Production and Multimodal Characterization of Decellularized Extracellular Matrix from Porcine Prepubertal Tunica Albuginea as Additive to Polymeric Scaffolds for Testicular Organoid Growth

Preservation of spermatogonial cells is of critical importance for male patients undergoing gonadotoxic therapies. Testicular organoids generated by 3D polymeric scaffolds filled with decellularized extracellular matrix (dECM) have the potential to promote stem cell growth. We propose a protocol to produce dECM from porcine prepubertal tunica albuginea for use in polymeric scaffolds. Spectroscopic analysis, molecular biology techniques, and histo-morphological assessment were used to evaluate the morphology and mechano-chemistry of the dECM at each phase of the process. The results obtained from this study demonstrate that the protocol can produce a high-purity product without causing significant alterations to protein conformation. The dECM obtained was then employed in the creation of a 3D scaffold for the cultivation of testis organoids. This was achieved by utilizing a mixture of alginate (A) and chitosan (C), which are natural polymers with a high degree of biocompatibility, that have extensive application in the field of biomedicine. Scaffold characterization demonstrated that the presence of dECM affects the scaffold's mechanical properties by tuning structural reorganization and reducing hygroscopicity. The cell viability assay demonstrates that the A/C scaffolds are non-cytotoxic after a pre-phase of immersion in the medium.