Recovery of Proliferative Fibroblast Cells from Refrigerated Sheep Skin after Different Postmortem Time Intervals and their Characterization

Author(s): Mahipal Singh, Xiaoling Ma

Background: Postmortem tissues are a potential source of stem/progenitor cells for cellular therapies, preservation of germplasm and revival of endangered and/or dead species by cloning. How long they can be recovered after animal death, however, is not known precisely. The objective of this study was to evaluate the window of postmortem interval (PMI) within which live and proliferative cells can be recovered from refrigerated sheep skin. Ear skin was procured from animals from slaughterhouse and stored at 4°C in the lab. Small explants (2-3 mm2) were then cultured in DMEM media supplemented with 10% FBS, 50 units/mL of penicillin, 50 µg/mL of streptomycin, and 2.5 µg/mL of fungizone after different PMI. Outgrowth of cells around the explants was scored after 10-12 days of culture at 37°C in a CO2 incubator and cells from selected PMI were sub-cultured for 3-5 times and characterized with respect to their growth profiles, genetic stability, cryopreservation ability and gene expression.

Results: A total of 474 explants adhered to dish surface, of which 369 (77.89%) exhibited outgrowth in various PMI including 34.79% of 65-days postmortem (dpm) interval. We observed recovery of proliferative cells up to a maximum of 65 days of PMI. Percent of explants exhibiting outgrowth as well as relative confluence of outgrowing cells decreased with increasing PMI. Comparative Growth Curves and GFP expression patterns, upon transfection with a GFP plasmid, were not significantly different in 0-dpm and 65-dpm cell populations (p<0.05). Recovered cells cryopreserved with >80% post-freezing cell-viability and were passaged up to 35 times in in vitro cultures. The cytogenetic analysis of 65-dpm tissue derived cells exhibited a normal female sheep karyotype without any genetic aberrations.

Conclusions: These results show that normal proliferative cells can be recovered from sheep skin up to about 2 months postmortem, if tissues are kept refrigerated. To our knowledge this is the first report of recovering proliferative cells from mammalian tissues up to such a long time of >2 months after death. The discovery has potential applications in preserving veterinary and livestock germplasm after death to revive in future by cloning as well as in cellular therapies in human and veterinary medicine.

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