KITS AND METHODS FOR PROCESSING STEM CELLS FROM BONE MARROW OR UMBILICAL CORD BLOOD

• US 20140271569A1
• Filed: 03/10/2014
• Published: 09/18/2014
• Est. Priority Date: 03/13/2013
• Status: Active Grant

• Current Assignee: Mingqi Tang
• Original Assignee: Mingqi Tang
• Inventors: TANG, Mingqi
• Granted Patent: US 9,241,959 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 35/51 Umbilical cord; Umbilical cord blood; Umbilical stem cells
• 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: C12N 5/0665 Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood

Abstract

Provided herein are kits for processing stem cells from bone marrow or umbilical cord blood, comprising: a) a precipitation reagent comprising an aqueous solution of 0.1-30% hydroxyethyl starch or 0.1-30% methyl cellulose, and 0.1-20% of cerebroprotein hydrolysate; and b) a separation reagent comprising an aqueous solution comprising Ficoll and diatrizoate and having a density of 1.0-1.2 g/ml, and methods of use. Further provided herein is a collection of stem cells obtained by the kits and methods disclosed herein comprising stem cells from bone marrow or umbilical cord blood, and uses thereof.

Claims

1. A kit for processing stem cells from bone marrow or umbilical cord blood, comprising:

a) a precipitation reagent comprising an aqueous solution of 0.1-30% hydroxyethyl starch or 0.1-30% methyl cellulose, and 0.1-20% of cerebroprotein hydrolysate; and

b) a separation reagent comprising an aqueous solution comprising Ficoll and diatrizoate and having a density of 1.0-1.2 g/ml.

2. The kit of claim 1, wherein the precipitation reagent comprises 0.3-15% of cerebroprotein hydrolysate.

3. The kit of claim 1, wherein the precipitation reagent comprises 0.8-6% of cerebroprotein hydrolysate.

4. The kit of claim 1, wherein the precipitation reagent comprises about 1.5-4.5% of cerebroprotein hydrolysate.

5. The kit of claim 1, wherein the precipitation reagent comprises about 3% of cerebroprotein hydrolysate.

6. The kit of claim 1, wherein the precipitation reagent comprises 0.5-18% aqueous solution of hydroxyethyl starch or 0.2-8% aqueous solution of methyl cellulose.

7. The kit of claim 1, wherein the precipitation reagent comprises about 1.5-12% aqueous solution of hydroxyethyl starch or about 0.3-5% aqueous solution of methyl cellulose.

8. The kit of claim 1, wherein the precipitation reagent comprises about 6% aqueous solution of hydroxyethyl starch or about 0.5% aqueous solution of methyl cellulose.

9. The kit of claim 1, wherein the separation reagent comprises an aqueous solution comprising Ficoll and diatrizoate and having a density of about 1.01-1.088 g/ml.

10. The kit of claim 1, wherein the separation reagent comprises an aqueous solution comprising Ficoll and diatrizoate and having a density of about 1.035-1.08 g/ml.

11. The kit of claim 1, wherein the separation reagent comprises an aqueous solution comprising Ficoll and diatrizoate and having a density of about 1.075 g/ml.

12. The kit of claim 1, wherein the precipitation reagent comprises 0.5-18% aqueous solution of hydroxyethyl starch or 0.2-8% aqueous solution of methyl cellulose, and 0.8-6% of cerebroprotein hydrolysate, and wherein the separation reagent comprises an aqueous solution comprising Ficoll and diatrizoate and having a density of about 1.01-1.088 g/ml.

13. The kit of claim 1, wherein the precipitation reagent comprises about 1.5-12% aqueous solution of hydroxyethyl starch or about 0.3-5% aqueous solution of methyl cellulose, and about 1.5-4.5% of cerebroprotein hydrolysate, and wherein the separation reagent comprises an aqueous solution comprising Ficoll and diatrizoate and having a density of about 1.035-1.08 g/ml.

14. The kit of claim 1, wherein the precipitation reagent comprises about 6% aqueous solution of hydroxyethyl starch or about 0.5% aqueous solution of methyl cellulose, and about 3% of cerebroprotein hydrolysate, and wherein the separation reagent comprises an aqueous solution comprising Ficoll and diatrizoate and having a density of about 1.075 g/ml.

15. The kit of claim 1, wherein the aqueous solution uses sterile water suitable for injection into a subject.

16. The kit of claim 1, wherein the subject is a human being.

17. The kit of claim 1, wherein the precipitation reagent and the separation reagent are sterilized under ultrafiltration conditions.

18. A method for separating stem cells using a kit for processing stem cells from bone marrow or umbilical cord blood comprising a precipitation reagent comprising an aqueous solution of 0.1-30% hydroxyethyl starch or 0.1-30% methyl cellulose, and 0.1-20% of cerebroprotein hydrolysate;

and a separation reagent comprising an aqueous solution comprising Ficoll and diatrizoate and having a density of 1.0-1.2 g/ml, which method comprises;

a) providing a bone marrow or umbilical cord blood sample;

b) adding the precipitation reagent to the bone marrow or umbilical cord blood sample, mixing and letting the solution to settle, followed by centrifugation of the top solution to obtain a concentrated sample; and

c) adding the concentrated sample from step b) on top of the separation reagent to obtain a collection of cells,

wherein the collection of cells comprises stem cells.

19. The method of claim 18, wherein step a) further comprises adding an anticoagulant to the bone marrow or umbilical cord blood.

20. The method of claim 18, wherein step a) further comprises adding an equal volume of saline (NaCl injection) to the bone marrow or umbilical cord blood sample to obtain a diluted sample.

21. The method of claim 18, wherein step c) further comprises adding the concentrated sample from step b) on top of the separation reagent followed by centrifugation.

22. The method of claim 18, wherein step a) is performed in a sterile environment.

23. The method of claim 18, wherein the bone marrow is obtained by a bone marrow needle using a posterior superior iliac spine method with local anesthesia.

24. The method of claim 21, wherein the ratio between the diluted sample and the precipitation reagent is 2:

1 by volume.

25. The method of claim 18, wherein the concentrated sample from step b) is diluted with saline.

26. The method of claim 18, wherein the bone marrow or umbilical cord blood sample is freshly collected from a mammal.

27. The method of claim 26, wherein the mammal is a human being.

28. The method of claim 18, wherein the bone marrow or umbilical cord blood sample has not been cultivated ex vivo.

29. A collection of stem cells obtained by the method of claim 18 comprising stem cells from bone marrow or umbilical cord blood.

30. The collection of cells of claim 29, wherein the recovery rate of the stem cells is at least 80%.

31. The collection of cells of claim 29, wherein the recovery rate of the stem cells is at least 85%.

32. The collection of cells of claim 29, wherein the recovery rate of the stem cells is at least 90%.

33. The collection of cells of claim 29, wherein the survival rate of the stem cells is at least 90%.

34. The collection of cells of claim 29, wherein the survival rate of the stem cells is at least 95%.

35. The collection of cells of claim 29, wherein the survival rate of the stem cells is at least 98%.

36. The collection of cells of claim 29, wherein the stem cells comprise hematopoietic stem cells (HSC).

37. The collection of cells of claim 29, wherein the stem cells comprise mesenchymal stem cells (MSC).

38. A method of treating a disease condition in a subject comprising administrating a collection of stem cells of claim 29 to said subject.

39. The method of claim 38, wherein the disease condition is selected from the group consisting of Parkinson'"'"'s, Alzheimer'"'"'s, cerebral complications, diabetes, diabetic foot, pediatric cerebral palsy, cirrhosis, and systemic lupus erythematosus.

40. The method of claim 38, which comprises injecting the collection of cells into the vein of the subject.

41. The method of claim 38, which comprises injecting the collection of cells into the lesion site of the subject through the femoral artery.

42. The method of claim 38, wherein at least 10⁷-10⁸ stem cells are used.

43. The method of claim 38, wherein at least 10⁶ stem cells are used.

44. The method of claim 38, wherein at least 10⁷ stem cells are used.

45. The method of claim 38, wherein at least 10⁸ stem cells are used.

46. The method of claim 38, wherein the collection of stem cells is from one or more other subjects.

47. The method of claim 38, wherein the collection of stem cells is from the subject.