Activating adipose-derived stem cells for transplantation

1. A method of preparing an adipose-derived mesenchymal stem cell preparation for treating a bone, ligament, tendon or cartilage injury in an animal, comprising:

a. collecting adipose tissue from a source animal, said adipose tissue including fat, connective tissue and mesenchymal stem cells;

b. contacting said adipose tissue with an enzyme preparation comprising 55-65 wt % collagenase from Clostridium histolyticum and 35-45 wt % protease from Bascillus thermoproteolyticus followed by incubation with a cell extraction medium;

c. isolating mesenchymal stem cells after step (b);

d. incubating isolated mesenchymal stem cells from step c with a platelet rich plasma derived from the source animal to form a mixture;

e. photoactivating the mixture from step d; and

f. administering the mixture to the animal.

2. The method of claim 1 further including providing a biologically inert protein stabilizer in said enzyme preparation wherein said biologically inert protein stabilizer is selected from a group of protein stabilizers consisting of a neutral salt, a sugar, sodium chloride, calcium chloride, glucose, fructose, sucrose, trehalose, mannose, dextrose mixtures thereof.

3. The method of claim 2 , wherein d further comprises:

(1) collecting blood from said source animal;

(2) preparing the platelet rich plasma; and

(3) mixing the platelet rich plasma from 2 with the isolated mesenchymal stem cells from c.

4. The method of claim 3 wherein said platelet rich plasma is prepared by separating said blood into a plasma layer and a blood cell layer, collecting said plasma layer, separating said plasma layer into a platelet poor plasma layer and a platelet rich plasma and collecting said platelet rich plasma.

5. The method of claim 4 , further comprising activating said platelet rich plasma by mixing said platelet rich plasma with an activator solution including calcium chloride.

6. The method of claim 5 , further comprising mixing said platelet rich plasma with the activator solution prior to f.

7. A method of treating an animal for a bone, ligament, tendon or cartilage injury, comprising:

administering a treatment animal a composition including a therapeutically effective amount of said stem cell preparation of claim 6 .

8. The method of claim 7 wherein said source animal is said treatment animal.

9. The method of claim 1 , wherein said enzyme preparation comprises about 60 wt % collagenase from Clostridium histolyticum and about 40 wt % protease from Bascillus thermoproteolyticus.

10. The method of claim 9 further including providing a biologically inert protein stabilizer in said enzyme preparation wherein said biologically inert protein stabilizer is selected from a group of protein stabilizers consisting of a neutral salt, a sugar, sodium chloride, calcium chloride, glucose, fructose, sucrose, trehalose, mannose, dextrose and mixtures thereof.

11. The method of claim 10 , wherein d further comprises:

(1) collecting blood from said source animal;

(2) preparing the platelet rich plasma; and

(3) mixing the platelet rich plasma from 2 with the isolated mesenchymal stem cells from c.

12. The method of claim 11 wherein said platelet rich plasma is prepared by separating said blood into a plasma layer and a blood cell layer, collecting said plasma layer, separating said plasma layer into a platelet poor plasma layer and a platelet rich plasma and collecting said platelet rich plasma.

13. The method of claim 12 , further comprising activating said platelet rich plasma by mixing said platelet rich plasma with an activator solution including calcium chloride.

14. The method of claim 13 , further comprising mixing said platelet rich plasma with the activator solution prior to f.

15. A method of treating an animal for a bone, ligament, tendon or cartilage injury, comprising:

administering to a treatment animal a composition including a therapeutically effective amount of said stem cell preparation of claim 14 .

16. The method of claim 15 wherein said source animal is said treatment animal.