82 VIABILITY OF SHEEP-SKIN FIBROBLASTS AFTER SLOW FREEZING

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 different cooling rates on viability of frozen–thawed sheep fibroblasts for conservation of biodiversity so that it might be used in the future to provide nuclear donors (Table 1). Skin samples collected from 10 adult sheep were cut on 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, 95% relative humidity, 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 presence of Dulbecco’s phosphate buffered saline + 0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300g for 10 min. For cryoconservation, fibroblast samples 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 modeling clay and maintained at +5°C for 120 min before freezing. Cryopreservation of fibroblasts was carried out by 2 procedures: (1) straws were frozen in programmable freezer Kryo Planer 360-3,5 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; (2) cryovials were placed in a Styrofoam box and loaded into a freezer at –70°C for 24 h, and then samples were 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 300g 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 frozen–thawed fibroblast samples was detected using the Trypan Blue staining method. The values obtained (Table 1) are expressed as mean standard error of the mean (SEM). Statistical analysis was done using Student’s test. Results indicated that there was a significant difference in viability between fresh and cryopreserved fibroblasts. However, there were no differences between the cooling procedures. Importantly, our data suggest that the use of 1.5-M ethylene glycol reduced the toxic elements contained in the cryopreservation solution while maintaining a similar ability to produce viable fibroblasts after cryoconservation. Table 1.Effect of 2 cryoprotectant agent (CPA) on the viability of frozen–thawed ovine fibroblasts