In vitro complete differentiation of human spermatogonial stem cells to morphologic spermatozoa using a hybrid hydrogel of agarose and laminin

Spermatogenesis refers to the differentiation of the spermatogonial stem cells (SSCs) located in the base seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix (ECM), paracrine factors, and testicular somatic cells which play a supporting role for SSCs. Thus, the present study evaluated the potential of co-culturing Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin, the main components of the ECM. Following the three–week conventional culture of human testicular cells, the cells were cultured in agarose hydrogel or agarose/laminin one (hybrid) for 74 days. Then, immunocytochemistry, real-time PCR, electron microscopy, and morphological staining methods were applied to analyze the presence of SSCs, as well as the other cells of the different stages of spermatogenesis. Based on the results, the colonies with positive spermatogenesis markers were observed in both culture systems. The existence of the cells of all three phases of spermatogenesis (spermatogonia, meiosis, and spermiogenesis) was confirmed in the two groups, while morphological spermatozoa were detected only in the hybrid hydrogel group. Finally, a biologically improved 3D matrix can support all the physiological activities of SSCs such as survival, proliferation, and differentiation.