In Vitro Spermatogenesis on Human Decellularized Testicular Matrix Plates Following Exosome Treatment in a Dynamic Culture System.

Testicular tissue engineering for in vitro spermatogenesis aims to restore fertility, focusing on challenges like efficiency, ethical concerns, and the need for a deeper biological understanding. The use of decellularized scaffolds led to better cell seeding and differentiation, and exosomes led to enhanced spermatogenesis. Also, the dynamic culture systems are being explored to replicate in vivo conditions more accurately. In this study, we aimed to utilize a perfusion mini-bioreactor for the dynamic culture of mouse spermatogonial stem cells on decellularized testicular matrix plates supplemented with exosomes. Our goal was to assess the progression of the spermatogenesis process through histological, immunohistochemical, and molecular analyses over four weeks. Human testicular tissues were decellularized using 1% sodium dodecyl sulfate and were then fabricated into thin plates using a cryostat. Sertoli and spermatogonial stem cells were isolated from neonate mouse testis and seeded onto the decellularized testicular matrix plates. A mini-perfusion bioreactor was employed to create dynamic culture conditions. Also, MSCs-derived exosomes were introduced to the culture medium, alone or in combination with a spermatogenic medium containing numerous chemical factors. The histological, IHC, and molecular analyses were performed at the end of the experiment. Our decellularization procedure successfully preserved the ECM components, while eliminating native cells. The isolated cells expressed PLZF and VIMENTIN markers, confirming the presence of SSCs and Sertoli cells. The seeded scaffolds exhibited proper homing, viability, proliferation, and differentiation of the cells towards in vitro spermatogenesis. Also, exosome treatment is capable of enhancing the spermatogenic potential of SSCs. Our findings indicate that the dynamic culture system significantly promoted the proliferation and differentiation of SSCs into mature spermatozoa. The use of exosomes further enhanced these effects, as evidenced by improved cellular viability, reduced apoptosis, and advanced spermatogenesis to the elongated spermatid stage. The combined treatment of exosomes and spermatogenic medium showed a synergistic effect, yielding superior outcomes in terms of sperm cell maturity and functionality. This study underscores the potential of combining decellularized testicular matrices with exosome therapy in a dynamic culture set up to advance the field of reproductive biology and fertility restoration.