast growth factor (bFGF), wherein the culture medium comprises serum replacement, wherein said culture medium is capable of maintaining pluripotent stem cell in an undifferentiated state for at least 5 passages.

16. The culture medium of claim 15, wherein said serum replacement comprises albumin or albumin substitutes, amino acids, vitamins, transferrins or transferrin substitutes, antioxidants, insulin or insulin substitutes, collagen precursors and trace elements.

17. The culture medium of claim 15, wherein the culture medium is substantially free of animal contaminants.

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

19. A culture medium comprising transforming growth factor β

₁ (TGFβ

₁), and basic fibroblast growth factor (bFGF), animal-free albumin and being devoid of serum, wherein said culture medium is capable of maintaining pluripotent stem cell in an undifferentiated state for at least 5 passages.

20. A culture medium comprising transforming growth factor β

₁ (TGFβ

₁), and basic fibroblast growth factor (bFGF), wherein the culture medium comprises albumin or albumin substitutes, amino acids, vitamins, transferrins or transferrin substitutes, antioxidants, insulin or insulin substitutes, collagen precursors and trace elements, wherein said culture medium is capable of maintaining pluripotent stem cell in an undifferentiated state for at least 5 passages.

21. A culture medium comprising transforming growth factor β

₁ (TGFβ

₁), and basic fibroblast growth factor (bFGF) and being devoid of serum, wherein the culture medium comprises serum replacement, wherein said culture medium is capable of maintaining pluripotent stem cell in an undifferentiated state for at least 5 passages.

22. The culture medium of claim 21, wherein said serum replacement comprises albumin or albumin substitutes, amino acids, vitamins, transferrins or transferrin substitutes, antioxidants, insulin or insulin substitutes, collagen precursors and trace elements.

23. A culture medium comprising transforming growth factor β

₁ (TGFβ

₁), and basic fibroblast growth factor (bFGF) and being devoid of serum, wherein the culture medium comprises albumin or albumin substitutes, amino acids, vitamins, transferrins or transferrin substitutes, antioxidants, insulin or insulin substitutes, collagen precursors and trace elements, wherein said culture medium is capable of maintaining pluripotent stem cell in an undifferentiated state for at least 5 passages.

24. The culture medium of claim 23, further comprises serum replacement.