Orthotopic grafting of cryopreserved prepubertal testicular tissue: in search of a simple yet effective cryopreservation protocol

Objective To investigate whether solid-surface vitrification (SSV) is an effective cryopreservation strategy regarding the integrity and function of prepubertal mouse testicular tissue. Design Prospective experimental study. Setting Academic research unit. Animal(s) Mice. Intervention(s) Testicular tissue from 5- to 10-day-old GFP+ mice was cryopreserved with the use of a conventional uncontrolled slow freezing (USF) technique and SSV before intratesticular grafting in busulfan-treated GFP− mice. Main Outcome Measure(s) Ultrastructural cryoinjury to spermatogonial stem cells (SSCs) and somatic cells was assessed by electron microscopy. Tubular structure was evaluated by histology, and graft survival and spermatogenic recovery by immunohistochemistry. Result(s) The tubular morphology and the proportion of ultrastructural cryodamage were similar between vitrified and slow-frozen testicular fragments. Allografting of tissue after both USF and SSV resulted in a recovery of spermatogenesis similar to fresh samples. Conclusion(s) SSV resulted in success rates similar to USF in maintaining testicular cell ultrastructure, tubular morphology, and tissue function. These data provide further evidence that vitrification, being an inexpensive and simple technique, can be considered as an alternative for cryopreservation of prepubertal testicular tissue.