Poster CC3 - Incorporating a tissue stromal compartment into a cervical microphysiological model

Introduction: The cervicovaginal region of the female reproductive tract (FRT) plays a pivotal role in reproductive health and immune defense mechanisms. Its anatomical and physiological features are key in a range of biological functions including pregnancy, menstruation, menopause, and infection clearance. Specifically, the cervix goes through drastic changes during these events. For instance, the median cervical elastic modulus decreases from 243 kPa in early pregnant people to 5 kPa in those at term (1). These changes drastically alter the function of the tissue; the composition and organization of the extracellular matrix (ECM) greatly contributes to the mechanical properties and function of the tissue (2), and its mechanical cues influence cell behavior and gene expression (3). Modeling the cervicovaginal region poses significant challenges due to its dynamic and complex nature; the interplay between the host immune response, microbiota, tissue structure, and physiological environment all present technical hurdles. These obstacles underscore the need for innovative approaches aimed at understanding better the role the critical role the cervix plays in gynecological health. Therefore, we developed a microphysiological model of the cervix that recapitulates the stromal ECM of the tissue for controlled in vitro study of complex interactions in this region.

Materials and

Methods: Microphysiological devices were made using a 3-part silicone insert clamped into a cassette. The insert was made by cutting a channel on three separate silicone sheets. The bottom (endothelial) and middle (stromal) sheets were adhered with double sided adhesive with a polycarbonate membrane separating the channels. The middle channel was treated with polydopamine to enable hydrogel attachment. Rat tail Collagen-I (5 mg/mL) with encapsulated 3T3 fibroblasts was pipetted into the middle channel and left to polymerize for ~20 mins at 37°C. The top silicone sheet (epithelial) was then adhered to create a vertically stacked system with channels on either side of the collagen gel in the middle layer. Calcein AM/Ethidium Homodimer III were used to determine cell viability of the encapsulated fibroblast after 7 days of culture. Human endocervical epithelial cells (A2EN) were seeded onto the top surface of the collagen gel and cultured in static conditions for 3 days. After A2EN monolayer formation, the insert was clamped in the acrylic cassette that interfaces with a pump to introduce fluid flow into the system. After 2 days of dynamic culture, A2EN cells were immunofluorescently stained and imaged with confocal microscopy.

Results, Conclusions, and Discussions: Results and discussion: We developed an assembly process that allows a hydrated stromal compartment to be incorporated into an in vitro model of the cervicovaginal region. By supporting a hydrogel layer on a polycarbonate membrane, we are able to separate the endothelial and epithelial compartments with a stromal compartment for separate cell culture (Figure 1A). The stromal compartment can be seeded with and supports viable culture of stromal cells (fibroblasts, Figure 1B) and with a monolayer of epithelial (Figure 1CD) and endothelial cells. The insert can be cultured under flow by clamping an acrylic cassette around it that includes inlet and outlet ports (Figure 1E). The cassette was connected to a pump system and media was perfused in the separate endothelial and epithelial channels. Once the insert was clamped into the cassette and flow initiated, the hydrogel maintained its integrity under the shear stress (~0.11 dyn/cm2) produced by the fluid flow for 2 days. These data demonstrate the potential of our microphysiological cervicovaginal model to better recapitulate the tissue mechanical properties of the cervix and be used to study physiological and pathological microenvironments with increased mechanical and structural complexity. By incorporating stromal cells, which influence the function of the cervix, our device will enable research into complex interactions between epithelial, endothelial, and stromal cervical cells.

Conclusion: We have successfully embedded a tissue stromal compartment of the cervicovaginal tissue into an in vitro device that enables the study of microenvironment and cellular interactions in physiologically relevant conditions. This model was designed with modularity and ease of use at the forefront, enabling our model to be highly translatable to different studies of of health and diseases in the female reproductive tract

Acknowledgements (Optional):

References: 1. Hee et al., PLoS One. (2014), 2. Karamanos et al., FEBS J. (2021), 3. Natale et al., Stem Cells Dev. (2023).