Claims for Patent: 8,093,053

Summary for Patent: 8,093,053

Title:	Methods and compositions for culturing of neural precursor cells
Abstract:	The present invention provides methods and compositions for the propagation and expansion of neural precursor cells (NPCs). NPCs may be used in the clinical implementation of stem cell therapy to treat disorders such as Parkinson\'s disease, Huntington\'s disease, neuropathic pain and other diseases of the central nervous system. The large-scale production of NPCs in bioreactors allows for the generation of clinical quantities of these cells.
Inventor(s):	Baghbaderani; Behnam A. (Calgary, CA), Sen; Arindom (Calgary, CA), Kallos; Michael S. (Calgary, CA), Behie; Leo A. (Calgary, CA)
Assignee:	UTI Limited Partnership (Calgary, CA)
Application Number:	12/405,736
Patent Claims:	1. A culture medium comprising, dissolved or dispersed in base culture media and water, the following components: (a) nutrients; (b) glutamine; (c) glucose; (d) heparin (e) Hepes; (f) recombinant human EGF at about 10 to about 40 .mu.g/mL; (g) recombinant human bFGF about 10 to about 40 .mu.g/mL; (h) recombinant hLIF at about 5 to about 25 .mu.g/mL; (i) DHEA at about 0.1 to about 5 .mu.mol/L; (j) serum albumin; (k) lipids; and (l) a mixture of hormones comprising apo-transferrin, insulin, putrescine, selenium and progesterone. 2. The medium of claim 1, wherein the base culture medium is DMEM. 3. The medium of claim 1, wherein nutrients comprises Ham's F12 nutrient mixture. 4. The medium of claim 1, wherein serum albumin is human or bovine serum albumin. 5. The medium of claim 1, wherein said medium is filter sterilized. 6. The medium of claim 1, wherein recombinant human EGF is present at about 20 .mu.g/mL, recombinant human bFGF is present at about 20 .mu.g/mL, recombinant hLIF is present at about 10 .mu.g/mL, and/or DHEA is present at about 1 .mu.mol/L. 7. The medium of claim 1, wherein apo-transferrin is present at about 0.01 to about 100 mg/L, insulin is present at about 0.01 to about 100 mg/L, putrescine is present at about 0.01 to about 100 mg/L, selenium is present at about 0.0001 to about 100 .mu.M, and progesterone is present at about 0.0001 to about 100 .mu.M. 8. The medium of claim 7, wherein apo-transferrin is present at about 10 to about 40 mg/L, insulin is present at about 10 to about 40 mg/L, putrescine is present at about 5 to about 20 mg/L, selenium is present at about 0.01 to about 1 .mu.M, and progesterone is present at about 0.01 to about 1 .mu.M. 9. The medium of claim 8, wherein apo-transferrin is present at about 25 mg/L, insulin is present at about 23 mg/L, putrescine is present at about 9 mg/L, selenium is present at about 0.027 .mu.M, and progesterone is present at about 0.018 .mu.M. 10. A bioreactor comprising at least one cell and the medium of claim 1. 11. The bioreactor of claim 10, wherein said cell is a neural precursor cell. 12. The bioreactor of claim 10, wherein said bioreactor is a dish, flask, vessel, bottle or multi-well plate. 13. A method of culturing a neural precursor cell (NPC) comprising the steps of: (a) providing an isolated NPC or NPC-containing population in culture medium according to claim 1 in a bioreactor; and (b) culturing said NPC or NPC-containing cell population under conditions that (i) produce cell aggregates having an average size of about 20-2000 .mu.m in diameter after 4 days of culture, (ii) wherein said culture medium is maintained at a pH of about 7.0-7.8; and (iii) wherein said NPC or NPC-containing cell population is cultured in batch mode, semi-fed batch mode or perfusion mode. 14. The method of claim 13, wherein culturing comprises conditions that (i) produce cell aggregates having an average size of about 100-1000 .mu.m in diameter after 5 days of culture, and (ii) wherein said culture medium is maintained at a pH of about 7.2-7.4. 15. The method of claim 13, wherein said NPC or NPC-containing population retains a neural stem or progenitor cell marker. 16. The method of claim 13, wherein said NPC or NPC-containing population is obtained from forebrain, ventral mesencephalon, brain stem or spinal cord. 17. The method of claim 13, wherein said NPC or NPC-containing population is passaged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 times. 18. The method of 13, wherein said NPC or NPC-containing population is maintained in culture for 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 100, 110, 120, 130 or 140 days. 19. The method of claim 13, further comprising inducing differentiation of said NPC or NPC-containing population. 20. The method of claim 19, wherein said NPC or NPC-containing population differentiates into a CNS cell. 21. The method of claim 19, wherein said NPC or NPC-containing population differentiates into an astrocyte(s) or a neuronal cell(s). 22. The method of claim 13, wherein said NPC or NPC-containing population is maintained at about 75-95% viability. 23. The method of claim 13, wherein said NPC or NPC-containing population is cultured in a stationary phase. 24. The method of claim 13, wherein said NPC or NPC-containing population is cultured in suspension with an agitation rate of greater than about 50 rpm and less than about 130 rpm. 25. The method of claim 24, wherein agitation is produced by a stir bar, an impeller or by movement of said bioreactor. 26. The method of claim 24, wherein said agitation rate is about 80 to about 90 rpm. 27. The method of claim 26, wherein said agitation rate is about 85 rpm. 28. The method of claim 13, wherein said NPC or NPC-containing population is cultured in about 0.1-5000 mL volume of culture medium. 29. The method of claim 28, wherein said NPC or NPC-containing population is cultured in about 100 mL, 200 mL, or 500 mL volume of culture medium. 30. The method of claim 13, wherein more than 50% of said cell aggregates have a size of between about 300 to about 700 .mu.M between days 8 and 20 of culture. 31. The method of claim 13, wherein said cell aggregates have a mean size of between about 400 to about 600 .mu.M. 32. The method of claim 13, wherein semi-fed batch mode comprises replacement of 30-50% of said culture medium each 3-6 days. 33. The method of claim 13, wherein said bioreactor is a dish, flask, bottle or multi-well plate. 34. The method of claim 13, wherein oxygenation of said culture medium is maintained at about 1-20% dissolved oxygen. 35. The method of claim 34, wherein oxygenation of said culture medium is maintained at about 5-20% dissolved oxygen. 36. The method of claim 13, wherein oxygenation of said culture medium is maintained at about 14% dissolved oxygen. 37. The method of claim 13, wherein cell-fold expansion per passage is 5-50 for the first 140 days. 38. The method of claim 13, wherein cell-fold expansion per passage is 10-40 for the first 140 days.

Details for Patent 8,093,053

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Grifols Therapeutics Llc	ALBUKED, PLASBUMIN-20, PLASBUMIN-25, PLASBUMIN-5	albumin (human)	For Injection	101138	10/21/1942		2028-03-17
Baxalta Us Inc.	BUMINATE, FLEXBUMIN	albumin (human)	Injection	101452	03/03/1954		2028-03-17
Csl Behring Ag	ALBURX	albumin (human)	Injection	102366	07/23/1976		2028-03-17
Grifols Biologicals Llc	ALBUTEIN	albumin (human)	Injection	102478	08/15/1978		2028-03-17
Instituto Grifols, S.a.	HUMAN ALBUMIN GRIFOLS	albumin (human)	Injection	103352	02/17/1995		2028-03-17
Instituto Grifols, S.a.	HUMAN ALBUMIN GRIFOLS	albumin (human)	Injection	103352	06/11/2003		2028-03-17