METHODS OF PREPARING FEEDER CELLS-FREE, XENO-FREE HUMAN EMBRYONIC STEM CELLS AND STEM CELL CULTURES PREPARED USING SAME

• US 20110189767A1
• Filed: 04/11/2011
• Published: 08/04/2011
• Est. Priority Date: 12/16/2002
• 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
• Granted Patent: US 8,222,034 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• 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), said culture medium being capable of maintaining pluripotent stem cell in an undifferentiated state.

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

3. The culture medium of claim 1, further comprises serum.

4. The culture medium of claim 1, substantially free of animal- and/or feeder cells contaminants.

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

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

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

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

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

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

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

10. The culture medium of claim 9, wherein said LIF is provided at a concentration of at least 500 u/ml.

11. The culture medium of claim 9, wherein said LIF is provided at a concentration range of 500-2000 u/ml.

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

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

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

15. The culture medium of claim 3, wherein said serum is provided at a concentration of 15%.

16. The culture medium of claim 3, wherein said serum is human serum.

17. A cell culture comprising the culture medium of claim 1 and pluripotent stem cells, wherein the culture medium being capable of maintaining said pluripotent stem cell in an undifferentiated state.

18. The cell culture of claim 17, wherein said pluripotent stem cells are human embryonic stem cells.

19. The cell culture of claim 17, wherein the culture medium being capable of maintaining said pluripotent stem cell in an undifferentiated state for at least 56 passages.

20. The cell culture of claim 18, wherein said human embryonic stem cells maintain a doubling time of 25 hours.