Decellularized testicular bio-scaffolds drive in vitro differentiation of chemically reprogrammed fibroblasts

Spermatogenesis is a highly orchestrated and complex biological process that, to date, has only been partially reproduced in vitro, primarily in murine models. Despite the growing prevalence of human male infertility, its etiology remains poorly understood, largely due to the limited availability of physiologically relevant experimental models and the intrinsic inaccessibility of human testicular tissue. The development of advanced 3D in vitro systems capable of reproducing the structural, biomechanical, and biochemical features of the testicular microenvironment represents a critical step toward modeling human spermatogenesis and dissecting the molecular mechanisms underlying male infertility. Here, we describe a novel bioengineering strategy that integrates chemical reprogramming cues with tissue-specific mechanical signals to generate biomimetic 3D testicular models. To this purpose, testicular bio-scaffolds were produced from bovine testes using a three-step decellularization protocol consisting of a freeze-thaw cycle followed by sequential treatments with 0.3% sodium dodecyl sulfate (SDS) for 12 h and 1% Triton X-100 for 6 h. The generated decellularized testicular bio-scaffolds preserved the gross morphology of the native tissue. Histological and DAPI staining, together with DNA quantification analyses, confirmed the effective removal of cellular material, with low residual DNA levels, compared with untreated testis. Histochemical analyses demonstrated the preservation of the key extracellular matrix (ECM) components collagen, elastin, and glycosaminoglycans, ensuring retention of the tissue-specific microarchitecture. SEM confirmed preservation of the 3D ultrastructure of the extracellular matrix, while quantification of residual SDS demonstrated efficient detergent removal. The resulting acellular scaffolds were subsequently repopulated with 1 x 10⁶ chemically reprogrammed human skin fibroblasts/mm³. Seven days after engraftment, cells had turned down the expression of pluripotency markers and actively transcribed for the testicular-associated markers SOX9, SS18L1, AMH, UCHL1, and PRM1. Immunofluorescence analyses further confirmed the expression of SOX9, AMH, and UCHL1 proteins, supporting the acquisition of testicular-associated phenotypes within the repopulated bio-scaffolds. Overall, these findings demonstrate that the generated decellularized testicular bio-scaffold represents a promising biomimetic artificial niche able to recapitulate key aspects of the native microenvironment. This platform may provide a reliable and versatile tool for modeling human spermatogenesis, investigating the pathophysiology of male infertility, and supporting future regenerative and translational applications.