Testis-derived extracellular matrix scaffolds: Evaluation of fresh and frozen tissue decellularization for applications in male infertility therapy

Differentiation of spermatogonial stem cells (SSCs) on decellularized biological scaffolds has emerged as a novel approach in tissue engineering and male infertility treatment. Testis-derived extracellular matrix (ECM) scaffolds provide a natural microenvironment that can support SSC culture and differentiation. In this study, we compared the efficiency of different detergents for the decellularization of fresh and frozen human testicular tissues. Human testicular tissue samples were divided into 2 groups: fresh and frozen at -80 °C. Cellular decellularization was performed using concentrations of 0.5% and 1% sodium dodecyl sulfate (SDS) and Triton x100 alone and in combination. The extent of cell removal and reduction in cell DNA content was examined using histological studies and one-way ANOVA statistical method. Frozen tissues exposed sequentially to 1% SDS for 24 h followed by 1% Triton X-100 for 24 h showed the highest efficiency in removing cellular components while maintaining ECM integrity, compared with fresh tissues. Moreover, frozen testicular matrices demonstrated greater resistance to detergent-induced damage than fresh matrices. Both detergent concentration and tissue condition (fresh vs. frozen) are critical determinants of scaffold quality in testicular decellularization. Optimizing these parameters may facilitate the development of natural testis-derived scaffolds for reproductive tissue engineering. In the future, seeding germ cells from patients with non-obstructive azoospermia onto these optimized scaffolds under differentiation-inducing conditions may provide a promising strategy for male infertility treatment.