CELL CULTURE MEDIUM

• US 20120156782A1
• Filed: 12/16/2011
• Published: 06/21/2012
• Est. Priority Date: 12/17/2010
• Status: Active Grant

• Current Assignee: BioLamina AB
• Original Assignee: BioLamina AB
• Inventors: Tryggvason, Karl, Rodin, Sergey
• Granted Patent: US 10,138,292 B2
• Time in Patent Office: Days
• US Class Current: 435/402
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/605 Nanog
• CPC Class Codes: C12N 2501/608 Lin28
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• 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
• CPC Class Codes: Y10T 428/13 Hollow or container type article [e.g., tube, vase, etc.]

Abstract

The present disclosure related to isolated laminin-521, methods for making recombinant laminin-521, host cells that express recombinant laminin-521, and compositions containing laminin-521. Laminin-521 can maintain stem cells in vitro pluripotency, enable self-renewal, and enable single cell survival of human embryonic stem cells. When pluripotent human embryonic stem cells are cultured on plates coated with recombinant laminin-521 (laminin-11), in the absence of differentiation inhibitors or feeder cells, the embryonic stem cells proliferate and maintain their pluripotency. It has also been discovered that human recombinant laminin-521 (laminin-11) provides single cell survival of stem cells after complete dissociation into a single cell suspension. Useful cell culture mediums containing at most 3.9 ng/ml of beta fibroblast growth factor (bFGF) are also described herein.

Claims

1. A system for maintaining pluripotent stem cells, comprising:

a cell culture medium comprising from greater than zero to 3.9 ng/mL of basic fibroblast growth factor (bFGF); and

a substrate for providing support to the stem cells.

2. The system of claim 1, wherein the substrate contains laminin-521 or laminin-511.

3. The system of claim 2, wherein the substrate also contains a cadherin.

4. The system of claim 1, wherein the substrate comprises laminin-521 and wherein the amount of bFGF is 3.5 ng/mL or less.

5. The system of claim 1, wherein the cell culture medium comprises from 0.5 to 3.5 ng/mL of bFGF.

6. The system of claim 1, wherein the cell culture medium further comprises at least one additional growth factor, at least one trace mineral, and at least one lipid.

7. A cell culture medium that provides nutrition to pluripotent stem cells, comprising from greater than zero to 3.9 ng/mL of basic fibroblast growth factor (bFGF).

8. The cell culture medium of claim 7, comprising 3.5 ng/mL or less of bFGF.

9. The cell culture medium of claim 7, comprising from 0.5 to 3.5 ng/mL of bFGF.

10. The cell culture medium of claim 7, further comprising at least one inorganic salt.

11. The cell culture medium of claim 7, further comprising at least one trace mineral.

12. The cell culture medium of claim 7, further comprising at least one energy substrate.

13. The cell culture medium of claim 7, further comprising at least one lipid.

14. The cell culture medium of claim 7, further comprising at least one amino acid.

15. The cell culture medium of claim 7, further comprising at least one vitamin.

16. The cell culture medium of claim 7, further comprising at least one additional growth factor.

17. The cell culture medium of claim 7, further comprising at least one inorganic salt, at least one trace mineral, at least one energy substrate, at least one lipid, at least one amino acid, and at least one vitamin.

18. The cell culture medium of claim 7, wherein the cell culture medium does not contain any one of (1) albumin, (2) insulin or an insulin substitute, or (3) transferrin or a transferrin substitute.

19. The cell culture medium of claim 7, further comprising albumin, insulin, lithium chloride, GABA, TGF beta 1, pipecolic acid, L-glutamine, MEM non-essential amino acid solution, and DMEM/F12 solution.

20. The cell culture medium of claim 7, further comprising at least one additional growth factor, at least one trace mineral, and at least one lipid.