Scalable primate pluripotent stem cell aggregate suspension culture and differentiation thereof

• US 8,895,300 B2
• Filed: 11/08/2012
• Issued: 11/25/2014
• Est. Priority Date: 11/04/2008
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Schulz, Thomas C
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US13/672,688
• Publication Number:
  US 20130115695A1
• Time in Patent Office: 747 Days
• Field of Search: 435/363
• US Class Current: 435/363
• CPC Class Codes: C12M 27/12 Roller bottles; Roller tubes
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/195 Heregulin, neu differentiation factor
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 2511/00 Cells for large scale production
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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 5/0676 Pancreatic cells

Abstract

The present invention relates to methods for production of undifferentiated or differentiated embryonic stem cell aggregate suspension cultures from undifferentiated or differentiated embryonic stem cell single cell suspensions and methods of differentiation thereof.

Claims

1. A roller bottle comprising primate pluripotent stem cell (pPSC) aggregates in suspension in a physiologically acceptable medium, wherein the pPSC aggregates are formed in the roller bottle.

2. The roller bottle of claim 1, wherein the pPSC aggregates have a diameter of about 100 to 300 microns.

3. The roller bottle of claim 1, substantially free of pPSC agglomerations having a diameter of more than 300 microns.

4. The roller bottle of claim 1, wherein the pPSC aggregates express at least one marker selected from the group consisting of OCT4, NANOG, SSEA-3, SSEA-4, Tra-1-80 and Tra-1-60.

5. The roller bottle of claim 1, wherein the pPSC aggregates are human cells.

6. The roller bottle of claim 5, wherein the pPSC aggregates are selected from the group consisting of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC).

7. The roller bottle of claim 1, wherein the pPSC aggregates in suspension are generated by a method comprising:

a. seeding a roller bottle with undifferentiated pPSCs in a culture medium that supports undifferentiated growth of the pPSCs; and

b. agitating the pPSCs at low rotation speed to form pPSC aggregates, thereby generating a roller bottle comprising pPSC aggregates.

8. The roller bottle of claim 7, wherein the pPSC aggregates have a diameter of about 100 to 300 microns.

9. The roller bottle of claim 7, substantially free of pPSC agglomerations having a diameter of more than 300 microns.

10. The roller bottle of claim 7, wherein the pPSC aggregates express at least one marker selected from the group consisting of OCT4, NANOG, SSEA-3, SSEA-4, Tra-1-80 and Tra-1-60.

11. The roller bottle of claim 7, wherein the pPSC aggregates are human cells.

12. The roller bottle of claim 11, wherein the pPSC aggregates are cells selected from the group consisting of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC).

13. The roller bottle of claim 7, wherein the rotation speed ranges from 3 to 20 rpm.

14. The roller bottle of claim 7, wherein the rotation speed ranges from 5 to 12 rpm.

15. The roller bottle of claim 7, wherein the undifferentiated pPSCs in step a is a suspension of single cell pPSCs.

16. A method for preparing the roller bottle of claim 1, comprising:

a. seeding a roller bottle with a single cell suspension of undifferentiated pPSCs in a culture medium that supports undifferentiated growth of the pPSCs; and

b. agitating the single cell suspension at low rotation speed in a roller bottle to form pPSC cell aggregates,

thereby preparing a roller bottle comprising pPSC aggregates in suspension in a physiologically acceptable medium.

17. The method of claim 16, wherein the undifferentiated pPSCs is a suspension of thawed pPSCs.

18. The method of claim 16, wherein the rotation speed is about 3 rpm to about 20 rpm.

19. The method of claim 16, wherein the rotation is about 5 rpm to about 20 rpm.

20. The method of claim 16, wherein the rotation speed is about 5 rpm to about 12 rpm.

21. The method of claim 16, wherein the pPSC is a human cell.

22. The method of claim 21, wherein the pPSC is selected from the group consisting of hESC and iPSC.