Effect of postmortem time interval on in vitro culture potential of goat skin tissues stored at room temperature

Abstract

Animal cloning using somatic cell nuclear transfer technology has renewed the interest in postmortem tissue storage, since these tissues can be used to reintroduce the lost genes back into the breeding pool in animal agriculture, preserve the genetic diversity, and revive the endangered species. However, for successful cloning of animals, integrity of nuclear DNA is essential. Cell viability and their potential to in vitro culture ensure nuclear integrity. The aim of this study was to determine the limits of postmortem time interval within which live cells can be recovered from goat skin tissues. To test the postmortem tissue storage limits, we cultured 2-3 mm(2) skin pieces (n = 70) from the ears of three breeds of goats (n = 7) after 0, 2, 4, and 6 days of postmortem storage at 24°C. After 10 days of culture, outgrowth of fibroblast-like cells (>50 cells) around the explants was scored. All the explants irrespective of breed displayed outgrowth of cells on the dish containing fresh tissues (i.e., day 0 of storage). However, the number of explants exhibiting outgrowth reduced with increasing time interval. Only 53.85% explants displayed outgrowth after 2 days of tissue storage. The number of explants displaying outgrowth was much smaller after 4 (16.67%) and 6 days (13.3%) of storage. In general, the number of outgrowing cells per explant, on a given day, also decreased with increasing postmortem storage time interval. To test the differences between cell cultures, we established secondary cultures from one of the goats exhibiting outgrowth of cells after 6 days of tissue storage and compared them to similar cells from fresh tissues. Comparison of both the cell lines revealed similar cell morphology and growth curves and had doubling times of 23.04 and 22.56 h, respectively. These results suggest that live cells can be recovered from goat (and perhaps other animal) tissues stored at room temperature even after 6 days of their death with comparable growth profiles and, thus, can be used for tissue banking for preservation of superior genetics, genetic diversity, and cloning of animals.