Testicular cell freezing for fertility preservation in pre-pubertal boys undergoing gonadotoxic therapy

Fertility preservation prior to gonadotoxic therapies is an important emerging field. This technology seeks to safeguard future chances of fatherhood in prepubertal patients facing fertility compromising regimens (e.g. chemo- or radiotherapy) by cryopreserving spermatogonial stem cell (SSC)- containing testicular tissue (TT). This procedure is currently offered as an innovative experimental method. This thesis started with an overview of the efficiency of existing TT and testicular cell suspension (TCS) cryopreservation protocols based on post-thaw recovery of viable cells (RVC) and SSC function. Efficient cryopreservation protocols for human TT exist and these are applied in the clinic. For TCSs, cryopreservation and cell functionality remained suboptimal. We optimized the cryopreservation protocol for TCSs with immature mouse TCSs aiming for a better RVC and fertility restoration after SSCT. The use of an insulate-controlled freezing device and DMEM supplemented with 1.5M DMSO, 10% FCS and 60µM of anti-apoptotic factor (Z-VAD(Oe)-FMK) as cryomedium resulted in 49% survival of SSCs. Next, we translated this protocol to human primary testicular cells. Upon freezing, the highest RVC was achieved by freezing cells using DMEM supplemented with 1M DMSO, 20% serum, 30 µM Z-VAD(Oe)-FMK and 200 mM of trehalose in an insulate-controlled slow freezing device. Controlled-rate thawing improved the viability even more. Unfortunately, due to the difficult replicability of the in vitro propagation culture, no conclusions could be drawn. Finally, we compared the ability to restore fertility by SSCT after cryopreserving TCSs with that after cryopreserving TT. Our results highlighted the superiority of TT cryopreservation, as currently applied, to preserve functional SSCs