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Background: Effective culture systems for attachment, migration, proliferation, and differentiation of spermatogonial stem cells (SSCs) can be a promising therapeutic modality for preserving male fertility. Decellularized extracellular matrix (ECM) from native testis tissue creates a local microenvironment for testicular cell culture. Furthermore, platelet-rich plasma (PRP) contains various growth factors for the proliferation and differentiation of SSCs. Methods:In this study, human testicular cells were isolated and cultured for 4 weeks, and SSCs were characterized using immunocytochemistry (ICC) and flow cytometry. Human testicular tissue was decellularized (0.3% SDS, 1% Triton), and the efficiency of the decellularization process was confirmed by histological staining and DNA content analysis. SSCs were cultured on the human decellularized testicular matrix (DTM) for 4 weeks. The viability and the expression of differentiation genes were evaluated by MTT and real-time polymerase chain reaction (PCR), respectively.Results: Histological evaluation and DNA content analysis showed that the components of ECM were preserved during decellularization. Our results showed that after 4 weeks of culture, the expression levels of BAX, BCL-2, PLZF, and SCP3 were unchanged, while the expression of PRM2 significantly increased in the cells cultured on DTM supplemented with PRP (ECM-PRP). In addition, the expression of GFRA1 was significantly decreased in the ECM group compared to the control and PRP groups. Furthermore, the MTT test indicated that viability was significantly enhanced in cells plated on DTM supplemented with PRP. Conclusion:Our study demonstrated that DTM supplemented with PRP can provide an effective culture system for the differentiation and viability of SSCs.

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Background Spermatogenesis refers to the differentiation of spermatogonial stem cells (SSCs) located in the basal part of seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix, a culture medium containing factors influencing the survival, proliferation and differentiation of spermatogenic cells, and testicular somatic cells that play a supporting role for SSCs. Accordingly, in this paper, the potential of co-culture of Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin as one of the main components of the extracellular matrix of the basal compartment of seminiferous tubules in the presence of culture medium containing growth factors was investigated. Methods After three–week conventional culture of the cellular suspension of enzymatic digestion of human testicular tissue, the cells were cultured in agarose hydrogel alone or hybrid hydrogel (agarose/laminin) for 74 days. Then, the presence of spermatogonial cells and other cells of the different stages of spermatogenesis were analysed by immunocytochemistry, real-time PCR, electron microscopy and morphological staining. Results The results showed that colonies with positive spermatogenesis markers were observed in both culture systems. The presence of different levels of spermatogenesis in two groups media was confirmed, but morphological spermatozoa were observed only in hybrid hydrogel group. Conclusions Based on the results of this paper, it can be concluded that an agarose and extracellular matrix-based scaffold which maintains the mechanical strength of the scaffold during culture and also increases the biological activity of the scaffold, can support from all physiological activities of SSCs such as survival, proliferation and differentiation.