Improvement of in vitro culture methods of Spermatogonial Stem Cells (SSCs) is known to be an effective procedure for further study of the process of spermatogenesis and can offer effective therapeutic modality for male infertility. Tissue decellularization by providing natural 3D and extracellular matrix (ECM) conditions for cell growth can be an alternative procedure to enhance in vitro culture conditions. In the present study, the testicular tissues were taken from brain death donors. After enzymatic digestion, the tissue cells were isolated and cultured for four weeks. Then the identity of the SSCs was confirmed using anti-GFRα1 and anti-PLZF antibodies via immunocytochemistry (ICC). The differentiation capacity of SSCs were evaluated by culture of them on a layer of decellularized testicular matrix (DTM) prepared from sheep testis, as well as under two-dimensional (2D) culture with differentiation medium. After four and six weeks of the initiation of differentiation culture, the pre-meiotic, meiotic and post- meiotic genes at the mRNA and protein levels was examined via qPCR and ICC methods, respectively. The results showed that pre-meiotic, meiotic and post-meiotic genes expressions were significantly higher in the cells cultured in DTM substrate (P ≤ 0.01).The present study indicated that, the natural structure of ECM prepare the suitable conditions for further study of the spermatogenesis process in the in vitro and contributes to the maintenance and treatment of male infertility.

已知改进精原干细胞 (SSC)体外培养方法是进一步研究精子发生过程的有效方法，可以为男性不育提供有效的治疗方式。通过为细胞生长提供天然 3D 和细胞外基质 (ECM) 条件的组织去细胞化可以成为体外增强的替代程序培养条件。在本研究中，睾丸组织取自脑死亡供体。酶消化后，分离组织细胞并培养4周。然后通过免疫细胞化学 (ICC) 使用抗 GFRα1 和抗 PLZF 抗体确认 SSC 的身份。SSCs 的分化能力通过将它们在一层由绵羊睾丸制备的脱细胞睾丸基质 (DTM) 上培养以及在具有分化培养基的二维 (2D) 培养下进行评估。分化培养开始 4 周和 6 周后，分别通过 qPCR 和 ICC 方法检查了 mRNA 和蛋白质水平的减数分裂前、减数分裂和减数分裂后基因。结果表明，减数分裂前，