Isolation of germ cells from human testicular tissue for low temperature storage and autotransplantation

Objective: To develop a new protocol for conserving fertile potential in men undergoing sterilizing chemotherapy by low temperature banking of germ cells which can be returned to the patient's testes after thawing. Design: Isolation of human and murine germ cells for comparing cellular viability after cooling to liquid nitrogen temperatures by the use of different cryoprotective agents and for infusion into the testis. Setting: Laboratory research environment. Patient(s): Men undergoing routine surgery in a urology department. Intervention(s): Testicular biopsy. Main Outcome Measure(s): Cellular viability and infusion of seminiferous tubules. Result(s): After isolation using a two-step enzymatic disaggregation protocol, 66% to 87% of germ cells from human and murine specimens, respectively, were still viable. Cell survival was similar in four commonly used cryoprotective agents after cooling to liquid nitrogen temperatures. Seminiferous tubules infused by back flow with dye solution via the rete testis were filled with an efficiency of 55%. Conclusion(s): Judging from the high viability of unfractionated germ cells, it is feasible to isolate germ cells from testicular biopsies for low temperature banking with the aim of attempting to restore fertility after iatrogenic sterilization. (C) 2001 by American Society for Reproductive Medicine.