49 Viability of snow leopard (Panthera Uncia) fibroblasts after vitrification

Skin cells cryopreservation can be applied for biodiversity conservation. The proper cells preservation from a wide range of different wild animal species is of paramount importance because these cell samples could be used to molecular genetic analyses and for animal biotechnology by assisted reproductive technology (ART) and cloning. The aim of this work was to investigate the effect of vitrification on viability of snow leopard (Panthera uncia) fibroblasts for conservation of biodiversity (Scientific program BR18574058, Ministry of Education and Science Republic of Kazakhstan, The United Nations Development Programme in the Republic of Kazakhstan (UNDP) in the framework of the UNDP-GEF Project#00101043 ‘Conservation and sustainable management of key globally important ecosystems for multiple benefits’). Snow leopard (Panthera uncia) is listed as “vulnerable” on the IUCN Red Book, in the Red Book of Kazakhstan, as well as in the protection documents of other countries. Skin samples collected from seven adult animals were cut into small pieces (1 × 1 mm), placed into culture flasks (25 cm²) containing Delbucco’s Modified Eagle Medium (DMEM) supplemented with 20% (vol./vol.) fetal bovine serum, antibiotics 1%, and followed by incubation at 5% CO₂, 95% RH, and 37°C. During culture, fibroblasts left skin samples and proliferated. Culture medium was changed every 5 days. After 25 to 30 days of incubation, a fibroblast monolayer was observed, culture medium was removed, and cells were incubated for 7 to 10 min in the 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 vitrification, fibroblast samples were then diluted at a concentration of 2 × 10⁶ cells mL⁻¹ in vitrification solution (DMEM + 20% ethylene glycol, 20% dimethyl sulfoxide, and 0.5 mol L⁻¹ of sucrose). The fibroblasts were then exposed to 50% and 100% vitrification solution (VS) at 37°C for 5 min and 30 s, respectively. Fibroblasts after saturation in VS were transferred and placed into 0.25-mL plastic straws. Straws were sealed and plunged into LN. 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 to 10 min. Supernatants were removed, and cells were diluted with DMEM at a concentration of 2 × 10⁶ cells mL^–1. Viability of vitrified-thawed fibroblast samples was detected using the Trypan Blue staining method (vitrified-thawed: 49.3 ± 2.8%; control (fresh): 96.1 ± 1.9%). The values obtained are expressed as mean ± standard error of the mean. Statistical analysis was done using Student’s t-test. Results indicated that there was a significant difference in viability between fresh and vitrified fibroblasts. Although further work on the viability of snow leopards skin fibroblast with the vitrification method is needed, these data suggest that vitrification should be further evaluated as a routine mechanism for cryopreservation of snow leopard fibroblasts.