Efficient cryopreservation of testicular tissue: Effect of age, sample state, and concentration of cryoprotectant

Objective: To study the effect of age and sample state on cryopreservation of testicular tissue, evaluate toxicity of commonly used cryoprotectants (CPs), and determine their optimal concentration for use. Design: Prospective experimental study. Setting: Academic research unit. Patient(s): Patients with prostate carcinoma undergoing orchidectomy. We also studied immature and adult male Holtzman rats. Intervention(s): Toxicity of CPs before freezing, morphology, and relative viability after freezing were evaluated for rat testicular cell suspensions (CS) and tubular fragments (TUB). Relative viability of adult human testicular CS and TUB after thaw was evaluated. Human TUB were cultured after thaw for 48 hours in medium containing epidermal growth factor (EGF), and effects on viability, morphology, and gene expression were determined. Main Outcome Measure(s): Viability and ploidy were measured with flow cytometry, postthaw cryodamage of immature rat tissue was studied by transmission electron microscopy, cell proliferation and differentiation were evaluated by immunohistochemistry and by real-time polymerase chain reaction. Result(s): Immature testicular tissue was more susceptible to toxic assault by CP than adult tissue and displayed cell-specific sensitivity to CP, with glycerol, dimethyl sulfoxide and ethylene glycol being effective in protecting spermatid (1N), spermatogonia (2N) and spermatocyte (4N) populations respectively. Preservation as TUB may be preferred over CS and DMSO is an effective CP for immature and ethylene glycol for adult testicular tissue. Conclusion(s): Differential sensitivity of immature testicular tissue to CPs warrants judicious selection of CP on the basis of end application for prepubertal tissue.