Methods and compositions for feeder-free pluripotent stem cell media containing human serum

• US 10,246,680 B2
• Filed: 02/22/2016
• Issued: 04/02/2019
• Est. Priority Date: 10/19/2007
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Robins, Allan J, Schulz, Thomas C
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US15/050,069
• Publication Number:
  US 20160222346A1
• Time in Patent Office: 1,135 Days
• 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 composition comprising human pluripotent stem cells and a medium comprising an effective amount of a soluble human serum attachment component, wherein the human pluripotent stem cells are in contact with the medium in a culture vessel that is not pre-coated with an extracellular matrix and wherein the effective amount of the soluble human serum attachment component promotes growth of the human pluripotent stem cells.

2. The composition of claim 1, wherein the attachment component is a fraction of human serum having a molecular weight greater than 100 kDa, greater than 150 kDa, greater than 200 kDa, greater than 250 kDa, or greater than 300 kDa.

3. The composition of claim 1, wherein the effective amount of the soluble human serum attachment component is about 0.5% to about 10% of the medium.

4. The composition of claim 1, wherein the human pluripotent stem cells are human embryonic stem cells.

5. The composition of claim 1, wherein the composition further comprises definitive endoderm cells and the medium further comprises Activin A.

6. The composition of claim 1, wherein the medium further comprises a TGFβ

superfamily member.

7. The composition of claim 1, wherein the medium is feeder-free.

8. The composition of claim 1, wherein the medium is not conditioned.

9. The composition of claim 1, wherein the human pluripotent stem cells proliferate in an undifferentiated state.

10. The composition of claim 1, wherein the human pluripotent stern cells are differentiable in vitro.

11. The composition of claim 1, wherein the human pluripotent stem cells are dissociated into a single cell suspension.

12. The composition of claim 1, wherein the culture vessel is a bioreactor.

14. A method of culturing human pluripotent stem cells comprising culturing the composition of claim 1.

15. The method of claim 14, wherein the attachment component is a fraction of human serum having a molecular weight greater than 100 kDa, greater than 150 kDa, greater than 200 kDa, greater than 250 kDa, or greater than 300 kDa.

16. The method of claim 14, wherein the human pluripotent stem cells are human embryonic stem cells.

17. The method of claim 14, wherein the composition is feeder-free.

18. The method of claim 14, wherein the medium is not conditioned.

19. The method of claim 14, wherein the medium comprises glucose.

20. The composition of claim 1, wherein the effective amount of the soluble human serum attachment component promotes growth of the human pluripotent stem cells by reducing deterioration in morphological quality of the human pluripotent stem cells.

13. A composition comprising human pluripotent stem cells and an effective amount of a soluble human serum attachment component to promote growth of the human pluripotent stem cells in a medium, wherein the cells, attachment component, and medium are in an uncoated culture vessel.