Recovery of fibroblast cells upto 65 days of postmortem storage of sheep ear skin at 4°C

Abstract Text:

Animal cloning technology has renewed interest in tissue storage, since these tissues can be used to reintroduce lost genetics back into the breeding pool in animal agriculture, preserve genetic diversity, and revive endangered species. Several studies have demonstrated that cell survival decreases with increasing postmortem tissue storage. However, the limits of time interval within which live cells can be recovered from animal tissues postmortem, have not been adequately studied. The objective of this study was to evaluate the time limits of cell survival in sheep skin tissues stored at 4°C after the death of the animal. Outgrowth of cells around small tissue explants in cultures was used as a measure of cell recovery. Ear skin was procured from the university slaughter house from six random but healthy animals and stored at 4°C in the lab. We cultured 2-3 mm² explants (n=60) after 0, 10, 20, 27, 30, 35, 38, 41, 45, 50, 55, 60, 65 and 70 days of tissue storage. DMEM media supplemented with 10% FBS and 50 units/mL of penicillin and 50 µg/mL of streptomycin was used.  Twelve dishes (60 mm) for each time point were used. After 10 days of culture in a CO₂incubator, outgrowth of fibroblast-like cells around the explants was scored. Out of 481 explants that adhered to dish surface, 374 exhibited outgrowth. Our results showed outgrowth of cells upto 65 days of postmortem storage. In general, the number of outgrowing cells decreased with increasing postmortem storage time. To test the differences between cell cultures obtained from postmortem fresh and stored tissues, we established secondary cultures from primary cells of 0-dpm and 65-dpm time points from selected cell lines and studied their growth profile (p5 level) which showed similar morphology and growth curves. The karyotype analysis of 65 dpm tissue derived cells revealed a normal female karyotype without any genetic aberrations. Both cultures have been passaged upto 33 times which exhibit similar morphology; however, they grew very slowly. These results suggest that live cells can be recovered from skin tissues of sheep and perhaps other animals for more than 2 months after their death with comparable growth profiles. 

Keywords: Sheep; fibroblast cells; postmortem tissue storage; skin; cell culture