Особливості клітинного циклу мезенхімальних стовбурових клітин з жирової тканини собаки за різних пасажів культивування

Abstract

The features of the cell cycle of culture of adipose-derived mesenchymal stem cells from the for different cultivating passages were studied. Mesenchymal stem cells were obtained from the adipose tissue of the dog under a laminar flow hood by an explant method in our modification. Cell cultivation was carried out at 37 °C, 100% moisture and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic. The culture medium was changed 2–3 times per week and the cells were selected by their capacity to attach to the flask surface. When culture flasks became 80% confluence, cells were detached with 0.25% trypsin containing 1 mmol/L EDTA and subsequently replayed at a concentration of 104 cells/cm2 for next passaging. A cells culture of adipose derived mesenchymal stem cells was obtained on the 2nd, 7th and 12th passages. The method of flow cytometry determined the level of aneuploid cells and the distribution in the cell cycle phases. The morphology of cells of different passages was studied using an inverted microscope Axiovert 40. It was investigated that the culture of mesenchymal stem cells from adipose tissue in the 2nd passage contains a significant number of the proliferative pool (S + G2/M) cells and it was 29.51 ± 3.56% of the total number of diploid cells. The number of aneuploid cells was 1.55 ± 0.43%. All cells had fibroblast-like morphology. It was established that in the middle passages (7th) in the culture of mesenchymal stem cells from the adipose tissue of the dog no significant changes were found in the distribution of cells in the phases of the cell cycle. The number of diploid cells of the proliferative pool S + G2/M and the G0/G1 pre-synthetic period remains unchanged. The level of aneuploidy increases only within the tendency. Morphologically, cells had fibroblast-like form. It was determined on 12th passage of cultivation, a significant decrease in the number of cells of the proliferative pool (S+G2/M), which was 18.93 ± 0.66% of the total number of diploid cells compared to the 2nd passage. The number of aneuploid cells increased and it was 3.49 ± 0.38%. Morphologically, separate cells had processes. The indicator of the effect of cells cultivation on the content of diploid cells of the proliferative pool (S+G2/M) in culture is ɳ2x = 70% (P < 0.05). So, first characteristic properties of the aging of the culture of canine adipose-derived mesenchymal stem cells appear on the 12th passage of cultivation.