# US 10,500,231 B2 · Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,500,231 B2 - Document ID: US10500231B2 - Status: Granted - Kind: B2 - Application number: 14/775,382 - Filed: 2014-03-12 - Published: 2016-06-30 - Granted: 2019-12-10 - Earliest claimed: 2013-03-13 - Assignees: University of Miami - Applicants: University of Miami - Inventors: Evangelos V. Badiavas; Arsalan Q. Shabbir; Stephen C. Davis - Web page: https://patentgrep.com/patent/US10500231B2 ## CPC - A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells - A61K 8/981: {of mammals or bird} - A61K 8/983: {Blood, e.g. plasma} - 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) - A61K 35/16: Blood plasma; Blood serum (umbilical cord blood A61K35/51) - A61K 35/22: Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland - A61P 7/06: Antianaemics - A61P 9/00: Drugs for disorders of the cardiovascular system - A61P 17/00: Drugs for dermatological disorders - A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like - A61P 25/00: Drugs for disorders of the nervous system - A61P 35/00: Antineoplastic agents - A61P 35/04: specific for metastasis - A61P 37/00: Drugs for immunological or allergic disorders - A61P 37/02: Immunomodulators - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - A61Q 19/00: Preparations for care of the skin - A61Q 19/08: Anti-ageing preparations - A61K 2800/74: Biological properties of particular ingredients - A61K 2800/86: Products or compounds obtained by genetic engineering - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - A61Q: SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY 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 ### Claim 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. ### Claim 2 (depends on 1) The method of claim 1, wherein the skin wound is a full-thickness wound. ### Claim 3 (depends on 1) The method of claim 1, wherein the skin wound is a burn. ### Claim 4 (depends on 1) 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. ### Claim 5 (depends on 1) The method of claim 1, wherein wrinkle formation or scar formation is reduced in the treated skin wound of the patient. ### Claim 6 (depends on 1) 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. ### Claim 7 (depends on 1) The method of claim 1, wherein the step of washing the precipitated microvesicles comprises centrifugal filtration. ### Claim 8 (depends on 1) The method of claim 1, wherein the isolated microvesicles have a molecular weight of at least 100 kDa. ### Claim 9 (depends on 1) The method of claim 1, wherein the PEG has an average molecular weight of 20,000 Da. ### Claim 10 (depends on 1) The method of claim 1, wherein the PEG is used at a percentage of between 5% and 25%. ### Claim 11 (depends on 1) The method of claim 1, wherein the mesenchymal stem cell is from bone marrow. ### Claim 12 (depends on 1) The method of claim 1, wherein the mesenchymal stem cell is allogeneic in origin. ### Claim 13 (depends on 1) The method of claim 1, wherein the isolated microvesicles have uncorrugated borders when examined by electron microscopy. ### Claim 14 (depends on 1) 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. ### Claim 15 (depends on 2) The method of claim 2, wherein the microvesicles are further processed to isolate microvesicles having a size of 2 nm to 200 nm. ### Claim 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. ### Claim 17 (depends on 16) The method of claim 16, wherein the burn wound is selected from a second-degree burn and a full-thickness burn. ### Claim 18 (depends on 16) 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. ### Claim 19 (depends on 16) The method of claim 16, wherein wrinkle formation or scar formation is reduced in the healed burn wound of the patient. ### Claim 20 (depends on 16) The method of claim 16, wherein the pharmaceutical composition of isolated microvesicles does not provoke a significant inflammatory response in the patient. ### Claim 21 (depends on 16) The method of claim 16, wherein the step of washing the precipitated microvesicles comprises centrifugal filtration. ### Claim 22 (depends on 16) The method of claim 16, wherein the isolated microvesicles have a molecular weight of at least 100 kDa. ### Claim 23 (depends on 16) The method of claim 16, wherein the PEG has an average molecular weight of 20,000 Da. ### Claim 24 (depends on 16) The method of claim 16, wherein the PEG is used at a percentage of between 5% and 25%. ### Claim 25 (depends on 16) The method of claim 16, wherein the mesenchymal stem cell is from bone marrow. ### Claim 26 (depends on 16) The method of claim 16, wherein the mesenchymal stem cell is allogeneic in origin. ### Claim 27 (depends on 16) The method of claim 16, wherein the isolated microvesicles have uncorrugated borders when examined by electron microscopy. ### Claim 28 (depends on 16) 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. ### Claim 29 (depends on 16) The method of claim 16, wherein the microvesicles are further processed to isolate microvesicles having a size of 2 nm to 200 nm. ### Claim 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. ### Claim 31 (depends on 30) The method of claim 30, wherein the burn wound is a full-thickness burn. ### Claim 32 (depends on 30) The method of claim 30, wherein the burn wound is a second degree burn. ### Claim 33 (depends on 30) The method of claim 30, wherein the isolated microvesicles have a molecular weight of at least 100 kDa. ### Claim 34 (depends on 30) The method of claim 30, wherein the mesenchymal stem cell is autologous in origin. ### Claim 35 (depends on 30) The method of claim 30, wherein the mesenchymal stem cell is allogeneic in origin. ### Claim 36 (depends on 30) 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.