Recovery of Fibroblast-Like Cells after 160 Days of Postmortem Storage of Goat Skin Tissues in Refrigerated Media

Abstract

Animals have been cloned from frozen decade old postmortem tissues preserved within few hours of animal death. Delay in tissue preservation may reduce cloning success due to compromised nuclear DNA integrity. In vitro culture of cells ensures nuclear integrity and is a preferred method of preparing somatic cells for cloning. However, the time limits of postmortem recovery of cells capable of in vitro culture are not precisely known. Here we show recovery of fibroblast-like cells after 160 days of postmortem storage of goat skin in culture media at 4?C. Forty skin explants were cultured at 10 days interval up to 160 days and the outgrowing fibroblast-like cells around them were observed under inverted microscope. Explants with a cluster of more than 50 cells, after 10-12 days of culture initiation, were considered positive. We observed the outgrowth in all the time points, however, the confluence level reduced with increasing postmortem time interval. Secondary cultures established from primary outgrowth of 0-, 90- and 160-dpm tissues exhibited similar fibroblast-like morphology. Growth curves of 0- and 90-dpm cells were similar but 160-dpm cells grow slightly slower.Cytogenetic analysis performed on twenty G-banded metaphase cells of 160-dpm cell line revealed an apparently normal male goat karyotype with 60 chromosomes and their post-freezing cell-viability was >67%. Potential of using these cells to clone the goats remains to be seen in future. These findings may be useful in decisions for preservation of tissues for future cloning of animals and cell therapy.