Abstract
Different cell types demonstrate an individual response to the doses of DMSO in freezing media that cannot be calculated empirically. Such situation requires experimental adjustment of the dose of the main cryoprotectant for each cell type. The purpose of the work was to optimize the basic composition of the cryopreservation medium for the rat spermatogonial cell lines, to investigate the time period of the initial storage of the cells at –80° C, as well as the effect of the ratio of volume of the freezing medium to the number of cells on their subsequent survival after recovery. Our data demonstrate that the 8% DMSO is the most effective concentration of the cryoprotectant. Furthermore, we tested different time of initial storage of cryovials in a commonly used “Mr. Frosty” Freezing Container at –80ºC. Our results suggest that 12 hours period (overnight) provides enough time for primary freezing step at –80ºC. Optimization of the volume of freezing medium revealed, that 0.5 ml, compared with 1.0 ml of medium for rat spermatogonial stem cells allows more effective preservation and long-time cryostorage of low numbers of cells (3×104) with successful recovery of up to 50% of the frozen cell population. Additionally, we attempted to improve the freezing medium composition by supplementing its base formulation with sucrose and/or trehalose. The use of optimal concentration of DMSO (8%) in the medium in combination with a 200mM of trehalose resulted in an increase of spermatogonia viability after recovery by more than 12% compared to the original composition of SG (Spermatogonia Growth) medium containing 10% of DMSO.
Highlights
Rats represent the most widely applied non-primate mammals to model disease processes (Chapman et al, 2011; Syvyk et al, 2017)
Taking into account constantly improving freezing medium composition we tested abilities of trehalose and sucrose additives to enhance the viability of rat spermatogonia after recovery from cryopreservation (Buchanan et al, 2004; Lee et al, 2013; Motta et al, 2014; Rodrigues et al, 2008)
Cells suspended in each respective freezing medium at a density of 106 cells/ml were aliquoted into vials, pre-cooled at –80°C for 48 hours and transferred into the liquid nitrogen storage for cryopreservation
Summary
Rats represent the most widely applied non-primate mammals to model disease processes (Chapman et al, 2011; Syvyk et al, 2017). Apart from studying spermatogenesis itself, male germ stem cell can be utilized for generation of random or predetermined genetic alterations and transfer those alterations onto a whole organism via direct germline transmission (Brinster and Nagano, 1998; Chapman et al, 2011; Wu et al, 2012; Syvyk et al, 2018). In this case, a robust and reproducible methodology for primary culture preparation and storage with subsequent line derivation is needed. The purpose of presented study was optimization of freezing medium composition and conditions for cryopreservation of rat spermatogonial stem cell
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