Development and Characterization of a Stratified Vaginal Epithelial Microphysiological Model

• Isabella Doncel, Biomedical Engineering, University of Delaware

• Jason Gleghorn, Biomedical Engineering, University of Delaware

The cervicovaginal region of the female reproductive tract (FRT) plays a pivotal role in reproductive health and immune defense. Sexually transmitted infections such as human papillomavirus (HPV) target this region; although most infections resolve spontaneously, persistent infection can progress to cervical or vaginal cancer. HPV’s life cycle depends on epithelial differentiation, requiring a stratified epithelium for viral replication and assembly. Within the FRT, epithelial tissues are exposed to mucus flow, immune cell trafficking, cytokine signaling, and shear stress, influencing barrier function, host-pathogen interactions, viral movement, immune response, and pathogen clearance. Therefore, standard cell culture methods are not well suited to accurately model the complex biology of the tissue. However, microphysiological systems (MPS) offer a more physiological relevant alternative system.

In this project, we worked to develop a stratified vaginal epithelium model using VK2 vaginal epithelial cells co-cultured with BJ fibroblasts within our  MPS, benchmarking performance against transwell culture. Unlike transwells, which provide an established platform for static epithelial barriers, the MPS confines cells within a long, narrow microfluidic channel rather than a large open membrane, altering cell distribution. Initial attempts using the MPS geometry produced sparse VK2 attachment; transitioning to a smaller channel with increased seeding density enabled sufficient adhesion across the membrane. Air-liquid interface (ALI) culture was applied to induce stratification, and barrier formation was assessed via FITC-dextran permeability and confocal imaging of cell layers. In transwell controls, cell-seeded membranes showed significantly lower permeability than no-cell controls over time, consistent with barrier formation. In the MPS, fibroblast attachment and VK2 adhesion were confirmed on both channels, with preliminary evidence of stratification by day 4. Ongoing work will evaluate the effects of estradiol and varying seeding densities on stratification. Establishing a reproducible stratified vaginal epithelium model within this MPS platform lays the groundwork for future studies of HPV infection.