Tuning the Testicular Microenvironment for Enhancing Human Sertoli Cells Maturation and Functionality In Vitro

Spermatogenesis relies on the highly specialized interaction between germ cells and a supportive somatic niche, yet replicating this environment in vitro remains a major challenge. Primary human Sertoli cells (hSCs), key architects of this niche, often lose their phenotype under conventional culture conditions, limiting the establishment of a stable blood-testis barrier (BTB)-associated phenotype and their ability to support germ cells. Here, we present a structurally organized, human ECM-derived connective tissue equivalent (CTE) designed to support long-term maintenance and organization of hSCs in vitro. The CTE, generated from fibroblast-secreted matrix enriched in laminin, fibronectin, and collagen IV, reproduces key biochemical and physical features of a supportive microenvironment. hSCs introduced into the CTE as single cells or pre-formed spheroids were evaluated for survival, structural organization, phenotypic stability, and ECM remodeling. Both configurations supported progressive expression and organization of BTB-associated proteins (ZO-1, OCLDN) together with upregulation of Sertoli cell-associated markers, including SOX9 and ABP, with the spheroid-based model showing improved structural cohesion, integration within the construct, and more evident junctional organization over time. Overall, this bioactive human-derived platform supports long-term maintenance of hSC phenotype and barrier-associated features in vitro, providing a promising basis for future human testis models and co-culture studies.