Claims
- 1. A method for obtaining ex vivo human stem cell division comprising culturing human hematopoietic stem cells contained in a human bone marrow composition in a liquid culture medium in the presence of human bone marrow stromal cells to obtain ex vivo human stem cell division therein, wherein the culture medium is replaced, either continuously or periodically, at a rate of about 1 ml of medium per ml of culture per about 24 to about 48 hour period.
- 2. The method of claim 1, wherein the stromal cells are present in the cell culture in an amount of approximately 10−3 to 10−1 stromal cells/total cells.
- 3. The method of claim 1, wherein the stromal cells secrete GM-CSF.
- 4. The method of claim 3, wherein the amount of GM-CSF secreted by the stromal cells is 300 centograms/ml/day to 200 picograms/ml/day.
- 5. The method of claim 1, wherein the stromal cells secrete IL-6.
- 6. The method of claim 1, wherein the amount of IL-6 secreted by the stromal cells is 1-2 ng/ml/day to 2-4 ng/ml/day.
- 7. The method of claim 1, wherein said cultured human stem cells produce hematopoietic progenitor cells.
- 8. The method of claim 1, wherein IL-3 and GM-CSF are added continuously to said medium, each at a rate of 0.1 to 100 ng ml−1 day−1.
- 9. The method of claim 8, wherein Epo is added to said medium at a rate of 0.001 to 10 U ml−1 day−1, mast cell growth factor is added to said medium at a rate of from 1 to 100 ng ml−1 day−1, or IL-1 (α or β) is added to said medium at a rate of from 10 to 100 U ml−1 per 3 to 5 day period.
- 10. The method of claim 9, wherein at least one member selected from the group consisting of G-CSF, basic fibroblast growth factor, Il-6, Il-7, IL-8, IL-9, IL-10, IL-11, PDGF and EGF, is added to said medium.
- 11. The method of claim 8, wherein Epo is added to said medium at a rate of 0.001 to 10 U ml−1 day−1.
- 12. The method of claim 8, wherein Epo is added to said medium at a rate of from 0.05 to 0.15 U ml−1 day−1.
- 13. The method of claim 1, wherein IL-3 and GM-CSF are added continuously to said medium, each at a rate of 0.5 to 10 ng ml−1 day−1.
- 14. The method of claim 1, wherein IL-3 and GM-CSF are added continuously to said medium, each at a rate of 1 to 2 ng ml−1 day−1.
- 15. The method of claim 1, wherein said medium comprises animal sera or plasma.
- 16. The method of claim 1, wherein said media comprises a corticosteroid.
- 17. A method for expanding a human hematopoietic stem cell pool, comprising culturing a human bone marrow composition comprising said human stem cell pool in a liquid culture medium in the presence of human bone marrow stromal ells to obtain ex vivo human stem cell expansion therein, and removing metabolic product and replenishing depleted nutrients while maintaining said culture under physiologically acceptable conditions, wherein the culture medium is replaced, either continuously or periodically, at a rate of about 1 ml of medium per ml of culture per about 24 to about 48 hour period.
- 18. The method of claim 17, wherein the stromal cells are present in the cell culture in an amount of approximately 10−3 to 10−1 stromal cells/total cells.
- 19. The method of claim 17, wherein the stromal cells secrete GM-CSF.
- 20. The method of claim 19, wherein the amount of GM-CSF secreted by the stromal cells is 300 centograms/ml/day to 200 picograms/ml/day.
- 21. The method of claim 17, wherein the stromal cells secrete IL-6.
- 22. The method of claim 21, wherein the amount of IL-6 secreted by the stromal cells is 1-2 ng/ml/day to 2-4 ng/ml/day.
- 23. The method of claim 17, wherein IL-3 and GM-CSF are added continuously to said medium, each at a rate of 0.1 to 100 ng ml−1 day−1.
- 24. The method of claim 23, wherein Epo is added to said medium at a rate of 0.001 to 10 U ml−1 day−1, mast cell growth factor is added to said medium at a rate of from 1 to 100 ng ml−1 day−1, or IL-1 (α or β) is added to said medium at a rate of from 10 to 100 U ml−1 per 3 to 5 day period.
- 25. The method of claim 24, wherein at least one member selected from the group consisting of G-CSF, basic fibroblast growth factor, Il-6, Il-7, IL-8, IL-9, IL-10, IL-11, PDGF and EGF, is added to said medium.
- 26. The method of claim 23, wherein Epo is added to said medium at a rate of 0.001 to 10 U ml−1 day−1.
- 27. The method of claim 23, wherein Epo is added to said medium at a rate of from 0.05 to 0.15 U ml−1 day−1.
- 28. The method of claim 17, wherein IL-3 and GM-CSF are added continuously to said medium, each at a rate of 0.5 to 10 ng ml−1 day−1.
- 29. The method of claim 17, wherein IL-3 and GM-CSF are added continuously to said medium, each at a rate of 1 to 2 ng ml−1 day−1.
- 30. The method of claim 17, wherein said medium comprises animal sera or plasma.
- 31. The method of claim 17, wherein said media comprises a corticosteroid.
- 32. The method of claim 17, comprising maintaining glucose concentration in said medium in the range of from 5 to 20 mM, lactate concentration in said medium below about 35 mM, glutamine concentration in said medium in the range of from 1 to 3 mM, and ammonia concentration in said medium below 2.5 mM.
- 33. A method for culturing human hematopoietic progenitor cells comprising culturing a human bone marrow composition comprising said human hematopoietic progenitor cells in a human liquid hematopoietic culture medium in the presence of human bone marrow stromal cells to obtain ex vivo human progenitor cell division therein and removing metabolic products and replenishing depleted nutrients while maintaining said culture under physiologically acceptable conditions, wherein the culture medium is replaced, either continuously or periodically, at a rate of about 1 ml of medium per ml of culture per about 24 to about 48 hour period.
- 34. The method of claim 33, wherein the stromal cells are present in the cell culture in an amount of approximately 10−3 to 10−1 stromal cells/total cells.
- 35. The method of claim 33, wherein the stromal cells secrete GM-CSF.
- 36. The method of claim 35, wherein the amount of GM-CSF secreted by the stromal cells is 300 centograms/ml/day to 200 picograms/ml/day.
- 37. The method of claim 33, wherein the stromal cells secrete IL-6.
- 38. The method of claim 37, wherein the amount of IL-6 secreted by the stromal cells is 1-2 ng/ml/day to 2-4 ng/ml/day.
- 39. The method of claim 33, wherein IL-3 and GM-CSF are added continuously to said medium, each at a rate of 0.1 to 100 ng ml−1 day−1.
- 40. The method of claim 39, wherein Epo is added to said medium at a rate of 0.001 to 10 U ml−1 day−1, mast cell growth factor is added to said medium at a rate of from 1 to 100 ng ml−1 day−1, or IL-1 (α or β) is added to said medium at a rate of from 10 to 100 U ml−1 per 3 to 5 day period.
- 41. The method of claim 39, wherein Epo is added to said medium at a rate of 0.001 to 10 U ml−1 day−1.
- 42. The method of claim 39, wherein Epo is added to said medium at a rate of from 0.05 to 0.15 U ml−1 day−1.
- 43. The method of claim 33, wherein IL-3 and GM-CSF are added continuously to said medium, each at a rate of 0.5 to 10 ng ml−1 day−1.
- 44. The method of claim 33, wherein IL-3 and GM-CSF are added continuously to said medium, each at a rate of 1 to 2 ng ml−1 day−1.
- 45. The method of claim 33, wherein at least one member selected from the group consisting of G-CSF, basic fibroblast growth factor, Il-6, Il-7, IL-8, IL-9, IL-10, IL-11, PDGF and EGF, is added to said medium.
- 46. The method of claim 33, wherein said medium comprises animal sera or plasma.
- 47. The method of claim 33, wherein said media comprises a corticosteroid.
- 48. The method of claim 33, comprising maintaining glucose concentration in said medium in the range of from 5 to 20 mM, lactate concentration in said medium below about 35 mM, glutamine concentration in said medium in the range of from 1 to 3 mM, and ammonia concentration in said medium below 2.5 mM.
- 49. A method for obtaining red blood cells, comprising culturing a human bone marrow composition comprising human stem cells in a liquid culture medium in the presence of human bone marrow stromal cells to and adding continuously to said medium, IL-3 at a rate of 0.1 to 100 ng ml−1 day−1, GM-CSF at a rate of 0.1 to 100 ng ml−1 day−1, and Epo at a rate of 0.001 to 10 U ml−1 day−1, and removing metabolic products and replenishing depleted nutrients while maintaining said culture under physiologically acceptable conditions, thereby obtaining a cell culture containing said red blood cells, wherein the culture medium is replaced, either continuously or periodically, at a rate of about 1 ml of medium per ml of culture per about 24 to about 48 hour period.
- 50. A method for obtaining granulocytes, comprising culturing a human bone marrow composition comprising human stem cells in a liquid culture medium in the presence of human bone marrow stromal cells, and continuously adding to said medium, IL-3 at a rate of 0.1 to 100 ng ml−1 day−1 and GM-CSF at a rate of 0.1 to 100 ng ml−1 day, and removing metabolic products and replenishing depleted nutrients while maintaining said culture under physiologically acceptable conditions, thereby obtaining a cell culture containing said granulocytes, wherein the culture medium is replaced, either continuously or periodically, at a rate of about 1 ml of medium per ml of culture per about 24 to about 48 hour period.
- 51. The method of claim 50, comprising adding continuously to said medium IL-6 or G-CSF, or both, each in an amount of from 1 to 100 ng ml−1 day−1.
Priority Claims (1)
Number |
Date |
Country |
Kind |
PCT/US90/03438 |
Jun 1990 |
WO |
|
Parent Case Info
This is a Continuation of application Ser. No. 08/401,978 filed on Mar. 10, 1995, now U.S. Pat. No. 5,646,043, which is a continuation of Ser. No. 08/164,779 filed Dec. 10, 1993, now U.S. Pat. No. 5,437,994; which is a continuation of Ser. No. 07/737,024 filed Jul. 29, 1991, now abandoned; which is a continuation-in-part of Ser. No. 07/628,343 filed Dec. 17, 1990, now abandoned; which is a continuation-in-part of Ser. No. 07/366,639 filed on Jun. 15, 1989, now abandoned; which was filed as International Application No. PCT/US90/03438 filed Jun. 14, 1990.
Non-Patent Literature Citations (6)
Entry |
Fause et al. Blood 52(6): 1243, 1978.* |
Gactre et al DNAS 77(8): 4756, 1980.* |
Dekte Nature 309: 746, 1984.* |
Sief Science 230: 1171, 1985.* |
Magliaccio Exp. Hematol 18: 1049, 1990.* |
Stephenson PNAS 68(7): 1542, 1971. |
Continuations (3)
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Number |
Date |
Country |
Parent |
08/401978 |
Mar 1995 |
US |
Child |
08/797730 |
|
US |
Parent |
08/164779 |
Dec 1993 |
US |
Child |
08/401978 |
|
US |
Parent |
07/737024 |
Jul 1991 |
US |
Child |
08/164779 |
|
US |
Continuation in Parts (2)
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Number |
Date |
Country |
Parent |
07/628343 |
Dec 1990 |
US |
Child |
07/737024 |
|
US |
Parent |
07/366639 |
|
US |
Child |
07/628343 |
|
US |