Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics

Objective To assess whether testicular cell dynamics are altered during long-term culture after testicular tissue cryopreservation. Design Experimental basic science study. Setting Reproductive biology laboratory. Patient(s) Testicular tissue with normal spermatogenesis was obtained from six donors. Intervention(s) None. Main Outcome Measure(s) Detection and comparison of testicular cells from fresh and frozen tissues during long-term culture. Result(s) Human testicular cells derived from fresh (n = 3) and cryopreserved (n = 3) tissues were cultured for 2 months and analyzed with quantitative reverse-transcription polymerase chain reaction and immunofluorescence. Spermatogonia including spermatogonial stem cells (SSCs) were reliably detected by combining VASA, a germ cell marker, with UCHL1, a marker expressed by spermatogonia. The established markers STAR, ACTA2, and SOX9 were used to analyze the presence of Leydig cells, peritubular myoid cells, and Sertoli cells, respectively. No obvious differences were found between the cultures initiated from fresh or cryopreserved tissues. Single or small groups of SSCs (VASA+ /UCHL1+ ) were detected in considerable amounts up to 1 month of culture, but infrequently after 2 months. SSCs were found attached to the feeder monolayer, which expressed markers for Sertoli cells, Leydig cells, and peritubular myoid cells. In addition, VASA− /UCHL1+ cells, most likely originating from the interstitium, also contributed to this monolayer. Apart from Sertoli cells, all somatic cell types could be detected throughout the culture period. Conclusion(s) Testicular tissue can be cryopreserved before long-term culture without modifying its outcome, which encourages implementation of testicular tissue banking for fertility preservation. However, because of the limited numbers of SSCs available after 2 months, further exploration and optimization of the culture system is needed.