Postmortem recovery of proliferative fibroblasts up to three days in livestock ear skin stored at 35°C

Mahipal Singh,Venkata N Degala

doi:10.5539/ijb.v9n2p9

Abstract

Effect of elevated temperature (35°C) on postmortem survival of cells in goat, sheep and bovine tissues was studied. Skin explants (n = 30; 2-3 mm2) were cultured in petri dishes after 2, 24, 48, 72, 96 and 120 hours of postmortem interval in DMEM media supplemented with 10% FBS, 50 units/mL of penicillin, 50 µg/mL of streptomycin and 2.5 µg/mL of fungizone. Outgrowth of cells around the explants was observed up to 72 h in sheep, 48 h in bovine and 24 h in goats. In general, the number of explants exhibiting outgrowth as well as the level of confluence decreased with increasing postmortem time interval. Secondary cultures established from primary cells for 72 h postmortem interval show cytogenetically stable chromosomes with 54XX[17] normal female sheep karyotype, comparable cell morphology, and growth curve to that of fresh tissue derived cells. Till date these cells have been passaged 21 times and show normal growth. These results suggest that normal, proliferative cells can be recovered from skin tissues stored at an elevated temperature of 35°C in livestock from 24-72 h of postmortem interval. Reprogramming of these cells to clone the dead animals or their use for cell therapies remain to be seen in future.

Highlights

Advances in somatic cell nuclear transfer (SCNT) aka cloning has allowed for the production of live animals from frozen postmortem tissues (Hoshino et al, 2009, Wakayama et al, 2008)
How long the live and proliferative cells can be recovered by culturing them from mammalian tissues after their death and, how different these cells are compared to fresh tissue derived cells, is not precisely known
We show in vitro culture of fibroblasts up to 3 days in livestock skin tissues incubated at 35°C after the animal death, simulating the summer field environment

Summary

Introduction

Advances in somatic cell nuclear transfer (SCNT) aka cloning has allowed for the production of live animals from frozen postmortem tissues (Hoshino et al, 2009, Wakayama et al, 2008). We show in vitro culture of fibroblasts up to 3 days in livestock skin tissues incubated at 35°C after the animal death, simulating the summer field environment. 20,000 cells were cultured in each well in 500 μl of media and the growing cells were harvested and counted in triplicate wells after different time intervals using trypan blue dye exclusion assay (Strober, 2001).

Results

Conclusion