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Spermatogenesis is a highly coordinated process occurring within the seminiferous tubules, where germ cells develop in close association with somatic Sertoli cells (SCs). These cells provide essential structural, metabolic, and regulatory support to developing germ cells, and their functionality is tightly regulated by the surrounding extracellular matrix (ECM). However, replicating the complex human testicular niche in vitro remains a major obstacle for advancing in vitro spermatogenesis (IVS), particularly with respect to long-term SC function and proper spatial organization. This study presents the development and characterization of a three-dimensional (3D) connective tissue equivalent (CTE), enriched with key ECM components, as a biomimetic platform to support human SC (hSC) function and maturation.
We employed a two-step tissue engineering strategy to fabricate an ECM-rich CTE using human stromal fibroblast-derived microtissues (μTPs). Porous gelatin microbeads, produced through double emulsion and crosslinked with glyceraldehyde, served as scaffolds for stromal cell attachment. These μTPs were dynamically cultured for 9–11 days in spinner flask bioreactors to promote matrix deposition, and subsequently fused in silicone molds under dynamic conditions to generate dense, collagen-rich CTEs containing testis-relevant ECM proteins such as laminin and fibronectin. hSCs were introduced either as single cells or pre-formed 3D spheroids onto matured CTEs and maintained under air-liquid interface (ALI) culture with hormone supplementation (FSH and testosterone) for up to 28 days.
The CTE-supported cultures demonstrated progressive cellular integration and ECM remodeling. Histological analyses revealed more efficient retention and 3D organization of hSC spheroids compared to single-cell seeded constructs. Masson’s Trichrome staining confirmed increased collagen deposition over time, particularly around spheroids. Immunofluorescence revealed progressive upregulation and spatial organization of tight junction proteins ZO-1 and OCLN, with more pronounced barrier integrity observed in the spheroid-based group by day 28. Gene expression analyses via qRT-PCR supported these findings, showing significantly higher expression of Sertoli-specific (SOX9, ABP, GATA4) and blood-testis barrier-related markers (CLDN11, ZO-1, FSHR) in the spheroid-seeded constructs at 28 days. These results underscore the synergistic role of ECM composition and spatial cell pre-organization in promoting SC maturation and niche remodeling.
In conclusion, the engineered CTE provides a functionally supportive testicular microenvironment that sustains hSC viability, phenotype, and barrier formation in vitro. The use of pre-formed hSC spheroids proved superior to single-cell approaches, enabling more effective recapitulation of the in vivo-like 3D architecture and function. This platform holds strong potential for future applications in IVS research, disease modeling, and fertility preservation strategies by offering a reliable and physiologically relevant model of the human testicular niche.
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