PS9-20. Evaluation of Post-cryopreservation Recovery of Mammalian Cells Frozen at Four Different Temperatures for a Month.

Abstract Effective cryopreservation of biological samples is vital for species conservation, especially at a time when climate change is visible. It safeguards the genetic material of cells, tissues, sperms, eggs and embryos from extinction, which can be used for breeding or cloning in future or to rescue endangered species. Traditionally, biological samples are stored in liquid nitrogen (LN2; -196 °C) in media containing cryoprotecting agents. But maintaining LN2 tanks is prohibitive at smaller academic institutions. It is known that lowering temperature increases shelf life of biological samples, however, the effect of different freezing temperatures on recovery of cells is not precisely known. The objective of this study was to evaluate the effect of different cryopreserving temperatures on post-freezing recovery of postmortem tissue derived primary fibroblast cells in goat and sheep. We cultured 2-3 mm2 skin explants (n = 10) from postmortem goat and sheep ears separately in DMEM media supplemented with 10% FBS, 50 units/mL of penicillin, 50 µg/mL of streptomycin and 2.5 µg/mL of fungizone. Outgrowing cells were recovered, washed, and dispensed in cryovials (50,000 live cells/vial) in 1.0 mL media with 10% DMSO. The cryovials were stored at 4 different freezing temperatures (-20 °C, -80 °C, LN2, and -80 °C/LN2), in triplicate, for each group. After four weeks the cells were thawed at 37 °C, cultured in 12-well microtiter plates in 5% CO2 incubator, and analyzed for cell recovery by measuring comparative growth confluence and the # of live cells recovered. Mean ± SD of live cells recovered after 5 days of in vitro culture in goat 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 and in sheep was 0.50 ± 0.10 x 105, 126.67 ± 30.24 x 105, 10.00 ± 12.17 x 105 and 158 ± 56.03 x 105 cells / mL for -20 °C, -80 °C, LN2, and -80 °C /LN2 group, respectively. Recovery was significantly higher in -80 °C/LN2 and -80 °C groups (p < 0.05) compared to LN2 and -20 °C groups in both species. Recovery was least upon freezing cells directly into LN2. These results agree with cell confluence images, captured prior to recovering cells. We conclude that mammalian cells can be effectively cryopreserved at -80 °C rather than requiring costly maintenance of LN2 system, although highest recovery was through step cooling (-80 °C overnight followed by LN2).