76 TESTING


Day 3


Day 6


Day 11


Day 14


Day 18


Figure 1: Light microscopy images of paraffin-embedded HVE stained with hemalum/eosin. Day 3 to 18: representative images after three, six, 11, 14 or 18 days at the air/liquid interface, respectively. Magnification 20x


this later stage of reconstruction. Consistent histological features were observed from one batch of reconstructed tissues to another, confirming the observed evolution of the 3D model with time of culture.


Characterization of the 3D HVE model To further characterize the structure and organization of this new multilayered tissue, the expression and localization of vaginal


biomarkers were analysed all along the 3D reconstruction of HVE. Functional and structural vaginal biomarkers were monitored by immunofluorescence in paraffin-embedded HVE after six, 11, 14 and 18 days at the air/liquid interface. Monoclonal antibodies targeting the water-


channel proteins Aquaporin-3 proteins (AQP3) that plays a key role in hydration, were used to evaluate the functionality of the reconstructed tissue. A double staining of the cytokeratins


13 (CK13) and 14 (CK14) was also performed to follow the expression of those structural proteins. A third immunofluorescence staining was also


conducted to track the expression of involucrin (IVL) since IVL, CK13 and CK14 proteins are typical epithelial differentiation markers of the basal layer. In parallel to the specific AQP3, CK13/CK14 and IVL immunostaining, cellular nuclei were labelled with DAPI. Fluorescence microscopy images from AQP3


staining revealed the widespread expression of this hydration protein from the sixth to the 14th day, with a sudden reduction of fluorescence in HVE cultured for 18 days. Likewise, the monitoring of the IVL


biomarker revealed its increasing expression in the cytoplasm of the epithelial cells with the length of the air/liquid tissue culture (Figure 2), up to 14 days. IVL immunostaining in HVE tissues after 18 days in culture displayed less specificity, confirming the loss of functionality consequent to the loss of structure observed by hemalum/eosin morphological staining. Finally, the pattern of CK13/CK14 cytokeratin


expression was typical of basal stratified squamous epithelium with a limited expression of CK13 compared to a widespread expression of CK14 from day six to day 18, attesting on cell differentiation and functionality up to 14 days in this reconstructed tissue. Altogether, histological analyses allowed


Figure 2: Fluorescent microscopy images of Aquaporin-3 (AQP3) and Involucrin (IVL) immunostaining, and of Cytokeratin 13 and 14 (CK13/CK14) co-immunostaining of HVE. Cell nuclei are stained with DAPI. Day 6 to 18: representative image after six, 11, 14 and 18 days at the air/liquid interface, respectively. Magnification 40x


PERSONAL CARE November 2025


to conclude that the herein reconstructed HVE replicates the stratified architecture and barrier properties as early as after 11 days at the air/ liquid interface. The disorganized morphology observed at day 18 confirmed that the robustness of the 3D model declines over time of culture, suggesting its highest and optimal functionality between the 11th and the 14th day of culture. Based on those conclusions, 11 days of


culture was considered as the ideal time of air/ liquid maintenance to generate a reproducible 3D model replicating the human vaginal


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