METHOD FOR EXTRACTING MESENCHYMAL STEM CELLS FROM ORBITAL ADIPOSE TISSUE Russian patent published in 2017 - IPC C12N5/00 C12N5/95 C12N5/22 

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, tissue engineering, specifically to the allocation of mesenchymal stem cells (MSC) from orbital adipose tissue (OAT), and can be used in medicine. Method comprises grinding of OAT into fragments, cleavage of fragments with collagenase solution, sedimentation of cells by centrifugation for 5 minutes, their transferring into a plastic culture flask. Cleavage of OAT fragments is carried out with a solution of the second type collagenase in a balanced Hanks solution at a ratio of 1 mg/ml to 0.1 g of adipose tissue at a temperature of 37 °C in a shaker for 60 minutes. Resulting suspension is centrifuged at 1,300 rpm, then the fat fraction together with the cells precipitated to the bottom are transferred into a plastic culture flask, which is filled with 5 ml of nutrient medium having the following composition: 45 ml of the DMEM/F12 medium, 5 ml of fetal calf serum, 2 mmol of L-glutamine, 100 µl of a mixture of antibiotics, consisting of 10,000 IU/ml penicillin, 10,000 µg/ml streptomycin, 25 µg/ml amphotericin, and cultivate in CO₂-incubator at 37 °C and 5 % CO₂ in the atmosphere, on the second day when changing the nutritional medium in the fresh one of the same composition carry out the removal of erythrocytes and other cells of the stromal vascular fraction, and fragments of adipose tissue floating on the surface.

EFFECT: invention allows higher uniformity and quality of producing the higher-purity MSC population, effectively use the original tissue material.

1 cl, 2 dwg, 2 ex