Optimizing cryopreservation of human spermatogonial stem cells: Comparing the effectiveness of testicular tissue and single cell suspension cryopreservation

Objective: To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. Design: In vitro human testicular tissues. Setting: Academic research unit. Patient(s): Adult testicular tissues (n = 4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n = 3). Intervention(s): Testicular tissue versus single cell suspension cryopreservation. Main Outcome Measure(s): Cell viability, total cell recovery per milligram of tissue, as well as viable and SSEA-4+ cell recovery. Result(s): Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs, whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. Conclusion(s): Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient's age, type of samples cryopreserved, and end points of therapeutic applications.