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

Area of Science:

• Reproductive Biology
• Biomaterials Science
• Cell Biology

Background:

• Spermatogenesis requires a specialized niche involving germ cells and Sertoli cells.
• Replicating the in vitro Sertoli cell niche is challenging due to phenotype loss in conventional cultures.
• Human Sertoli cells (hSCs) are crucial for the blood-testis barrier (BTB) but difficult to maintain ex vivo.

Purpose of the Study:

• To develop a human extracellular matrix (ECM)-derived connective tissue equivalent (CTE) for long-term hSC maintenance.
• To assess the ability of the CTE to support hSC survival, organization, and phenotypic stability in vitro.
• To evaluate the CTE as a platform for creating in vitro human testis models.

Main Methods:

• Generated a CTE from fibroblast-secreted matrix (laminin, fibronectin, collagen IV).
• Cultured hSCs (single cells or spheroids) within the CTE.
• Assessed hSC survival, organization, BTB protein expression (ZO-1, OCLDN), and Sertoli cell markers (SOX9, ABP) over time.

Main Results:

• The CTE supported hSC survival and organization, mimicking a supportive microenvironment.
• Both single cells and spheroids showed progressive expression and organization of BTB proteins.
• Spheroid culture demonstrated enhanced structural cohesion and junctional organization within the CTE.
• Upregulation of key Sertoli cell markers (SOX9, ABP) was observed.

Conclusions:

• The human-derived CTE platform effectively supports long-term maintenance of hSC phenotype and BTB-associated features in vitro.
• This biomaterial offers a promising foundation for developing advanced human testis models.
• The CTE facilitates future co-culture studies crucial for understanding spermatogenesis and male infertility.