Cryopreservation of prepubertal mouse testicular tissue by vitrification

ObjectiveTo compare cryopreservation of prepubertal testicular tissue after vitrification (V) and slow-freezing (SF).DesignProspective experimental study.SettingAcademic research unit.Animal(s)Six-day-old mice.Intervention(s)After cryopreservation, viability tests (n = 10) and short-term culture (1 and 3 days) (n = 5) were performed. A comparison was made with fresh (FR) and noncultured controls (FR Ctrl).Main Outcomes Measure(s)Tissue viability was assessed by lactate dehydrogenase release assay. Apoptosis (caspase-3) and proliferation (Ki67) were evaluated by immunohistochemistry, and tubular diameter, integrity, and cell density by light microscopy.Result(s)Lactate dehydrogenase release was greater after SF than V (54.6% vs. 26.7%), whereas the mean number of apoptotic cells/tubule was higher after V than SF (2.13 vs. 0.07). On day 1, a decrease in cell density was noted in both cryopreserved groups, but this difference was not subsequently observed. On day 3, an increase in proliferation was seen in the SF and V groups versus FR tissue, and similar tubular diameter, integrity, and cell density were found in all cultured groups.Conclusion(s)This study shows that both SF and V protocols preserve survival, development, and integrity of prepubertal mouse testicular tissue in short-term organotypic culture. Additional investigation should now be conducted to assess tissue functionality.