METHODS FOR COLLECTING AND USING PLACENTA CORD BLOOD STEM CELLS

• US 20090123437A1
• Filed: 11/10/2008
• Published: 05/14/2009
• Est. Priority Date: 05/11/2006
• Status: Active Grant

• Current Assignee: Celularity, Inc.
• Original Assignee: Cord Blood Science Inc.
• Inventors: TAKEBE, Naoko
• Granted Patent: US 8,329,468 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/51 Umbilical cord; Umbilical cord blood; Umbilical stem cells
• CPC Class Codes: A61L 27/3834 Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

An innovative method of collecting cord blood stem cells from an isolated mammalian non-exsanguinated or partially exsanguinated placenta by placental perfusion is described and also an easy method for safe long duration cold storage of the placenta. Placental perfusion can include perfusing the isolated placenta with a pulsatile flow of perfusion solution, for example, using a pulsatile or peristaltic pump or device. The stem cells can then be isolated from the perfusate. Significantly increased amounts of CD133+ stem cells can be collected from the perfusate. The perfusion solution can include an anticoagulant. The isolated mammalian placenta need not be treated with an anticoagulant prior to perfusing. The isolated placenta can be free from an anticoagulant prior to perfusing.

Claims

1. A method of isolating cord blood stem cells, comprising:

providing an isolated mammalian placenta comprising cord blood comprising cord blood stem cells, wherein said isolated mammalian placenta is non-exsanguinated or partially exsanguinated;

perfusing the isolated mammalian placenta with a pressure mediated flow of a perfusion solution to produce a perfusate comprising cord blood comprising cord blood stem cells;

collecting the perfusate; and

isolating the cord blood stem cells from the perfusate to produce isolated cord blood stem cells.

2. The method according to claim 1, wherein said providing an isolated mammalian placenta comprises providing an isolated non-exsanguinated mammalian placenta.

3. The method according to claim 1, wherein said providing an isolated mammalian placenta comprises providing an isolated partially exsanguinated mammalian placenta.

4. The method according to claim 1, wherein said providing an isolated mammalian placenta comprises:

providing said isolated mammalian placenta comprising cord blood comprising cord blood stem cells, wherein said isolated mammalian placenta is non-exsanguinated; and

partially exsanguinating said isolated non-exsanguinated mammalian placenta to produce a partially exsanguinated placenta and a volume of cord blood comprising cord blood stem cells.

5. The method according to claim 4, wherein said partially exsanguinating comprises:

collecting cord blood by partially exsanguinating said isolated mammalian placenta by venipuncture to produce a partially exsanguinated placenta and a volume of cord blood comprising cord blood stem cells.

6. The method according to claim 4, wherein said partially exsanguinating comprises:

collecting cord blood by partially exsanguinating said isolated mammalian placenta by gravity drainage to produce a partially exsanguinated placenta and a volume of cord blood comprising cord blood stem cells

7. The method according to claim 4, further comprising prior to isolating, pooling the volume of cord blood and the perfusate.

8. The method according to claim 1, wherein the pressure-mediated flow of perfusion solution comprises a pulsatile flow of perfusion solution.

9. The method according to claim 8, wherein perfusing is carried out under conditions sufficient to produce a mammalian placenta substantially free from cord blood stem cells.

10. The method according to claim 8, wherein perfusing is performed using a peristaltic pump.

11. The method according to claim 1, wherein the isolated cord blood stem cells comprise hematopoietic stem cells.

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

cooling the isolated mammalian placenta to produce a cooled mammalian placenta;

said cooling the isolated mammalian placenta being performed prior to said perfusing.

13. The method according to claim 12, further comprising:

maintaining the cooled isolated mammalian placenta at a temperature of from about >

0°

C. to about 15°

C. prior to said perfusing.

14. The method according to claim 12, further comprising:

maintaining the cooled isolated mammalian placenta at a temperature of from about >

0°

C. to about 6°

C.

15. The method according to claim 12, further comprising:

maintaining the cooled isolated mammalian placenta at a temperature of from about >

0°

C. to about 15°

C. for a period of time of up to about 40 hours.

16. The method according to claim 12, further comprising:

maintaining the cooled isolated mammalian placenta at a temperature of from about >

0°

C. to about 15°

C. for a period of time of from 6 to 40 hours.

17. The method according to claim 12, further comprising:

maintaining the cooled isolated mammalian placenta at a temperature of from about >

0°

C. to about 15°

C. for a period of time of from 10 to 40 hours.

18. The method according to claim 12, further comprising:

maintaining the cooled isolated mammalian placenta at a temperature of from about >

0°

C. to about 15°

C. for a period of time of from 20 to 40 hours.

19. The method according to claim 12, wherein said cooling comprises placing said isolated mammalian placenta in ice.

20. The method according to claim 1, wherein the placenta is not administered or injected with an anticoagulant prior to perfusing.

21. The method according to claim 1, wherein the perfusion solution comprises a physiologically-compatible solution comprising an anticoagulant.

22. The method according to claim 1, wherein the perfusion solution comprises one or more anticoagulants selected from the group consisting of heparin, creatine phosphate dextrose (CPDA), or a combination thereof.

23. The method according to claim 10, wherein said perfusing comprises perfusing via one or more of the umbilical arteries and the umbilical vein.

24. The method according to claim 23, wherein said perfusing further comprises cannulating one or more of the umbilical arteries and the umbilical vein to produce a cannulated placenta;

and placing the cannulated placenta in a closed perfusion circuit.

25. The method according to claim 24, wherein said perfusing is carried out at a pulse setting of from about 15 beats/min to about 60 beats/min.

26. The method according to claim 24, wherein said perfusing is carried out at a pulse setting of about 60 beats/min.

27. The method according to claim 25, wherein said perfusing is carried out at a systolic pressure of from about 30 mm Hg to about 70 mm Hg.

28. The method according to claim 27, wherein said perfusing is carried out for a period of time of from about 15 min. to about 35 min.

29. The method according to claim 1, wherein:

the isolated cord blood stem cells comprise a defined percentage of CD133+ cells.

30. The method according to claim 29, wherein the isolated cord blood stem cells further comprise cells other than CD133+ cells, wherein said cells other than CD133+ cells are selected from the group consisting of embryonic stem cell (ES)-like stem cells, hematopoietic stem cells, mesenchymal stem cells and combinations thereof.

31. The method according to claim 29, wherein:

said defined percentage of CD133+ cells exceeds a control percentage of CD133+ cells in a control sample of isolated stem cells;

wherein said control sample has been isolated by partially exsanguinating a control non-exsanguinated mammalian placenta without performing perfusion on said control non-exsanguinated mammalian placenta.

32. The method according to claim 29, wherein the isolated cord blood stem cells further comprise cells selected from the group consisting of CD34+/CD38-cells, CD133+/CD34+ cells, CD133+/CD34−

cells, CD117+ cells, CD90+ cells, CD59+ cells, Thy1+ cells, Lin−

cells, CXCR4+ cells, ALDH^high cells, side population (SP) cells, SSEA-3+ cells, SSEA-4+ cells, TRA-1-60 cells, TRA-1-81 cells, and combinations thereof.

33. The method according to claim 29, wherein the isolated cord blood stem cells further comprise stem cells that can differentiate into cells other than CD34+/CD38−

cells, CD133+ cells, CD133+/CD34+ cells, or CD133+/CD34−

cells, CD117+ cells, CD90+ cells, CD59+ cells, Thy1+ cells, Lin−

cells, CXCR4+ cells, ALDH^high cells, side population (SP) cells, SSEA-3+ cells, SSEA-4+ cells, TRA-1-60 cells, or TRA-1-81 cells.

47. A method for treating a mammal in need of hematopoietic reconstitution or regeneration of damaged tissue, comprising:

(a) optionally culturing in vitro isolated cord blood stem cells, thereby producing differentiated cells or expanded stem cells,

said isolated cord blood stem cells being obtained according to the method of claim 1; and

(b) introducing into the mammal a composition comprising a therapeutically effective amount of cells selected from the group consisting of;

i) said isolated cord blood stem cells;

ii) said differentiated cells or expanded stem cells, and

iii) combinations thereof;

whereby hematopoietic reconstitution or tissue regeneration is effected.

34. A method for treating a mammal in need of hematopoietic reconstitution or regeneration of damaged tissue, comprising:

a) providing isolated stem cells by;

i) perfusing an isolated non-exsanguinated or partially exsanguinated mammalian placenta comprising cord blood stem cells with a perfusion solution to produce a perfusate comprising cord blood stem cells; and

ii) isolating the cord blood stem cells from the perfusate to produce isolated stem cells; and

b) introducing into the mammal a composition comprising a therapeutically effective amount of cells selected from the group consisting of the isolated stem cells and cells derived therefrom.

35. A method according to claim 34, wherein said perfusing comprises:

perfusing an isolated non-exsanguinated or partially exsanguinated mammalian placenta comprising cord blood stem cells with a pressure mediated flow of a perfusion solution to produce a perfusate comprising cord blood stem cells.

36. A method according to claim 34, wherein said introducing comprises:

introducing into the mammal a composition comprising a therapeutically effective amount of the isolated stem cells.

37. A method according to claim 34, wherein said introducing comprises:

obtaining said cells derived therefrom by culturing in vitro the isolated stem cells, thereby producing said cells derived therefrom; and

introducing into the mammal a composition comprising a therapeutically effective amount of said cells derived therefrom.

38. The method according to claim 37, wherein said cells derived therefrom are differentiated cells or expanded stem cells.

39. The method according to claim 34, wherein:

the mammal is in need of hematopoietic reconstitution; and

said isolated stem cells comprise hematopoietic stem cells.

40. The method according to claim 39, wherein the mammal in need of hematopoietic reconstitution has aplastic anemia, a hematopoietic malignancy, an autoimmune disease, a genetic disorder, an immunodeficiency, a malignant solid tumor, or a combination thereof.

41. The method according to claim 40, in which the mammal in need of hematopoietic reconstitution has a hematopoietic malignancy selected from leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome.

42. The method according to claim 40, in which the mammal in need of hematopoietic reconstitution has an immunodeficiency resulting from irradiation, chemotherapy, infection by a pathogenic microorganism, or a combination thereof.

43. The method according to claim 39, wherein said introducing comprises introducing into the mammal intravenously or intraosseously a composition comprising a therapeutically effective amount of cells selected from the group consisting of the isolated stem cells and cells derived therefrom.

44. The method according to claim 34, wherein:

the mammal is in need of regeneration of damaged tissue.

45. The method according to claim 44, wherein:

said introducing comprises introducing into the mammal intravenously or by direct injection into the target organ a composition comprising a therapeutically effective amount of cells selected from the group consisting of isolated stem cells and cells derived therefrom

46. The method according to claim 45, wherein the tissue comprises one or more of cardiac tissue, muscle tissue, liver, skin, neural tissue, bone tissue, epithelia, stroma, or endothelium.