| CPC C12N 5/0634 (2013.01) [A61K 35/28 (2013.01)] | 6 Claims |
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1. A method for producing an exosome containing therapeutic agent, comprising:
separating mononuclear cells from a bone marrow sample of a healthy donor;
treating the separated mononuclear cells in a culture media at 37° C. and at 5% CO2 for 48-72 hours;
separating an exosome composition from the culture media; and
dissolving the separated exosome composition in a phosphate buffer to obtain the exosome containing therapeutic agent;
wherein the culture media comprises a 1:1 mixture of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) and N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 2 mmol/mL or 3.65 mg/10mL of L-glutamine, 100 units/mL of penicillin, 100μg/mL of streptomycin, and 10 wt % of fetal bovine serum
wherein separating the mononuclear cells comprises:
i) applying the mononuclear cells in the culture media to a saccharose-epichlorohydrin-copolymer, sodium amidotrizoate and meglumine amidotrizoate solution with a density of 1.077 g/cm3, wherein the ratio of separated mononuclear cells in the culture media to the saccharose-epichlorohydrin-copolymer, sodium amidotrizoate and meglumine amidotrizoate solution is 1:3-1:4, followed by centrifugation for 20 min at 1500 rpm;
ii) collecting an interphase ring with mononuclear cells after centrifugation;
iii) mixing the interphase ring with a Hanks salt solution in a 1:1 ratio and centrifuging the mixture for 3 min at 1500 rpm; and
iv) optionally repeating step iii), wherein after each centrifugation mononuclear cells are separated.
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