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Womens Health

Women's Health - Poster Session B

Poster L22 - Design and finite element modeling of a multicompartment human endocervix model capable of longitudinal in situ imaging

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The human endocervix is the exit of the uterus and creates mucus which flows into the vaginal canal and acts as both lubrication and a defense mechanism1. The cervix is a largely understudied organ, especially in regard to sexually transmitted infections (STIs) and resulting cell-cell and cell-extracellular matrix (ECM) interactions2. While animal models provide the complexity of a complete physiological system, many STIs pathogens are human specific and cannot be investigated in animal models. However, limited in vitro models exist that are able to integrate the complex physiology of the endocervix, including the multiple cellular compartments and the ECM. Further, even fewer have the ability for live imaging in situ, which is necessary to understand mechanisms of pathogenesis. The design of a new in vitro model that incorporates endocervical cells, vasculature, immune cells, and stromal cells within a three dimensional ECM would enable mechanistic understanding of STIs and the host-pathogen response. We developed a modular multicompartment microphysiological model in which multiple cell types can be incorporated with an ECM hydrogel that can be live imaged in situ. Dynamic control of fluid flows was achieved across the cervical lumen surface and the vascular surface without additional manipulation of the model. Computational modeling of fluid flow over the suspended collagen gel was used for device design and characterization. Our in vitro model coupled with the computational model can be used to model STIs and the resulting impact on tissue function.

Materials and

Methods:  The modular endocervix model contains an “insert” and “cassette” that clamps against the insert to create sealed fluidic channels. The insert consists of three layers of silicone. The top and bottom layers provide paths for fluid while the middle layer (ECM) allows for placement of a collagen-I gel, creating a 3D stromal ECM compartment. The acrylic cassette consists of a lid with fluid ports and a base with three pieces that are welded together (Figure 1A). NIH3T3 fibroblasts (1x104 cells/device) were incorporated within the collagen gel and polymerized in the insert. The cells were stained with Calcein AM and imaged in the cassette at 5x using an epifluorescent microscope to perform a live/dead assay. We performed a fluid seal experiment on the assembly in which blue or yellow dyed water was perfused through each channel to ensure there were no leaks or mixing. FEBio software was used to develop a model of the fluidic channels to study the effect of shear stress and pressure differentials on the suspended collagen-I gel. We used FEBio to model the channel geometry, establish material properties and boundary conditions to analyze the deformations of the gel. Mooney-Rivlin material was selected for the 500 μm thick hydrogel with a density of 1 tonne/mm3 and a bulk modulus of 2.15 kPa as determined experimentally3. With a flow rate of 500 μL/min, the applied force on the top surface of the hydrogel was 0.062 N. A 0.0465 N load was applied to the bottom surface of the gel.

Results, Conclusions, and Discussions: We successfully created a 3D cervicovaginal model with the capability of in situ time lapse imaging (Figure 1B). The cassette base was manufactured using varying thickness, clear acrylic and cover glass to ensure compression/fluid seal within the insert and to reduce the distance from the embedded gel to the objective. Clear images of cells embedded within hydrogel were taken while the device was assembled, indicating that the system can be imaged in situ and that the geometry of the cassette is compatible with an inverted microscope (Figure 1C). To ensure that the thin cassette base still resulted in a sealed system, fluid flow was initiated through the assembly and after four hours, we found that the top and bottom channels did not mix and there were no leaks from visual inspection of the colored streams (Figure 1D) and quantitative measurements. This result indicates that the collagen-I gel maintained integrity under flow conditions and the cassette could successfully clamp the insert creating fluid channels. We simulated the hydrogel’s response to fluid flow and varying pressures by parametrically testing fluid flow profiles to determine deformation and fluid-induced loading of the cellular compartment. As expected, maximum deformation of the hydrogel occurred at the center with a displacement of 36 μm and a maximum effective Lagrange strain of 6.89x10-4 (Figure 1E). Together these data validate a device design that stably incorporates an extracellular stromal compartment and enables in situ time lapse imaging of a human endocervical model. Our modular endocervix model, consisting of an insert and the cassette, allows for leak-free perfusion and longitudinal in situ imaging. In future studies, the physiological response of the endocervix to mucus flow and the circulating immune cells can be studied using this controlled microphysiological system. Additionally, we have created a computational model to be able to model the fluid-induced forces applied to the hydrogel stromal compartment to reproduce relevant tissue properties and fluid forces for mechanotransduction studies. The incorporation of endocervical cells, vasculature, and stromal cells within an ECM results in a model to study the immune response of STIs.

Acknowledgements (Optional):