Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids

• US 10,500,231 B2
• Filed: 03/12/2014
• Issued: 12/10/2019
• Est. Priority Date: 03/13/2013
• Status: Active Grant

• Current Assignee: University of Miami
• Original Assignee: University of Miami
• Inventors: Badiavas, Evangelos V., Shabbir, Arsalan Q., Davis, Stephen C.
• Primary Examiner(s): Wu, Julie
• Assistant Examiner(s): White, Douglas F
• Application Number:
  US14/775,382
• Publication Number:
  US 20160184363A1
• Time in Patent Office: 2,099 Days
• Field of Search: None
• CPC Class Codes: A61K 2800/74 Biological properties of particular ingredients
• CPC Class Codes: A61K 2800/86 Products or compounds obtained by genetic engineering
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/16 Blood plasma; Blood serum umbilical cord blood A61K35/51
• CPC Class Codes: A61K 35/22 Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
• 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 8/981 of mammals or bird
• CPC Class Codes: A61K 8/983 Blood, e.g. plasma
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61Q 19/00 Preparations for care of the skin
• CPC Class Codes: A61Q 19/08 Anti-ageing preparations

Abstract

The present invention relates to the fields of medicine, cell biology, molecular biology and genetics. In particular, the present invention provides methods to isolate and purify microvesicles from cell culture supernatants and biological fluids. The present invention also provides pharmaceutical compositions of microvesicles to promote or enhance wound healing, stimulate tissue regeneration, remodel scarred tissue, modulate immune reactions, alter neoplastic cell growth and/or mobility, or alter normal cell growth and/or mobility. The present invention also provides compositions of microvesicles to be used as diagnostic reagents, and methods to prepare the compositions of microvesicles.

Claims

1. A method of treating a skin wound in a patient in need thereof, the method comprising:

administering a pharmaceutical composition of isolated microvesicles to the skin wound of the patient,

wherein the pharmaceutical composition of isolated microvesicles is prepared comprising the steps of;

(a) obtaining a biological fluid containing mammalian microvesicles from a mesenchymal stem cell;

(b) precipitating the microvesicles by adding polyethylene glycol (PEG) to the biological fluid;

(c) washing the precipitated microvesicles to remove the PEG;

(d) isolating the microvesicles to obtain isolated microvesicles; and

(e) providing a therapeutically effective amount of the precipitated isolated macrovesicles as a pharmaceutical composition,

wherein the precipitated microvesicles range in size from 2 nm to 5000 nm,

wherein the isolated microvesicles result in increased migration of non-proliferating cultured fibroblasts in a cell migration assay when administered to the non-proliferating cultured fibroblasts, and

wherein administering the pharmaceutical composition of isolated microvesicles to the skin wound of the patient results in regeneration of at least one skin tissue in the skin wound.

2. The method of claim 1, wherein the skin wound is a full-thickness wound.

3. The method of claim 1, wherein the skin wound is a burn.

4. The method of claim 1, wherein the at least one skin tissue regenerated in the skin wound is selected from the group consisting of epithelial tissue, stromal tissue, nerve tissue, vascular tissue, and adnexal tissue.

5. The method of claim 1, wherein wrinkle formation or scar formation is reduced in the treated skin wound of the patient.

6. The method of claim 1, wherein the pharmaceutical composition of isolated microvesicles does not provoke a significant inflammatory response in the skin wound of the patient.

7. The method of claim 1, wherein the step of washing the precipitated microvesicles comprises centrifugal filtration.

8. The method of claim 1, wherein the isolated microvesicles have a molecular weight of at least 100 kDa.

9. The method of claim 1, wherein the PEG has an average molecular weight of 20,000 Da.

10. The method of claim 1, wherein the PEG is used at a percentage of between 5% and 25%.

11. The method of claim 1, wherein the mesenchymal stem cell is from bone marrow.

12. The method of claim 1, wherein the mesenchymal stem cell is allogeneic in origin.

13. The method of claim 1, wherein the isolated microvesicles have uncorrugated borders when examined by electron microscopy.

14. The method of claim 1, wherein the microvesicles are further processed to isolate microvesicles having at least one characteristic selected from the group consisting of:

a particular size, a particular size range, a particular molecular weight, a particular weight range, and containing a specific molecule.

15. The method of claim 2, wherein the microvesicles are further processed to isolate microvesicles having a size of 2 nm to 200 nm.

16. A method of treating a burn wound in a patient in need thereof, the method comprising administering a pharmaceutical composition of isolated microvesicles to the burn wound of the patient,

wherein the isolated microvesicles are prepared comprising the steps of:

(a) obtaining a biological fluid containing mammalian microvesicles from a mesenchymal stem cell;

(b) precipitating the microvesicles by adding polyethylene glycol (PEG) to the biological fluid;

(c) washing the precipitated microvesicles to remove the PEG,

(d) isolating the microvesicles to obtain isolated microvesicles; and

(e) providing a therapeutically effective amount of the precipitated isolated microvesicles as a pharmaceutical composition,

wherein the precipitated microvesicles range in size from 2 nm to 5000 nm,

wherein the isolated microvesicles result in increased migration of non-proliferating cultured fibroblasts in a cell migration assay when administered to the non-proliferating cultured fibroblasts, and

wherein administering the pharmaceutical composition of isolated microvesicles to the burn wound of the patient results in in regeneration of at least one skin tissue in the burn wound.

17. The method of claim 16, wherein the burn wound is selected from a second-degree burn and a full-thickness burn.

18. The method of claim 16, wherein the skin tissue regenerated in the burn wound of the patient is selected from the group consisting of epithelial tissue, stromal tissue, nerve tissue, vascular tissue, and adnexal tissue.

19. The method of claim 16, wherein wrinkle formation or scar formation is reduced in the healed burn wound of the patient.

20. The method of claim 16, wherein the pharmaceutical composition of isolated microvesicles does not provoke a significant inflammatory response in the patient.

21. The method of claim 16, wherein the step of washing the precipitated microvesicles comprises centrifugal filtration.

22. The method of claim 16, wherein the isolated microvesicles have a molecular weight of at least 100 kDa.

23. The method of claim 16, wherein the PEG has an average molecular weight of 20,000 Da.

24. The method of claim 16, wherein the PEG is used at a percentage of between 5% and 25%.

25. The method of claim 16, wherein the mesenchymal stem cell is from bone marrow.

26. The method of claim 16, wherein the mesenchymal stem cell is allogeneic in origin.

27. The method of claim 16, wherein the isolated microvesicles have uncorrugated borders when examined by electron microscopy.

28. The method of claim 16, wherein the microvesicles are further processed to isolate microvesicles having at least one characteristic selected from the group consisting of:

a particular size, a particular size range, a particular molecular weight, a particular weight range, and containing a specific molecule.

29. The method of claim 16, wherein the microvesicles are further processed to isolate microvesicles having a size of 2 nm to 200 nm.

30. A method of treating a burn wound in a patient in need thereof, the method comprising

administering a pharmaceutical composition of isolated microvesicles directly to the burn wound of the patient,

wherein the isolated microvesicles are prepared comprising the steps of:

(a) obtaining culture medium conditioned using human bone marrow-derived mesenchymal stem cells, the culture medium containing microvesicles;

(b) precipitating the microvesicles by adding polyethylene glycol (PEG) to the biological fluid;

(c) washing the precipitated microvesicles to remove the PEG;

(d) isolating the microvesicles to obtain isolated microvesicles; and

(e) providing a therapeutically effective amount of the isolated microvesicles as a pharmaceutical composition,

wherein the precipitated microvesicles range in size from 2 nm to 5000 nm,

wherein the isolated microvesicles result in increased migration of non-proliferating cultured fibroblasts in a cell migration assay when administered to the non-proliferating cultured fibroblasts, and

wherein administering the pharmaceutical composition of isolated microvesicles to the burn wound of the patient results in in regeneration of at least one skin tissue in the burn wound of the patient.

31. The method of claim 30, wherein the burn wound is a full-thickness burn.

32. The method of claim 30, wherein the burn wound is a second degree burn.

33. The method of claim 30, wherein the isolated microvesicles have a molecular weight of at least 100 kDa.

34. The method of claim 30, wherein the mesenchymal stem cell is autologous in origin.

35. The method of claim 30, wherein the mesenchymal stem cell is allogeneic in origin.

36. The method of claim 30, wherein administering a pharmaceutical composition of isolated microvesicles directly to the burn wound of the patient is by local injection.