METHOD OF IMPROVING CELL PROLIFERATION OF PANCREATIC PROGENITOR CELLS IN A PANCREATIC CELL CULTURE

• US 20100311166A1
• Filed: 01/18/2007
• Published: 12/09/2010
• Est. Priority Date: 01/20/2006
• Status: Active Grant

• Current Assignee: ReNeuron Limited (ReNeuron Group Plc)
• Original Assignee: ReNeuron Limited (ReNeuron Group Plc)
• Inventors: Florio, Monica, Tsang, Wen-Ghih, Francki, Aleksandar
• Granted Patent: US 9,745,554 B2
• Time in Patent Office: Days
• US Class Current: 435/375
• CPC Class Codes: A61K 2035/126 Immunoprotecting barriers, e.g. jackets, diffusion chambers
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/48 Regulators of apoptosis
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

The invention relates to the discovery that the proliferation and survival of pancreatic progenitor cells can be enhanced by contacting the cells with, (1) a caspase inhibitor sufficient to reduce apoptosis in the pancreatic endocrine cells; and, (2) a growth factor in an amount sufficient to increase the level of activated Akt in the pancreatic endocrine cells.

Claims

1. A method of improving cell proliferation and survival of pancreatic endocrine cells in a pancreatic cell culture by contacting the cells with, (1) an exogenous caspase inhibitor in an amount sufficient to reduce apoptosis in the pancreatic endocrine cells;

and (2) at least one exogenous growth factor in an amount sufficient to increase the level of activated Akt in the pancreatic endocrine cells.

2. The method of claim 1, wherein the pancreatic endocrine cells are insulin producing aggregates.

3. The method of claim 1 wherein the caspase inhibitor is selected from the group consisting of Q-VD-OPH, Z-VAD (OMe)-FMK, Ac-VAD-CHO, Boc-D-FMK, BACMK, BI-9B12, Ac-LDESD-CHO, and DEVD-CHO CPP32/Apopain Inhibitor.

4. The method of claim 1, wherein the concentration of caspase inhibitor is from about 1 μ

m to about 100 μ

m.

5. The method of claim 1, wherein the caspase inhibitor is an irreversible pan caspase inhibitor, selected from the group consisting of Q-VD-OPH and Z-VAD (OMe)-FMK.

6. The method of claim 5, wherein the caspase inhibitor is Q-VD-OPH.

7. The method of claim 1, wherein the growth factor is selected from the group consisting of EGF, IGF-I, IGF-II, heregulin, and PDGF-BB.

8. The method of claim 1, wherein the growth factor is PDGF-BB and one or more members of the group consisting of IGF-I and IGF-II.

9. The method of claim 8, wherein the concentration of each growth factor is from about 10 ng/ml to about 100 ng/ml of culture medium.

10. A cell culture media composition for proliferation and survival of a pancreatic endocrine cell comprising:

1) a culture media containing the pancreatic endocrine cell,

     2) an exogenous caspase inhibitor in an amount sufficient to reduce apoptosis in the pancreatic endocrine cells; and

     3) at least one exogenous growth factor in an amount sufficient to increase the level of activated Akt in the pancreatic endocrine cells.

11. The composition of claim 10, wherein the pancreatic endocrine cells are insulin producing aggregates.

12. The composition of claim 10 wherein the physiologically acceptable culture media is selected from the group consisting of:

CMRL 1066, RPMI 1640, DMEM/F12, and SM95.

13. The composition of claim 10, wherein the caspase inhibitor is selected from the group consisting of:

Ac-VAD-CHO, Boc-D-FMK, BACMK, BI-9B12, Ac-LDESD-CHO, and DEVD-CHO CPP32/Apopain Inhibitor.

14. The composition of claim 10, wherein the concentration of caspase inhibitor is from about 1 μ

M to about 100 μ

M.

15. The composition of claim 10, wherein the caspase inhibitor is an irreversible pan caspase inhibitor selected from the group consisting of Q-VD-OPH and Z-VAD (OMe)-FMK.

16. The composition of claim 15, wherein the caspase inhibitor is Q-VD-OPH.

17. The composition of claim 10, wherein the concentration of caspase inhibitor is from about 1 μ

M to about 100 μ

M.

18. The composition of claim 10, wherein the growth factor is selected from the group consisting of EGF, IGF-I, IGF-II, heregulin, and PDGF-BB.

19. The composition of claim 10, wherein the growth factor is PDGF-BB, and one or more members of the group consisting of IGF-I and IGF-II.

20. The composition of claim 10, wherein the concentration of each growth factor is from about 10 ng/ml to about 100 ng/ml of culture medium.