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.