Most of modern mammalian cell cryopreservation protocols use dimethyl sulfoxide solutions as cryoprotectant. That is why we studied the effect of dimethyl sulfoxide on cell culture quality after interaction and cryopreservation. Dermal papilla vibrissa is a source of pluripotent cells neural crest derivatives. Because of pluripotency this type of cell is of interest for transplantation, which is associated with the need for their long-term cultivation and storage. In this work, we studied the pathological changes in the cells of the diploid culture of the dermal papilla of the rabbit vibrissa that occur at the interphase and persist for 2 passages after exposure with cryoprotective media based on different concentrations of dimethyl sulfoxide. Material and methods. The cell culture of dermal papilla was obtained by the method of explants from hair follicles of vibrissa of newborn rabbits. Passaging of received adhesive cultures was performed every 7-10 days. Contact with dimethyl sulfoxide was performed for 20 minutes, after which the cells were placed in standard culture medium for further growth. The calculation of interphase pathologies was carried out on fixed cytological preparations stained with hematoxylin-eosin. Results and discussion. The incubation in a cryoprotective medium containing 5 and 7.5% was safe from the point of view of inducing cytological disorders. Dimethyl sulfoxide in concentrations exceeding 10% led to a dose-dependent increase in cell pathologies. Among the pathologies, violations most often occurred in the association with defects in membranes and the cytoskeleton such as vacuolization of the cytoplasm, the formation of microvesicles on the membrane. There were also disorders affecting the genetic and protein-synthesizing apparatus: polyploid, multinucleated cells, cells with abnormal nuclei, with micronuclei, at various terminal stages of apoptosis. Based on a comparison of cultures at passages 1 and 2 after exposure to dimethyl sulfoxide, it was found that the detected violations were not eliminated during cultivation, but were stored and accumulated. Conclusion. The study showed that protein-peptide additives were capable of exerting a protective effect on the background of high concentrations of dimethyl sulfoxide, which contributed to the preservation of cell viability, but led to the accumulation of anomalies in the culture during further cultivation.
Full text: PDF (Rus) 390K