Culture media, cell cultures and methods of culturing pluripotent stem cells in an undifferentiated state

• US 9,018,010 B2
• Filed: 11/11/2010
• Issued: 04/28/2015
• Est. Priority Date: 11/12/2009
• 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): Fox, Allison
• Assistant Examiner(s): Pyla, Yvonne
• Application Number:
  US13/508,991
• Publication Number:
  US 20120225480A1
• Time in Patent Office: 1,629 Days
• Field of Search: None
• US Class Current: 435/404
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/92 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2500/99 Serum-free medium
• 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 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• 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 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Provided are serum-free culture media which comprise basic fibroblast growth factor (bFGF), transforming growth factor beta-3, ascorbic acid, xeno-free serum replacement and a lipid mixture. The media may also comprise an IL6R/IL6 chimera, or leukemia inhibitory factor (LIF); wherein the culture medium is capable of maintaining pluripotent stem cells in an undifferentiated state in the absence of feeder cell support. Also provided are cell cultures comprising pluripotent stem cells such as human embryonic stem cells and induced pluripotent stem (iPS) cells, and methods of using the same for expanding pluripotent stem cells in an undifferentiated state using two-dimensional or three-dimensional culture systems. Methods of expanding iPS cells in a suspension culture devoid of substrate adherence and cell encapsulation are also provided.

Claims

1. A culture medium being serum-free and devoid of non-human contaminants comprising ascorbic acid at a concentration range of about 400-600 μ

g/ml, basic fibroblast growth factor (bFGF) at a concentration range of about 50-200 ng/ml, serum replacement and a lipid mixture, wherein the culture medium is capable of maintaining pluripotent stem cells in an undifferentiated state and with a stable karyotype for at least 40 passages in the absence of feeder cell support.

2. A cell culture comprising a pluripotent stem cell and the culture medium of claim 1, wherein the cell culture is feeder cells free.

3. The cell culture of claim 2, wherein said stem cells are embryonic stem cells.

4. The cell culture of claim 3, wherein said embryonic stem cells are human embryonic stem cells.

5. The cell culture of claim 2, wherein said stem cells are induced pluripotent stem (iPS) cells.

6. The cell culture of claim 5, wherein said induced pluripotent stem cells are human induced pluripotent stem cells.

7. The culture medium of claim 1, wherein said culture medium is capable of expanding said pluripotent stem cells in an undifferentiated state when cultured in a suspension culture.

8. The culture medium of claim 1, wherein said culture medium is devoid of sodium-bicarbonate.

10. A cell culture system comprising the culture medium of claim 1 and a fibronectin matrix.

9. A culture medium being serum-free, devoid of non-human contaminants and devoid of TGFβ

1, comprising ascorbic acid at a concentration range of about 400-600 μ

g/ml, basic fibroblast growth factor (bFGF) at a concentration range of about 50-200 ng/ml, serum replacement and a lipid mixture, wherein the culture medium is capable of maintaining pluripotent stem cells in an undifferentiated state and with a stable karyotype for at least 40 passages in the absence of feeder cell support.