In vitro culture of fibroblast-like cells from postmortem skin of Katahdin sheep stored at 4°C for different time intervals

Abstract

Live animals have been produced recently from animal tissues preserved for decades at frozen temperatures with or without cryoprotectants. However, the tissues in these studies were cryopreserved within few hours of animal death to obtain culturable live cells as nuclear donors. How long the tissues can be left unfrozen after animal death, without losing the viability and potential to in vitro culture with comparable morphology and proliferative rate as the fresh tissues, is not completely understood. To understand this phenomenon, ear skin samples from individual sheep (n=3) were procured from slaughter plant and stored at 4 °C. After various intervals (2, 8, 24, 32, 48, and 56 h after slaughter), 2-3 mm(2) pieces (n=10) of skin samples were cultured for 12 d on two dishes (60 mm) for each sheep. Outgrowth of fibroblast-like cells was observed as early as day 4 of culture and was visible on dishes of all time points including 56 h by day 10. The number of outgrowing cells decreased with increasing time interval between animal slaughter and culture initiation. Secondary cultures were successfully established for all the time points. All cultures proliferated well and were apparently normal. Passage 2 cultures of 2 h and 56 h interval for one of the three sheep were compared for their growth pattern and proliferation rates. The population doubling time of 2 h and 56 h intervals was 33.12 and 34.8 h, respectively, and both the lines exhibited similar cell morphology and an "S"-shaped growth curve. These results suggest that skin tissues of sheep and perhaps other animal species with superior traits are effectively preserved at cellular level at least for 56 h at normal refrigerating conditions, without need of complicated cryopreservatives/cryotanks that are usually not available at small farms.

Use of Postmortem Human Dura Mater and Scalp for Deriving Human Fibroblast Cultures

“…In addition, Hjelm and colleagues generated iPSCs from fibroblasts using skin samples from a single autopsy case of a 75 year-old whole body donor [6]. Additionally, fibroblasts were recently cultured from postmortem ear tissue of sheep [7]. Culturing of postmortem tissue samples offers another avenue of study for human disease.…”

In vitro culture of somatic cells derived from ear tissue of collared peccary (Pecari tajacu Linnaeus, 1758) in medium with different requirements

“…Studies of the property of skin cells have differences in their behavior, as the growth curve in the days of culture under similar culture conditions. In cases of cooled tissue curve, cells reached the stationary phase on day 7 of culture as observed by Singh et al (2011) and Singh & Xiaoling (2014). Nevertheless, Mahesh et al (2012) observed the same culture conditions at the beginning of the stationary phase on day 4 of the curve.…”

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

“…Ear skin was cleaned, first with tape water and subsequently with 70% alcohol swabs, wrapped in clean sterile paper towels, and stored in the laboratory in an incubator set at 35°C. Small slices were excised aseptically from the ear skin, chopped into small explants (2-3 mm Primary outgrowing cells were trypsinized on day 10-12 of culture to recover outgrowing cells as described (Singh et al 2011). Briefly, the cells in dishes were washed twice with 1.0 mL of the balanced salt solution without calcium and magnesium (Gibco, Carlsbad, CA) and incubated with 1.0 mL of 0.125% trypsin for 5-7 min at 37°C followed by neutralization with 5 vol.…”