48 Effect of different cryoprotectants and slow freezing on viability of Saiga (Saiga tatarica) fibroblasts

The saiga antelope (Saiga tatarica tatarica) is a member of the Bovidae family, an endangered nomadic antelope found only in the steppes and deserts of Eurasia. In 2002, the International Union for Conservation of Nature (IUCN) classified this species as a critically endangered, which implies development and implementation of an urgent set of measures to conserve the saiga population, among which cryopreservation of animal germplasm is very relevant. The aim of this work was to study effect of various cryoprotectants on viability of frozen–thawed saiga cells in order to increase the efficiency of their cryopreservation to preserve the biodiversity of the animal world (Scientific program BR18574058, Ministry of Education and Science of the Republic of Kazakhstan). Fibroblasts were isolated using skin samples obtained from the auricle of 10 adult Saiga individuals. Skin samples were prerinsed in phosphate-buffered saline for several minutes and then cut with a scalpel under sterile conditions into 1 mm³ pieces in a small volume of culture medium. Next, tissue pieces were placed on the bottom of plastic cell culture flasks with a surface area of 25 cm² and covered with culture medium. Explants and further, cells, were cultured in a CO₂ incubator at 37°C, 5% CO₂ and 85% humidity until a monolayer was reached, replacing half of the volume of the culture medium with fresh one every 3 days. Cell dissociation and separation of monolayer from the surface was performed using a warm 0.25% trypsin solution. Cell viability was assessed using the “trypan blue dye exclusion” method. For balanced cell cryopreservation, the SF method was used with a Planer Kryo 330–3.3 programmable freezer (Planer). Fibroblasts were frozen in 2 different cryoprotectants: 1.5 M dimethyl sulfoxide (Sigma) and 1.5 M ethylene glycol (Sigma), both prepared in Dulbecco’s Modified Eagle Medium. When diluted with a cryoprotectant, a cell concentration of 2 × 10⁶ cells/mL was maintained. For cryopreservation and further storage, 0.5-mL straws (CryoBioSystem) were filled with samples with a cryoprotectant. For freezing, the following regimen was used: from +5°C to −40°C at a rate of −1°C/min, from −40°C to −85°C at a rate of −4°C/min, and then straws were plunged into liquid nitrogen for storage. Samples were thawed in a water bath at 37°C for 5 min. The values obtained (Table 1) are expressed as mean standard error of the mean. Statistical analysis was done using Student’s t-test. Results showed a significant difference in fibroblast viability between fresh and cryopreserved samples (P < 0.05), while no significant difference was observed in viability between different cryoprotectants (P > 0.05).