METHODS AND COMPOSITIONS FOR FEEDER-FREE PLURIPOTENT STEM CELL MEDIA CONTAINING HUMAN SERUM

• US 20100311164A1
• Filed: 08/15/2010
• Published: 12/09/2010
• 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 8,334,138 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

1-14. -14. (canceled)

15. A tissue culture composition consisting of:

a. a primate pluripotent stem cell;

b. a defined culture medium that supports proliferation of the primate pluripotent stem cell in an undifferentiated state; and

c. sufficient human serum to promote attachment of the primate pluripotent stem cell to a substrate,

wherein the composition does not comprise conditioned media and is essentially free of feeder cells.

16. The composition of claim 15, wherein the primate pluripotent stem cell is a human pluripotent stem cell.

17. The composition of claim 16, wherein the human pluripotent stem cell is a human embryonic stem cell (hESC).

18. The composition of claim 15, comprising human serum at a concentration of about 0.1% to about 20% of the medium.

19. The composition of claim 18, wherein the concentration of human serum in the medium is between about 0.5% and about 20%;

between about 0.5% and about 10%;

between about 0.5% and about 5%;

between about 0.5% and about 3%;

between about 0.5% and about 2%;

or between about 0.5% and about 1%.

20. The composition of claim 19, wherein the concentration of human serum in the medium is selected from the group consisting of about 0.5%, about 0.7%, about 1%, about 1.5%, about 2%, about 3%, about 5%, about 10%, and about 20%.

21. The composition of claim 15, wherein the substrate is a tissue culture vessel.

22. The composition of claim 21, wherein the substrate is uncoated and free of matrix.

23. The composition of claim 15, wherein the defined culture medium is free of non-human animal proteins.

24. A method of culturing a primate pluripotent stem cell comprising:

a. plating a primate pluripotent stem cell in a unconditioned and feeder-free combination consisting of;

i. a defined culture media medium that supports proliferation of the primate pluripotent stem cell in a undifferentiated state; and

ii. sufficient human serum to promote attachment of the primate pluripotent stem cell to the substrate,

b. incubating the primate pluripotent stem cell plated in step a, thereby culturing the primate pluripotent stem cell.

25. The method of claim 24, wherein the primate pluripotent stem cell is a human pluripotent stem cell.

26. The method of claim 24, wherein the human pluripotent stem cell is a human embryonic stem cell (hESC).

27. The method of claim 24, wherein the combination comprises human serum at a concentration of about 0.1% to about 20% of the medium.

28. The method of claim 27, wherein the concentration of human serum in the combination is between about 0.5% and about 20%;

between about 0.5% and about 10%;

between about 0.5% and about 5%;

between about 0.5% and about 3%;

between about 0.5% and about 2%;

or between about 0.5% and about 1%.

29. The method of claim 28, wherein the concentration of human serum in the combination is selected from the group consisting of about 0.5%, about 0.7%, about 1%, about 1.5%, about 2%, about 3%, about 5%, about 10%, and about 20%.

30. The method of claim 24, wherein the substrate is a tissue culture vessel.

31. The method of claim 30, wherein the substrate is uncoated and is free of matrix.

32. The method of claim 24, wherein the primate pluripotent stem cell is cultured for over one month and continues to proliferate in an undifferentiated state.

33. The method of claim 32, wherein the undifferentiated state of the primate pluripotent stem cell is determined by observing at least one characteristic selected from the group consisting of:

primate pluripotent stem cell morphology;

normal karyotype;

ability to differentiate into derivatives of endoderm, mesoderm, and ectoderm; and

expression of at least one pluripotent stem cell marker.

34. The method of claim 33, wherein the marker is selected from Oct4, SSEA-4, Tra-1-81, and Tra-1-60.

35. The method of claim 24, wherein the primate pluripotent stem cell continues to proliferate without differentiation for 2, 3, 4, 5, 6, 7, 8, 9, or 10 serial passages.

36. The method of claim 35, wherein the primate pluripotent stem cell is passaged by enzymatic dissociation and replating.

37. The method of claim 36, wherein the enzymatic dissociation generates a single cell suspension.