METHOD FOR OBTAINING BIOSAFE CULTURE OF MESENCHYMAL STEM CELLS FROM GELATIN OF WHARTON OF HUMAN UMBILICAL CORD

摘要

FIELD: biotechnology.;SUBSTANCE: invention relates to medicine, in particular to biotechnology, and can be used to obtain a biosafe culture of mesenchymal stem cells from gelatin of Wharton of human umbilical cord. This method includes isolation of primary culture of mesenchymal stem cells from gelatin of Wharton of umbilical cord tissue after cesarean section. Moreover, during the release of mesenchymal stem cells, the umbilical cord is freed from blood residues and washed in Versene solution with the addition of 100 U/ml of penicillin antibiotics, 100 mcg/ml of streptomycin, 0.25 mcg/ml of amphotericin for 1 hour at room temperature on a shaker, veins are cannulated from both sides and washed with saline (0.9 %). Disaggregation of the tissue is carried out with a solution containing 0.1 % collagenase type 2 and 0.15 % collagenase type 4 prepared in DMEM/F12 at a ratio of 1:1, incubated at 37 °C for 30 min, the separated cells are collected. It is further centrifuged at 800 rpm for 15 minutes to obtain a precipitate of cells, which is resuspended in DMEM/F12 growth medium containing 10 % fetal bovine serum, 100 U/ml of penicillin, 100 mcg/ml of streptomycin, 2 mM of glutamine, 1 mM of sodium pyruvate. Then, the precipitate of cells is resuspended in DMEM/F12 growth medium at a 1:1 ratio, transferred to culture flasks and cultured in atmosphere containing 5 % CO₂ before the formation of 3/4 monolayer, changing the growth medium 3 times a week. When reaching 3/4 of the monolayer, the cells are subcultured, obtaining the MSC culture, and after 1 passage, a part of the cells is used to detect the presence of AIDS, hepatitis B and C viruses and mycoplasma in the culture by PCR and in the case of a positive result of the test to detect these infections the culture is rejected.;EFFECT: use of this method allows to increase the yield and viability of mesenchymal stem cells obtained from gelatin of Wharton of human umbilical cord.;1 cl, 1 tbl