37 Viability of sheep skin fibroblasts after vitrification

Abstract

Both tissue and cell cryopreservation can be applied for biodiversity conservation. The proper preservation of tissues and cells from a wide range of animals of 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 vitrification on viability of vitrified sheep fibroblasts for conservation of biodiversity so that it might be used in the future to provide nuclear donors. Skin samples collected from 10 adult sheep were cut into small pieces (1 × 1 mm), placed into culture Petri dishes containing DMEM supplemented with 20% (vol/vol) fetal bovine serum, and covered with coverslips followed by incubation at 5% CO₂, 95% RH, and 37°C. During culture, fibroblasts left skin samples and proliferated. Culture medium was changed every 4 days. After 21 to 22 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 PBS + 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 DMEM +  20% ethylene glycol, 20% dimethylsulfoxide, 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 with modelling clay and plunged into LN. Viability of frozen-thawed fibroblast samples was detected using the Trypan Blue staining method (frozen-thawed: 53.0 ± 2.6%; control (fresh): 98.5 ± 1.2%). 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 cryopreserved fibroblasts. Importantly, our data suggest that the use of vitrification reduced the toxic elements contained in the cryopreservation solution while maintaining a similar ability to produce viable fibroblasts after cryopreservation. Although further work on the viability of sheep skin fibroblast with the vitrification method is needed, these data suggest that with vitrification a faster cooling rate and high level of cryoprotectants are able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving sheep fibroblasts.