Scalable primate pluripotent stem cell aggregate suspension culture and differentiation thereof

• US 10,138,463 B2
• Filed: 11/24/2014
• Issued: 11/27/2018
• 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:
  US14/552,390
• Publication Number:
  US 20150132846A1
• Time in Patent Office: 1,464 Days
• 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 single chamber rolling bottle comprising primate pluripotent-derived cell aggregates in suspension in a physiologically acceptable medium, wherein the primate pluripotent-derived cell aggregates are derived from a single cell suspension of primate pluripotent stem cells agitated by rotation at a speed between about 3 to about 20 rpm.

2. The single chamber rolling bottle of claim 1, wherein the primate pluripotent-derived cell aggregates are formed in the roller bottle.

3. The single chamber rolling bottle of claim 1, wherein the primate pluripotent-derived cell aggregates are substantially definitive endoderm lineage cell aggregates.

4. The single chamber rolling bottle of claim 3, wherein the definitive endoderm lineage cell aggregates are substantially pancreatic endoderm cells or are substantially definitive endoderm cells.

5. The single chamber rolling bottle of claim 3, wherein the definitive endoderm lineage cell aggregates express at least one marker selected from the group consisting of Sox17, HNF3β

, HNF1β

, PDX1, NKX6.1, PTF1A, NGN3 and NKX2.2.

6. The single chamber rolling bottle of claim 3, wherein the definitive endoderm lineage cell aggregates do not significantly express PAX6, SOX7 or ZIC1.

7. The single chamber rolling bottle of claim 1, further comprising an effective amount of a retinoic acid receptor (RAR) agonist.

8. The single chamber rolling bottle of claim 1, wherein the primate pluripotent-derived cell aggregates are also agitated by rotation at a speed between about 3 to about 20 rpm.

9. The single chamber rolling bottle of claim 1, wherein the primate pluripotent-derived-cell aggregates are substantially uniform in size and shape.

10. The single chamber rolling bottle of claim 1, wherein the primate pluripotent-derived cell aggregates expand after contact with an effective amount of noggin.

11. The single chamber rolling bottle of claim 3, wherein the definitive endoderm lineage cell aggregates are agitated by laminar or streamline flow.

12. The single chamber rolling bottle of claim 1, wherein the primate pluripotent-derived cell aggregates have a diameter from about 50 microns to about 250 microns.

13. A method for preparing a single chamber rolling bottle containing primate pluripotent-derived cell aggregates in suspension, comprising:

(a) providing a single chamber roller bottle containing primate pluripotent cell aggregates derived from a single cell suspension of primate pluripotent stem cells; and

(b) contacting the primate pluripotent cell aggregates with a differentiation agent thereby generating a rolling bottle comprising primate pluripotent-derived cell aggregates in suspension,

wherein the primate pluripotent-derived cell aggregates are agitated by rotation at a speed between about 3 to about 20 rpm.

14. The method of claim 13, wherein the primate pluripotent-derived cell aggregates express at least one marker selected from the group consisting of Sox17, HNF3β

, HNF1β

, PDX1, NKX6.1, PTF1A, NGN3 and NKX2.2.

15. The method of claim 13, wherein the primate pluripotent-derived-cell aggregates do not significantly express PAX6, SOX7 or ZIC1.

16. The method of claim 13, wherein the primate pluripotent-derived cell aggregates are generated by laminar or streamline flow.

17. The method of claim 13, wherein the primate pluripotent-derived cell aggregates are substantially definitive endoderm lineage cell aggregates.

18. The method of claim 17, wherein the definitive endoderm lineage cell aggregates are substantially pancreatic endoderm cells or are substantially definitive endoderm cells.

19. The method of claim 13, wherein the primate pluripotent-derived cell aggregates have a diameter from about 50 microns to about 250 microns.

20. The method of claim 13, wherein the primate pluripotent-cell aggregates are substantially uniform in size and shape.