Methods and Compositions for Feeder-Free Pluripotent Stem Cell Media Containing Human Serum

• US 20090104696A1
• Filed: 10/19/2007
• Published: 04/23/2009
• Est. Priority Date: 10/19/2007
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Schulz, Thomas C., Robins, Allan J.
• Granted Patent: US 8,623,650 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2506/02 from embryonic 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 5/068 Stem cells; Progenitors

Abstract

The present invention provides compositions and methods for the culture and maintenance of pluripotent stem cells. More particularly, the present invention provides for compositions and methods for culturing, maintaining, growing and stabilizing primate pluripotent stem cells in a feeder-free defined media further comprising human serum, or a soluble attachment component of the human serum, for promoting cell attachment.

Claims

1. A feeder-free primate pluripotent stem cell tissue culture composition comprising:

a) an undifferentiated human embryonic stem cell (hESC);

b) a defined culture media comprising human serum (hS) or a soluble attachment component thereof, wherein the human serum further comprises at least one soluble attachment component having at least 100 kDa molecular weight, and wherein the composition is essentially free of feeder cells.

2. The composition of claim 1, wherein the human serum is in a concentration of about 0.5 to about 2% of the media.

3. The composition of claim 1, wherein the size of the soluble attachment component is selected from the group consisting of at least 100 kDa, at least 150 kDa, at least 200 kDa, at least 250 kDa, and at least 300 kDa.

4. The composition of claim 1, wherein the component promotes attachment of the hESCs to a substrate.

5. The composition of claim 4, wherein the substrate is that from a tissue culture vessel.

6. A method of culturing primate embryonic stem cells in a feeder-free media comprising:

culturing the primate embryonic stem cells in a culture medium which can support stem cells, the culture medium being essentially free of feeder cells, and wherein the medium comprises human serum or a soluble attachment component thereof,

thereby promoting undifferentiated embryonic stem cell proliferation.

7. The method of claim 6, wherein the culture medium comprises about 0.5% to about 2% human serum or a soluble attachment component thereof.

8. The method of claim 7 further comprising enrichment of at least one embryonic stem cell soluble attachment component selected from the group consisting of 100 kDa, 150 kDa, 200 kDa, 250 kDa, and 300kDa enriched by fractionation.

9. The method of claim 8, wherein the enrichment is by fractionating the human serum using a size-exclusion device.

10. The method of claim 9, wherein the size-exclusion device has a 300 KDa molecular weight cut-off.

11. The method of claim 9, wherein said size-exclusion device has a 100KDa molecular weight cut-off.

12. The method of claim 9, wherein said size-exclusion device has a 30 KDa molecular weight cut-off.

13. The method of claim 9, wherein the primate embryonic stem cells are human embryonic stem cells (hESCs).

14. A method of culturing primate embryonic stem cells in a feeder-free defined media comprising:

culturing the primate embryonic stem cells in a culture medium which can support stem cells, the culture medium being substantially free of feeder cells and further comprising about 0.5% to about 2% human serum or a soluble attachment component thereof the culturing step being conducted for over one month with the embryonic stem cells proliferating in culture while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the stem cells.