ISOLATION AND USE OF AUTOGENOUS PLATELET DERIVED EXOSOMES
Disclosed are compositions and methods for the isolation of platelet derived exosomes. In some aspects the exosomes may be used to treat chronic orthopedic conditions without causing inflammation, may include extracting platelet-rich plasma (PRP) from whole blood; isolating platelets from the PRP; activating the platelets to induce the release of the exosomes; and isolating the exosomes by differential centrifugation.
1 . A method of isolating platelet derived exosomes, the method comprising:
a) extracting platelet-rich plasma (PRP) from whole blood;
b) isolating platelets from the PRP;
c) activating the platelets to induce the release of the exosomes; and
d) isolating the exosomes.
wherein the resultant exosomes have reduced red blood cells and leukocytes relative to whole blood to allow for use in the treatment of chronic orthopedic conditions without causing inflammation.
2 . The method of isolating platelet derived exosomes of claim 1 , wherein the PRP is extracted by density gradient centrifugation, differential centrifugation, or elutriation.
3 . The method of isolating platelet derived exosomes of claim 1 , wherein to extract the PRP from the whole blood, the whole blood is centrifuged at 250×g for 15 min at least one time.
4 . The method of isolating platelet derived exosomes of claim 1 , wherein the extracted platelet rich plasma comprises at least 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 1.1×10 6 , 1.2×10 6 , 1.3×10 6 , 1.4×10 6 , 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , or 2×10 6 platelets per microliter.
5 . The method of isolating platelet derived exosomes of claim 1 , wherein the platelets are isolated from the PRP by applying the PRP to a centrifuge at 250×g for 15 min at least one time.
6 . The method of isolating platelet derived exosomes of claim 1 , wherein the isolated platelets are activated by contacting the isolated platelets with CaCl 3 , thrombin, collagen, lipopolysaccharide, and/or Ca 2+ ionophores.
7 . The method of isolating platelet derived exosomes of claim 1 , wherein the released exosomes are separated from the platelets by filtration prior to isolating the exosomes.
8 . The method of isolating platelet derived exosomes of claim 1 , wherein the exosomes are isolated by centrifugation with a 30% sucrose-D2O cushion and ultra-centrifuged at 100,000×g for about 70 min.
9 . The method of isolating platelet derived exosomes of claim 1 , wherein the wherein the number of red blood cells and leukocytes contaminating the isolated platelet rich plasma derived exosomes is at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 -fold reduced relative to whole blood.
10 . A platelet rich plasma derived exosome preparation made by the method of claim 1 and comprising 100, 200, 300, 400, 500, 600, 700, 800, 900, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 -fold fewer red blood cells, lysate, and/or leukocytes relative to whole blood.
11 . A method of treating an orthopedic and spine pathology by administering to a subject a therapeutically effective amount of the isolated exosomes prepared by the method of claim 1 or administering to the subject a therapeutically effective amount of the exosome preparation of claim 10 .
12 . The method of treating an orthopedic and spine pathology of claim 11 , wherein the orthopedic or spine pathology comprises osteoarthritis, injured soft tissue, degenerated disc, or ankylosing spondylitis.