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Cryopreservation is an important technology to preserve livestock germplasm for future use. How different freezing temperatures affect the post-freezing recovery of cells is not precisely known. In this study, we report the isolation of live fibroblast cells from goat skin after 14 days of animal death and the effect of 4 different freezing temperatures on the post-cryopreservation cell recovery. The primary outgrowing cells were cultured for three passages and cryopreserved at -20°C, -80°C, Liquid nitrogen (LN2) (-156°C), and -80°C overnight followed by storage in LN2 (-80°C/LN2) group. After 4 weeks of storage, cryovials in triplicate for each group were thawed, cultured, and analyzed. Mean and standard deviation of live cells recovered after 5 days of in vitro culture was 1.0 ± 0.71 x 105, 7.60 ± 2.88 x 105, 1.40 ± 0.55 x 105, and 14.80 ± 1.30 x 105 cells/mL for -20°C, -80°C, LN2, and -80°C/LN2 groups, respectively. Recovery of cells stored at -80°C/LN2 group was significantly higher than that in -80°C, -20°C, and LN2 groups (P < 0.05). Recovery was least upon freezing cells directly into LN2 or in -20°C. Although, storing cells in -80°C was significantly higher than that in -20°C and LN2 groups, it was significantly less than that in -80°C/LN2 group (P < 0.05). Based upon these results, we conclud that step cooling (-80°C overnight followed by LN2) was the most effective method for cryopreservation of goat fibroblast and perhaps other mammalian cells. This study highlighted the importance of storing biological tissues and cells at -80°C, especially at small twoand four-year teaching institutions or at limited resource farms, where the cost of maintaining liquid nitrogen supply is prohibitive. [ABSTRACT FROM AUTHOR]

Recovery of proliferative stem cells from postmortem tissues is an important area of research due to its potential applications in cellular therapies in human and veterinary medicine, and preservation of germplasm for future revival of lost genetics. The objective of this study was to assess the postmortem time interval (PMI) within which proliferative cells could be recovered from the tissues of newborn goat kids after their death. Skin explants (3-4 mm2; n = 20) were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% Fetal bovine serum (FBS) after 0, 7, 14, 21, and 28 days of postmortem (dpm) storage at 4°C. Outgrowth of cells was seen only up to 14 days of PMI in all (100%) of explants but not beyond that. Outgrowth started on day 3, 5, and 8 in 0-dpm, 7-dpm, and 14-dpm time intervals, respectively. The level of confluence was inversely corelated with increasing PMI. The average number of cells recovered from each explant was 26.11 x 104, 7.10 x 104, and 2.40 x 104 cells/mL for 0-dpm, 7-dpm, and 14-dpm cultures, respectively. Subcultures of these primary cells, when plated in equal numbers, showed comparable growth profile to that of fresh tissue derived cell populations. This study showed that postmortem tissues retained proliferative cells for much longer time than it was previously thought, and thus, could be preserved even after several days of animal death to recover the lost genetics in future. [ABSTRACT FROM AUTHOR]

A simple agarose gel-based method for evaluating the homology between two DNA molecules has been developed. This procedure involves a simple in vitro assay where gene-targeting plasmid DNA vector and PCR amplified genomic fragment of the desired species are first denatured by heat treatment and later mixed in a solution to achieve gradual renaturation. The renatured DNA molecules are then resolved in an agarose gel to evaluate the degree of homology between desired DNA molecules. [ABSTRACT FROM AUTHOR]

Dimethyl sulfoxide is a preferred solvent for a wide range of compounds. It is used as a solvent of drugs for invitro toxicity testing using cell lines. Determination of safe solvent concentration for cell type to be used is a prerequirement. Here we show that 0.1% DMSO in growth media is the safest concentration for skin fibroblast cell line used in this study. The low concentrations (0.01-0.001%) enhance proliferation of cells when cultured in-vitro on plastic surfaces for a 4-day period, whereas higher concentrations (0.5-3%) lead to reduction in cell-viability on a dose dependent manner. No cell survived beyond 3% DMSO. [ABSTRACT FROM AUTHOR]