Modular 3D culture platform combining methylcellulose, testicular ECM, and Sertoli cells for in vitro spermatogenesis.

OBJECTIVES: We aimed to develop and characterize a 3D culture platform combining methylcellulose (MC) with decellularized testicular extracellular matrix (ECM) and Sertoli cell co-culture to support human spermatogonial stem cell (SSC) survival and differentiation. MATERIALS AND METHODS: Human testicular cells were isolated and characterized by immunofluorescence and flow cytometry. Sheep testicular tissue was decellularized using detergents and characterized by several histology techniques. ECM was solubilized and mixed with MC at various ratios to make hybrid hydrogels. Hydrogels were characterized for pore size, porosity, mechanical properties, gelation kinetics, and degradation kinetics using scanning electron microscopy, rheological analysis, turbidity analysis, and mass loss assays. Human SSCs and Sertoli cells were cultured in 3D hydrogels for four weeks under differentiation conditions. Cell viability was assessed using the MTT assay, and gene expression of SSC, meiotic, post-meiotic, and apoptotic markers was evaluated by RT-PCR. RESULTS: After four weeks of proliferation, PLZF-positive cells increased from 19% to 73%. Decellularization reduced DNA content by 96% while preserving key matrix components. Hybrid hydrogels displayed interconnected porous structures with pore sizes of about 65-181 µm. All functional hydrogels supported cell viability; however, ECM-rich hydrogels significantly up-regulated PRM2 compared to 2D controls, indicating enhanced post-meiotic differentiation. MC-only scaffolds displayed elevated pro-apoptotic BAX expression compared to ECM-rich matrices, suggesting that bioactive ECM ligands confer cytoprotection. CONCLUSION: The modular MC/ECM hybrid hydrogel provides a tunable, physiologically relevant 3D culture system that is superior to conventional approaches in supporting SSC maintenance and differentiation, offering promise for fertility preservation strategies.