Medium comprising transforming growth factor beta 1 and basic fibroblast growth factor

• US 10,590,382 B2
• Filed: 09/03/2018
• Issued: 03/17/2020
• 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): Ton, Thaian N.
• Application Number:
  US16/120,342
• Publication Number:
  US 20190093078A1
• Time in Patent Office: 561 Days
• 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 establishing a human pluripotent stem cell line capable of being maintained in an undifferentiated, pluripotent and proliferative state, the method comprising:

culturing human pluripotent stem cells under culturing conditions devoid of feeder cells and including a matrix and a culture medium supplemented with TGFβ

₁, bFGF and/or LIF to thereby obtain the human pluripotent stem cell line.

2. The method of claim 1, further comprising cloning a cell from the human pluripotent stem cell line under said culturing conditions.

3. The method of claim 1, wherein said matrix is selected from the group consisting of a fibronectin matrix, a laminin matrix, foreskin fibroblast matrix, a synthetic fibronectin matrix a mouse embryonic fibroblast (MEF) matrix and a basement membrane matrix extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

4. The method of claim 3, wherein said fibronectin matrix is a human-derived fibronectin matrix.

5. The method of claim 3, wherein said laminin matrix is a human-derived laminin matrix.

6. The method of claim 4, wherein said human derived fibronectin matrix is a plasma fibronectin or cellular fibronectin.

7. The method of claim 3, wherein said fibronectin matrix is a synthetic fibronectin matrix.

8. The method of claim 1, wherein the cells of the human pluripotent stem cell line maintain a doubling time of at least 25 hours.

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

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

11. The method of claim 9, wherein said serum is at a concentration of at least 10%.

12. The method of claim 10, wherein said serum replacement is at a concentration of at least 10%.

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 bFGF is at a concentration of at least 2 ng/ml.

15. The method of claim 1, wherein said LIF is at a concentration of at least 500 u/ml.

16. A xeno-free, feeder cells-free culture system comprising a matrix and a culture medium, wherein said culture medium comprises TGFβ

₁, bFGF and LIF.

17. A culture system comprising a matrix and a tissue culture medium, wherein said matrix is selected from the group consisting of s a fibronectin matrix, a laminin matrix, foreskin fibroblast matrix, a synthetic fibronectin matrix, a mouse embryonic fibroblast (MEF) matrix and a basement membrane matrix extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, wherein said culture medium comprises TGFβ

₁, bFGF and LIF, wherein the culture system being capable of maintaining human pluripotent stem cells cultured therein in a proliferative, pluripotent and undifferentiated state.

18. A culture system comprising a matrix and a tissue culture medium, wherein said matrix is a laminin matrix, wherein said culture medium comprises TGFβ

₁, bFGF and/or LIF, wherein the culture system being capable of maintaining human pluripotent stem cells cultured therein in a proliferative, pluripotent and undifferentiated state.

19. The culture system of claim 18, wherein said culture medium comprises TGFβ

₁, bFGF and LIF.