Trans-epithelial permeability is a critical functional parameter for reconstructed tissues, particularly in genitourinary tissue engineering, where urine leakage must be avoided. Although Franz diffusion cells are considered the gold standard for permeability measurements, their cost and limited accessibility restrict their widespread use. In parallel, the reliable quantification of urea in culture media remains challenging due to protein interference and assay cost. The Inexpensive Trans-Epithelial Permeability (I-TEP) test is a simple and a low-cost Franz-like permeability system which can be combined with an optimized diacetyl monoxime–thiosemicarbazide (DAMO–TSC) colorimetric urea assay. I-TEP system relies on readily available laboratory components to create physically separate donor and receiver compartments, with the tissue acting as the sole diffusion interface. The DAMO–TSC assay was optimized through systematic evaluation of deproteinization, incubation time, storage conditions, and serum interference. The I-TEP test showed a strong correlation with conventional Franz diffusion cells when testing similar tissue samples. Deproteinization was identified as a mandatory step for accurate urea quantification in serum-containing media. The combined approach was successfully applied on engineered genitourinary tissues, demonstrating sensitivity to tissue maturation and cellular composition. This protocol provides a proof of concept for an affordable, robust, and autonomous method for routine permeability assessment, bridging the gap between costly commercial systems and high-throughput experimental needs.

Cell identity within the human urogenital tract is strongly influenced by hormonal signaling, yet increasing evidence suggests that epithelial differentiation may also be shaped by microenvironmental cues. In this study, we investigated how hormonal and physical environments modulate epithelial identity and plasticity using a human tissue-engineered urethral model. Primary epithelial and stromal cells derived from vaginal and trans-women’s urethral tissues were combined in homologous and heterologous configurations and cultured under controlled conditions. Constructs were exposed to estradiol or dihydrotestosterone, and epithelial differentiation was assessed using histological staining and immunofluorescence targeting lineage-specific markers. Our results show that vaginal epithelial cells cultured on vaginal stroma maintain a stable vaginal phenotype characterized by epithelial stratification and glycogen accumulation, although the magnitude of these features was modulated by hormonal stimulation. In contrast, urethral epithelial cells displayed a high degree of plasticity, adopting distinct phenotypes depending on the physical-culture environment. Notably, constructs containing urethral epithelium developed a dual phenotype, with vaginal-like differentiation at the air–liquid interface and a more urethral-like organization in immersed conditions. Furthermore, urethral cells derived from long-term estrogen-exposed tissues exhibited persistent expression of both urethral and vaginal markers, suggesting incomplete or hybrid differentiation states. Overall, these findings demonstrate that epithelial identity in the human urethra is not fixed but dynamically regulated by an interplay between intrinsic cellular properties, stromal interactions, and physical microenvironmental cues, while hormonal signaling alone appears insufficient to dictate cell fate. This study provides new insights into epithelial plasticity and has important implications for both developmental biology and tissue-engineering approaches to urogenital reconstruction.

Urethral reconstruction is required for a variety of congenital and acquired conditions. Current grafting techniques rely on autologous tissues such as buccal mucosa, which are often limited by donor-site morbidity. Biomaterials could be an alternative. However, they may present immunological risks and inconsistent surgical outcomes. The tissue-engineering self-assembly approach, which uses autologous patient cells to generate tissue constructs without exogenous biomaterials, offers a promising alternative by improving graft integration. Such natural tissue-engineered constructs exhibit promising histological similarity to native urethral tissue and hold potential for clinical application. However, graft selection for surgery remains subjective, lacking standardized mechanical criteria to ensure consistent quality. Previous efforts to quantify graft properties through uniaxial tensile testing have proven informative but require cutting the tissue into standardized specimens and are therefore not readily suited to routine quality-control workflows. To address this, we investigated puncture testing as a simpler practical alternative that does not require tissue cutting and assessed its correlation with tensile testing and surgeon-based evaluation. Self-assembled dermal and urethral constructs were evaluated using uniaxial tensile and puncture tests, and results were correlated with the surgeon’s qualitative assessments during graft handling. Our findings reveal a strong linear relationship between two mechanical testing techniques. Puncture testing also showed a strong relationship with surgeon-based qualitative assessments of graft handling. This study highlights the potential of puncture testing as a practical mechanical assessment tool for characterizing graft mechanical behavior in the context of surgical handling, and offers a pathway toward the development of standardized, objective quality control protocols for tissue-engineered urethral grafts prior to implantation.