title: Recovery of fibroblast-like cells from refrigerated goat skin up to 41 d of animal death.
abstract: Successful cloning of animals using somatic cell nuclear transfer requires undamaged nuclear DNA from desired donor cell types. In vitro culture of cells is one way of ensuring nuclear integrity. The goal of this study was to evaluate the limits of postmortem cell survival/culture in refrigerated goat ear skin tissues which could be used for long-term storage and cloning of animals in future. To achieve this, 60 explants from 6 different goats were cultured after 0, 3, 6, 9, 13, 16, 20, 23, 27, 30, 33, 37, and 41 d postmortem and observed under inverted microscope for outgrowth of fibroblast-like cells, after 10-12 d of culture. Explants from all time points including 19% from 41-dpm tissues exhibited outgrowth. However, the percentage of outgrowth positive explants, as well as culture confluence, reduced with increasing postmortem time interval. Cell cultures established from primary outgrowth of 41-dpm tissues when compared for their growth profile with similarly obtained 0-dpm cultures revealed similar growth curve and cell morphology. Cytogenetic analysis of 41-dpm tissue-derived cell populations revealed a normal female karyotype with 60 XX homologous chromosomes indicating genetic stability of the cell population. In conclusion, these results show that refrigerated skin tissue remains alive for more than a month and that the cells derived from such tissues are normal and can be cryopreserved for long-term storage and future cloning of animals with desired genetics.
authors.affiliation: Animal Science Division, Agricultural Research Station, Fort Valley State University, 1005 State University Drive, Fort Valley, GA, 31030, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Okonkwo
authors.forename: Mahipal
authors.initials: M
authors.lastname: Singh
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods; D019976:Cloning, Organism / Q000379:methods; D015925:Cryopreservation / Q000379:methods; D020732:Cytogenetic Analysis; D005347:Fibroblasts / Q000166:cytology*; D006041:Goats; D012867:Skin / Q000166:cytology*; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors