COMPOSITIONS AND METHODS USEFUL FOR CULTURING DIFFERENTIABLE CELLS

• US 20080268534A1
• Filed: 02/23/2007
• Published: 10/30/2008
• Est. Priority Date: 02/23/2006
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Novocell, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: Schulz, Thomas C., Robins, Allan J.
• Granted Patent: US 8,153,429 B2
• Time in Patent Office: Days
• US Class Current: 435/363
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• 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: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

The present invention relates to cell culture methods and compositions that are essentially serum-free and comprise a basal salt nutrient solution and an ErbB3 ligand.

Claims

1. A composition comprising a basal salt nutrient solution and an ErbB3 ligand, said composition being essentially free of serum.

2. The composition of claim 1, wherein said composition is free of exogenous insulin and insulin substitutes.

3. The composition of claim 2, further comprising insulin-like growth factor or a functional fragment thereof.

4. The composition of claim 2, wherein said ErbB3 ligand is selected from the group consisting of Neuregulin-1, Heregulin-β

(HRG-β

), Heregulin-α

(HRG-α

), Neu differentiation factor (NDF), acetylcholine receptor-inducing activity (ARIA), glial growth factor 2 (GGF2), motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2β

(NRG2-β

), Epiregulin, Biregulin and functional fragments thereof.

5. The composition of claim 4, wherein said ErbB3 ligand is HRG-β

or a functional fragment thereof.

6. The composition of claim 5, further comprising transforming growth factor beta (TGF-β

), a TGF-β

family member or a functional fragment thereof.

7. The composition of claim 6, wherein said TGF-β

family member is selected from the group consisting of Nodal, Activin A, Activin B, bone morphogenic protein-2 (BMP2), bone morphogenic protein-4 (BMP4), and functional fragments thereof.

8. The composition of claim 7, comprising Activin A.

9. The composition of claim 8, further comprising insulin-like growth factor or a functional fragment thereof.

10. The composition of claim 9, wherein said composition is free of exogenous fibroblast growth factor.

11. The composition of claim 9, further comprising at least one fibroblast growth factor (FGF) selected from the group consisting of FGF-2, FGF-7, FGF-10, FGF-22 and variants functional fragments thereof.

12. The composition of claim 11, wherein said at least one FGF is FGF-7, FGF-10 and FGF-22.

13. The composition of claim 12, further comprising a serum albumin (SA).

14. The composition of claim 13, wherein the SA is bovine SA (BSA) or human SA (HSA).

15. The composition of claim 14, wherein the concentration of the SA is more than about 0.2%, volume to volume (v/v).

16. The composition of claim 14, wherein the concentration of SA is less than about 5% v/v.

17. A composition comprising a basal salt nutrient solution and a means for stimulating ErbB2-directed tyrosine kinase activity in differentiable cells.

18. The composition of claim 17, wherein said means for stimulating ErbB2-directed tyrosine kinase activity comprises at least one ligand that specifically binds ErbB3 in said differentiable cells.

19. The composition of claim 18, wherein the differentiable cells are primate embryonic stem cells.

20. A method of culturing differentiable cells, said method comprising

a) plating the differentiable cells on a cell culture surface;

b) providing a basal salt nutrient solution to said differentiable cells; and

c) providing a ligand that specifically binds ErbB3.

21. The method of claim 20, wherein the differentiable cells are primate embryonic stem cells.

22. The method of claim 21, wherein neither insulin nor an insulin substitute is provided to said embryonic stem cells.

23. The method of claim 21, further comprising providing insulin-like growth factor or a functional fragment thereof to said embryonic stem cells.

24. The method of claim 23, wherein said ligand that specifically binds ErbB3 is selected from the group consisting of Neuregulin-1, Heregulin-β

(HRG-β

), Heregulin-α

(HRG-α

), Neu differentiation factor (NDF), acetylcholine receptor-inducing activity (ARIA), glial growth factor 2 (GGF2), motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2β

(NRG2-β

), Epiregulin, Biregulin and functional fragments thereof.

25. The method of claim 24, wherein said ligand is HRG-β

or a functional fragment thereof.

26. The method of claim 25, further comprising providing to said cells transforming growth factor beta (TGF-β

), a TGF-β

family member or a functional fragment thereof.

27. The method of claim 26, wherein said TGF-β

family member is selected from the group consisting of Nodal, Activin A, Activin B, bone morphogenic protein-2 (BMP2), bone morphogenic protein-4 (BMP4), and functional fragments thereof.

28. The method of claim 27, further comprising providing insulin-like growth factor or a functional fragment thereof to said embryonic stem cells.

29. The method of claim 28, further comprising providing at least one fibroblast growth factor (FGF) selected from the group consisting of FGF-2, FGF-7, FGF-10, FGF-22 and functional fragments thereof to said embryonic stem cells.

30. The method of claim 29, wherein said at least one FGF is FGF-7, FGF-10 and FGF-22.

31. The method of claim 30, further comprising providing a serum albumin (SA) to said embryonic stem cells.

32. The method of claim 31, wherein the SA is bovine SA (BSA) or human SA (HSA).

33. The method of claim 32, wherein the concentration of the SA is more than about 0.2%, volume to volume (v/v).

34. The method of claim 33, wherein the concentration of SA is less than about 5% v/v.