55 Viability of Altai lynx (Lynx lynx wardi) skin fibroblasts after slow freezing

Cell cryopreservation can be applied for biodiversity conservation of wild animals. The proper preservation of cells from a wide range of animals of wild different species is of paramount importance because these cell samples could be used to reintroduce lost genes back into the breeding pool by somatic cell cloning. The aim of this work was to investigate the effect of different cryoprotectants on viability of frozen-thawed Altai Lynx (Lynx lynx wardi) fibroblasts for conservation of biodiversity so that it might be used in the future to provide nuclear donors (Scientific program BR10965224, Ministry of Education and Science Republic of Kazakhstan). Skin samples collected from three adult Altai Lynx (Lynx lynx wardi) were cut into small pieces (1 × 1 mm), placed into culture Petri dishes containing DMEM supplemented with 20% (v/v) fetal bovine serum (FBS), and covered with coverslips followed by incubation at 5% CO₂ and 95% relative humidity at 37°C. The study needs to be repeated with a larger sample size to confirm our preliminary findings. During culture, fibroblasts left skin samples and proliferated. The culture medium was changed every four days. After 21 to 22 days of incubation, a fibroblast monolayer was observed, the culture medium was removed, and cells were incubated for 7 to 10 min in presence of Dulbecco’s phosphate buffered saline + 0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300 g for 10 min. For cryoconservation, fibroblasts were then diluted at a concentration of 2 × 10⁶ cells mL⁻¹ in DMEM + 20% FBS and 10% dimethyl sulfoxide or 10% ethylene glycol and placed into 0.25-mL plastic straws or 2-mL cryovials. Straws were sealed with modelling clay and maintained at +5°C for 120 min before freezing. Cryopreservation of fibroblasts was carried out by the following procedures: straws were frozen in a programmable freezer (Kryo 360-3.5, Planer Limited) using the following freezing regimen: +5°C to −40°C at −1°C min⁻¹, −40°C to −85°C at −4°C min⁻¹, and then plunged into liquid nitrogen for storage. Samples were thawed for 1 min in a 37°C water bath. Frozen-thawed samples were diluted with DMEM (1:5) and centrifuged at 300 × g for 7–10 min. Supernatants were removed, and cells were diluted with DMEM at a concentration of 2 × 10⁶ cells mL⁻¹. Viability of frozen-thawed fibroblasts was detected using the Trypan Blue staining method. The values obtained (Table 1) are expressed as mean standard error of the mean (s.e.m.). Statistical analysis was done using Student’s t-test. Results indicated that there was a significant difference in viability between fresh and cryopreserved fibroblasts (ac P < 0.05); however, there were no differences between the different cryoptotectants (ab P > 0.05). Further research is needed on cryoconservation of somatic cells of Altai lynx (Lynx lynx wardi).

Table 1. Viability of frozen-thawed fibroblast cells