Testicular Cell–Conditioned Chitosan Thermosensitive Hydrogel as an Excellent Substrate for Culture and Proliferation of Spermatogonia Cells

Successful in vitro transplantation of spermatogonia cells (SCs) requires effective culture systems for the proliferation of SCs. The natural extracellular matrix (ECM) creates a suitable microenvironment for stem cell culture. In the present study, a temperature-sensitive hydrogel scaffold was fabricated from chitosan (TCTS), a biocompatible polysaccharide with the ability to turn into gel using <i>β</i>-glycerophosphate as a crosslinker at 37°C. Then, the surface of the hydrogel scaffold was covered with the ECM secreted from testicular cells (TC-TCTS) to provide an excellent substrate for the adhesion and proliferation of SCs toward sperm-producing cells. The synthesized scaffold had a uniform morphology with high biocompatibility and adhesion for both testicular cells and SCs. SEM micrographs, DNA content, and DAPI confirmed the presence of ECM and successful removal of the TC from the hydrogel after decellularization. TC-TCTS showed a higher rate of water absorption and weight loss (around 80%) compared to TCTS. The survival of SCs seeded on the TC-TCTS hydrogel showed a significant increase compared with TCTS. The SCs cultured on TC-TCTS were much more elongated and had a greater tendency to stick to the surface of the hydrogel compared with TC-TCTS. Molecular studies showed a significant increase in the expression of proliferation-associated genes (around 4.5-fold for <i>id4</i> and around 7-fold for <i>plzf</i>) in SCs cultured on the TC-TCTS compared with TCTS and control. Our findings confirmed that the TC-TCTS can provide an excellent microenvironment biomimicking the natural ECM of seminiferous tubules for adhesion and proliferation of SCs and possible production of sperm in vitro in the future investigations.