Development of a cryopreservation protocol for testicular interstitial cells with the account of temperature intervals for controlled cooling below −60 °С

A long course of anticancer therapy may lead to testicular steroidogenesis destruction. Cryopreservation of testicular interstitial cells (TIC) would be a strategy to protect hormonal and fertile potential of pre-pubertal boys treated with chemo – or radiotherapy. The aim of this research was to optimize protocols for freezing of TIC. Essential physical processes associated with the presence of dimethyl sulphoxide (Me2SO) in the cryoprotectant solution take place at the temperatures below −60 °С. These processes are the eutectic crystallization at the stage of freezing and the recrystallization before the melting of the eutectic mixture at the stage of heating. Both of the processes affect the viability of the cells subjected to cryopreservation. Temperature intervals when these processes take place were determined by the method of thermoplastic deformation for 10% Me2SO selected for cryopreservation of TIC. Rat TIC were cryopreserved using five different protocols which varied in cooling rates within the chosen temperature intervals. Post-thaw cell viability and metabolic activity were evaluated by Trypan Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining assays. Leydig cell recovery after cryopreservation was measured by 3-beta-hydroxysteroid dehydrogenase reaction. Based on the obtained results, the authors developed a cryopreservation protocol for TIC which makes it possible to achieve great cell viability due to using controlled cooling rates within the temperature intervals below −60 °С.