Poster CC19 - Development of A Primary In Vitro 3D Vaginal Tissue Model

Introduction: Women’s health remains an understudied field, warranting the need for physiologically relevant models to investigate this area, especially the vagina. Current models are not fully translatable as they typically employ animal systems, which are not directly comparable in physiology and bacterial composition to the human vaginal microenvironment and hence lack the ability to replicate native cues. For this reason, there is a need for an in vitro human vaginal model. Critical to such a model is a faithful vaginal epithelium since it is the outermost barrier of the tissue and is known to be impacted by important developmental transitions, such as puberty and menopause. Here, we investigate a confluent vaginal epithelium incorporating primary human vaginal cells (VK/E6E7). We report on the response of the model to estrogen cycling, including estradiol secretion, cell proliferation, cell stress markers, and epithelial integrity, to evaluate their utility in a 3D vaginal tissue equivalent model.

Materials and

Methods: VK/E6E7 cultures were seeded and brought to confluency in different media (KSFM complete or ReproLife™ complete media) and then allowed to incubate under a range of supplemental estradiol conditions (0, 1, 2, or 4 nM). After 4 days, the cultures were washed with PBS four times and incubated with clean (unsupplemented) media. Cell layer integrity was tracked using transepithelial electrical resistance (TEER) measurements. Live cell imaging was used to determine cell growth rates and population doubling times. After 6 days, immunohistochemistry was performed to quantify epithelial markers such as pan-CK, CK5, P63, and CK8/18, and cell supernatants were extracted and used to quantify estradiol secretion with an Estradiol Parameter Assay.

Results, Conclusions, and Discussions: Cell imaging shows consistent morphology and survival across all groups. The linear increases of estradiol secretion with respect to supplemented estradiol concentrations demonstrate epithelial cell responsiveness to hormone exposure in both media. However, cells in KSFM media show decreased estradiol secretion in comparison to ReproLife™ media. This can be attributed to the phenol red found in the KSFM, which acts as an estrogen agonist, suggesting that the media is incompatible with accurate hormonal testing with regard to estradiol levels. We also report on cell viability, proliferation dynamics, epithelial marker assessments, and TEER analyses. Future works following these experiments will include the establishment of a 3D organ tissue equivalent of the vagina that incorporates additional cell layers (e.g. fibroblasts) and design of a vagina-on-a-chip system for dynamic control of hormone cycling to mimic the 28-day in vivo cycle.

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