Method for generating protein rich conditioned medium

A method of creating a protein rich conditioned medium comprising:

• a) culturing mesenchymal stem cells in a container utilizing a first growth medium,
• wherein the first growth medium comprises:
• i) a serum from about 1 percent to about 10 percent by volume;
• ii) fibroblast growth factor from about 1 ng/ml to about 10 ng/ml;
• iii) an L-cysteine or a glutathione or an N Acetyl Cysteine from about 0.5 mM to about 5 mM;
• and
• iv) a serum free medium up to 99 percent volume and:
• 1) an epidermal growth factor from about 1 ng/ml to about 10 ng/ml;
• 2) a hydrocortisone from about 10 ng/ml to about 100 ng/ml;
• 3) a calcium chloride from about 0.01 mM to about 0.1 mM;
• 4) an insulin from about 0.5 mg/ml to about 5 mg/ml;
• 5) a bovine pituitary extract from about 10 μg/ml to about 100 μg/ml;
• 6) a selenium from about 0.1 μg/ml to about 1 μg/ml;
• 7) a stromal-derived factor from about 1 ng/ml to about 10 ng/ml;
• 8) a sodium pyruvate from about 2 mg/ml to about 20 mg/ml;
• and
• 9) a transferrin from about from about 0.1 mg/ml to about 1 mg/ml;
• b) allowing a time period for proliferation of the mesenchymal stem cells until a desired level of confluence is achieved in the container;
• c) discarding a supernatant from the container;
• d) adding a second serum-free medium or a salt buffered solution to the container;
• e) incubating the mesenchymal stem cells and the second serum-free medium or the salt buffered solution for a desired period of time, thereby creating a conditioned medium;
• and
• f) collecting the conditioned medium;
• and
• wherein the mesenchymal stem cells can be incubated for up to seven days in the second medium without change in mesenchymal stem cell characteristics.