What is the best cryopreservation protocol for human testicular tissue banking?

Is there a better alternative to the conventional cryopreservation protocols for human testicular tissue banking? Uncontrolled slow freezing (USF) using 1.5 M dimethylsulphoxide (DMSO) and 0.15 M sucrose as cryoprotectants appears to be a user-friendly and efficient method for the cryopreservation of human testicular tissue. Currently, time-consuming controlled slow freezing (CSF) protocols that need expensive equipment are commonly used for human testicular tissue banking. USF and vitrification are cryopreservation techniques that were successfully applied in several animal models but need further exploration with human tissue. Fragments (n 160) of testicular tissue from 14 patients undergoing vasectomy reversal were assigned to a fresh control group or one of the following cryopreservation procedures: CSF using DMSO at a concentration of 0.7 or 1.5 M in the presence (S) or absence of sucrose (S), USF using either 0.7 or 1.5 M DMSO combined with sucrose, solid-surface vitrification (SSV) or direct cover vitrification (DCV). Light microscopic evaluations were performed to study apoptosis, germ cell proliferation ability, spermatogonial survival, coherence of the seminiferous epithelium and integrity of the interstitial compartment after cryopreservation. Ultrastructural alterations were studied by scoring cryodamage to four relevant testicular cell types. The USF 1.5 M DMSO S protocol proved not solely to prevent cell death and to preserve seminiferous epithelial coherence, interstitial compartment integrity, SG and their potential to divide but also protected the testicular cell ultrastructure. A significant reduction in the number of SG per tubule from 21.4 5.6 in control tissue to 4.9 2.1, 8.2 5.4, 11.6 5.1, 8.8 3.9, 12.6 4.4 and 11.7 5.7 was observed after cryopreservation combined with at least one other form of cryoinjury when using CSF 0.7 M DMSO S, CSF 0.7 M DMSO S, CSF 1.5 M DMSO S, USF 0.7 M DMSO S, SSV and direct cover vitrification (DCV), respectively (P 0.001). Supplementary research is required to investigate the effect on tissue functionality and to confirm this studys findings using prepubertal tissue. An optimal cryopreservation protocol enhances the chances for successful fertility restoration. USF, being an easy and cost-effective alternative to CSF, would be preferable for laboratories in developing countries or whenever tissue is to be procured from a diseased child at a site distant from the banking facility. This study is supported by a PhD grant from the Agency for Innovation by Science and Technology and research grants from the Flemish League against Cancer-Public Utility Foundation, the Scientific Research Foundation Flanders, Methusalem grant and the Vrije Universiteit Brussel. E.G. is a postdoctoral fellow of the Scientific Research Foundation Flanders. The authors declare that no competing interests exist.