Cryopreservation of Skin Fibroblasts of the Snow Leopard (Panthera Uncia) From Kazakhstan: Passive Slow Freezing Versus Controlled-Rate Freezing With Dimethyl Sulfoxide or Ethylene Glycol

The snow leopard (Panthera uncia) is a Vulnerable felid, and cryobanking of its somatic cells is a practical way to preserve the genetic diversity of the Kazakhstan population. We compared the post-thaw viability of snow leopard skin fibroblasts after two slow-freezing protocols. Primary fibroblast lines were established from ear-skin biopsies of 16 animals and cryopreserved at passage 5. In experiment 1, cells were frozen passively in cryovials placed in a polystyrene block at −70 °C (about 1 °C/min) in fetal bovine serum with 10% dimethyl sulfoxide (DMSO). In experiment 2, cells were frozen in 0.5 mL straws in a programmable freezer with 1.5 M DMSO or 1.5 M ethylene glycol (EG). Viability was assessed by trypan blue exclusion. Passive freezing reduced viability from 98.5 ± 0.3% to 43.7 ± 12.5% (p < 0.001). Controlled-rate freezing yielded 57.9 ± 9.4% viable cells with DMSO and 62.9 ± 8.9% with EG; the difference between the two cryoprotectants was not significant (p = 0.13). Both controlled-rate variants outperformed passive freezing, by 14.2 and 19.2 percentage points (one-way ANOVA, F(2,45) = 14.7, p < 0.001; Tukey test, p ≤ 0.001). Thawed cells reattached, proliferated and reached confluence within 7 days. Controlled-rate freezing in straws is recommended for the snow leopard somatic cell bank, and DMSO and EG can be used interchangeably. The resulting fibroblast collection forms the first somatic cell cryobank of snow leopards in Kazakhstan.