The method of obtaining and concentration of micro-RNA-containing exosomes of multipotent mesenchymal-stromal cells for use in cosmetics and medicines to stimulate regenerative processes and slow down the aging process

摘要

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely biotechnology, and can be used to produce and concentrate micro-RNA-containing exosome multipotent mesenchymal-stromal cells. Method involves production of mesenchymal-stromal cells of umbilical cord, adipose tissue and tooth pulp, subsequent cultivation and collection of culture fluid enriched with exosomes, concentration of exosomes and depletion of large molecular proteins in order to reduce sensitization, freezing or drying of the obtained concentrate for long-term storage. Exosome source used is multipotent mesenchymal stromal cells (MMSC) 1 to 4 passage obtained from tested donors, wherein the cell sources are a dental pulp, adipose tissue, bone marrow, umbilical cord, to accumulate exosomes, MMSC are cultured in medium with low-glucose alpha-MEM or DMEM, 5 mM L-alanyl-glutamine and activated platelet plasma urea-cord plasma (UCPRP), culturing the cells in the medium described above is carried out up to 80–100 % of the monolayer of the culture, wherein culturing can be static or flowing: in static cultivation, cells are placed in a culture dish such as a bottle and a petri dish. When monolayer reaches 80–100 %, the culture fluid is changed every 24 hours, the collected culture medium is stored for exosome release, wherein from one culture is 5–7 removable medium, and in flow cultivation cells are placed in a flow type incubator on cellular carriers, such as microparticles, hollow-fiber structures, when cells reach the monolayer, the culture medium is completely changed to a new one, after which standard cultivation is performed for 7 days. When obtaining medium enriched with exosomes, its concentration is carried out with removal of protein fraction of more than 100 kDa, using a system of tangential filtration to remove coarse particles and concentration: first, the culture medium is centrifuged for 30 minutes at 2000g or filtered by 1–10 mcm filter to remove large cell debris, then the supernatant is filtered by a tangential filter with pores of 100 kDa or 500 kDa. As a result of filtration volume of liquid is reduced, and exosomes are concentrated in diafiltrate, the latter is further filtered by ultrafiltration to remove micro vesicles and other coarse particles using filters with size of 0.22 mcm and 0.1 mcm, obtained filtrate contains exosomes, which are measured by flow cytometry and measurement of RNA level or by other available methods. Obtained exosomes can be used for 2 weeks unchanged to produce cosmetic or dosage forms, can be frozen at -20 °C, -40 °C, -60 °C and -80 °C and stored for 6 months, or subjected to lyophilisation for long-term storage: up to 1 year at room temperature and more than 2 years at negative temperatures or +4 °C.EFFECT: use of the given method enables to obtain and concentrate micro-RNA-containing exosomes of multipotent mesenchymal-stromal cells for use in agents for stimulating regenerative processes.1 cl