Tangential flow filtration devices and methods for stem cell enrichment

• US 20050189297A1
• Filed: 11/18/2004
• Published: 09/01/2005
• Est. Priority Date: 11/24/2003
• Status: Active Grant

• Current Assignee: Northwest Biotherapeutics Incorporated
• Original Assignee: Northwest Biotherapeutics Incorporated
• Inventors: Bosch, Marnix L., Boynton, Alton L., Lodge, Patricia A., McEarchern, Julie Anna, Hugenholtz, Paul G.
• Granted Patent: US 7,790,039 B2
• Time in Patent Office: Days
• US Class Current: 210/651
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2500/35 Polyols, e.g. glycerin, inositol
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0663 Bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

The present invention provides methods for enriching a heterogenous mixture of bone marrow or blood constituents for stem cells by removal of non-stem cell constituents comprising separation of the non-stem cell constituents using a tangential flow filtration device.

Claims

1. A method for separating stem cells from a sample from a subject wherein the sample comprises stem cells and non-stem cell constituents, the method comprising separation on a tangential flow filtration device by:

(i) introducing the sample into a remover unit (1) comprising a cross-flow chamber (3) through an inlet (6) in the remover unit;

(ii) subjecting the sample to cross-flow substantially parallel to a filter (5) having a pore size of about 1 to about 10 microns;

(iii) subjecting the fluid to filtration through the filter; and

(iv) selectively removing non-stem cell constituents from the sample to form a cell population enriched for stem cells.

2. The method according to claim 1, further comprising:

preparing the sample from the subject by leukopheresis, density centrifugation, differential lysis, filtration, or preparation of a buffy coat, for introduction in the remover unit.

3. The method according to claim 1., wherein the sample is bone marrow, a tissue suspension, an organ suspension, or blood constituents.

4. The method according to claim 1, wherein the non-stem cell constituents are stroma, erythrocytes, plasma and platelets.

5. The method according to claim 1, further comprising repeating steps (i), (ii), and (iii) at least two times to form cell population enriched for stem cells.

6. The method according to claim 1, wherein the stem cells are hematopoietic stem cells, mesenchymal stem cells, or pluripotent stem cells.

7. The method according to claim 5, wherein the hematopoietic stem cells are CD34⁺cells.

8. The method according to claim 3, wherein the stem cells are hematopoietic stem cells, mesenchymal stem cells, or pluripotent stem cells.

9. The method according to claim 8, wherein the hematopoietic stem cells are CD34⁺cells.

10. The method according to claim 1, wherein the subject has undergone stem cell mobilization by administering a stem cell mobilization agent.

11. The method according to claim 10, wherein the stem cell mobilization agent is M-CSF, G-CSF, GM-CSF, or cyclophosphamide.

12. The method according to claim 1, wherein the tangential flow filtration device has a means for providing a predetermined input rate of the sample to the inlet of the cross-flow chamber;

. a means for controlling a filtration rate through the filter and into the filtrate chamber; and

wherein the filtration rate controlling means limits the filtration rate to less than the unopposed filtration rate for the filter.

13. The method according to claim 1, wherein the sample is preteated to induce cell shrinkage of cell populations with essentially the same size as the stem cells.

14. The method according to claim 13, wherein the cell population with essentially the same size are granulocytes.

15. The method according to claim 13, where the pretreatment comprises contacting the cells with a physiologically acceptable solution comprising dimethylsulfoxide (DMSO).

16. The method according to claim 15, wherein the final DMSO concentration is between 5% and 20%.

17. The method according to claim 16, wherein the final DMSO concentration is between 10% and 15%.

18. The method according to claim 17, wherein the final DMSO concentration is 12.5%.

19. The method according to claim 17, wherein the final DMSO concentration is 15%.

20. The method according to claim 15, wherein the physiologically acceptable solution is of low ionic strength.

21. The method according to claim 13, wherein the pretreatment comprises contacting the cells with a physiologically acceptable solution comprising glycerol.

22. The method according to claim 21, wherein the final glycerol concentration is between 0.5 mol/L and 2.5 mol/L.

23. The method according to claim 21, wherein the final glycerol concentration is 1 mol/L.

24. The method according to claim 14, wherein granulocytes are preferentially removed from the cell mixture through lysis.

25. The method according to claim 24, wherein lysis is effected through sequential contact of the sample to an effective amount of DMSO and glycerol.

26. The method according to claim 25, wherein contact of the sample is with a solution with low osmotic strength.

29. The method according to claim 1, further comprising the step of administering the cell population enriched for stem cells to an individual.

30. The method according to claim 29, wherein the sample is from a subject that has been treated with at least one stem cell mobilizing agent.

31. The method according to claim 30, wherein the stem cell mobilizing agent is M-CSF, G-CSF, GM-CSF or cyclophosphamide.

32. The method according to claim 29, wherein the stem cells are reinfused into a recipient.

33. The method according to claim 32, where the recipient is autologous to the subject.

34. The method according to claim 32, wherein the recipient is allogeneic to the subject.

35. The method according to claim 32, wherein the recipient is in need of bone marrow regeneration.

36. The method according to claim 32, wherein the recipient has undergone myeloablative therapy.

37. The method according to claim 32, wherein the recipient is in need of repair of cardiac tissue.

38. The method of claim 32, wherein the recipient has undergone a cardiac infarct.

39. The method according to claim 32, wherein the recipient is suffering from congestive heart failure.

40. The method of claim 32, where the recipient is suffering from cardiac insufficiency.

41. The method of claim 32, where the enriched stem cell population is infused into the circulation.

42. The method of claim 32, where the enriched stem cell population is infused into the coronary artery.

43. The method of claim 32, where the enriched stem cell population is applied directly to the injured heart tissue.

27. A method for enriching a sample of bone marrow or blood constituents for stem cells, comprising:

(i) introducing the sample into a tangential flow filtration (TFF) unit, the TFF unit comprising a cross-flow chamber, a filtrate chamber, and a filter in fluid communication with the cross-flow chamber and the filtrate chamber, the filter having a pore size of about 1 to about 10 microns;

(ii) recirculating the sample through the TFF unit at a predetermined input rate and a predetermined filtration rate, the predetermined input rate at least five times the predetermined filtration rate;

wherein the predetermined filtration rate is less than the unopposed filtration rate for the filter; and

(iii) isolating a cell population enriched for stem cells.

28. The method according to claim 27, wherein the enriched cell population is substantially free of non-leukocyte blood constituents.