Poster CC1 - A Mimetic Mucus with Independently Tunable Biophysical and Biochemical Properties for an in vitro Model of the Female Reproductive Tract

Introduction: Cervical mucus lines the cervical and vaginal epithelium of the female reproductive tract (FRT). Cervical mucus undergoes significant rheological and compositional changes over the menstrual cycle, pregnancy, and during infection. This mucus is thought to play a large role in barrier function and host-microbiome interactions; however, the mechanisms behind its function are poorly understood. The complexity of mucus – structurally and compositionally – is difficult to replicate and most in vitro mucosal models do not fully consider this biologically relevant physiological compartment. Although widely used mucins, such as bovine submaxillary mucin (BSM) and porcine gastric mucin (PGM), contain relevant cervical mucin glycoproteins (e.g., muc5b and muc5ac), their ability to form a mucus to recapitulate the dynamic range of the viscoelastic properties of cervical mucus is limited. To make a physiologically relevant in vitro model of lower FRT, mucus must be included in a way that is able to mimic the dynamic range of the biophysical and biochemical properties of mucus while supporting both optimal and non-optimal bacterial growth to investigate reproductive health and disease. Therefore, we created a mimetic mucus in which the biophysical and biochemical properties can be independently tuned to incorporate into in vitro models of the FRT.

Materials and

Methods: Mimetic mucus was created by combining methylcellulose (MC) with BSM or PGM. The MC concentration defines the viscoelastic properties of the mimetic system while the BSM and PGM define the biochemical properties. We mixed 0-3 w/v% MC and 2 w/v% PGM and/or BSM in a HEPES-based, neutral buffer. The viscoelasticity of the resulting reconstituted mucus was measured using a rheometer (TA Instrument, DHR3) using a 20 mm crosshatched parallel plate geometry with an amplitude sweep from 0.1% to 100% strain at 1 rad/s, followed by a frequency sweep from 0.1 to 10 rad/s at 5% strain. The functional diffusivity of the mimetic mucus of either 2 w/v% PGM or 2 w/v% BSM with varying MC concentrations was tested by tracking the movement of 500 nm fluorescent microspheres in the material. To confirm that the mucus was biocompatible with bacteria, two mucus formulations were made to mimic cervical mucus viscoelasticity and mucin content at different stages of the menstrual cycle: (1) menstruation (0.2 w/v% MC, 1.3 w/v% PGM, 0.7 w/v% BSM) and (2) ovulation (1.0 w/v% MC, 0.8 w/v% PGM, 1.2 w/v% BSM). We then cultured L. crispatus (LUCA103) within the mimetic mucus, MC only, or an HBSS++ control and tracked its growth and viability with PCR after 24 hours of culture.

Results, Conclusions, and Discussions: By mixing MC with commercial mucins, we were able to take advantage of the viscoelasticity properties of MC and the biochemical properties of mucin independently. Using rheological characterization, we found that the storage and loss modulus of the mimetic mucus was dominated by the concentration of MC used to create the mimetic mucus and was independent of mucin identity. By varying the amount of MC the mimetic mucus was made with, the viscoelastic properties could be matched to the dynamic range of cervical mucus properties over the menstrual cycle and in pregnancy (Figure 1). In addition, diffusivity of the microspheres in the mimetic mucus was inversely correlated to MC concentration with similar ranges between different mimetic mucus formulations with PGM or BSM (Figure 2). These data provide direct reference values to model drug or bacteria transport within the mucus and are consistent with bulk rheology properties. We further verified the mimetic mucus was able to support growth of L. crispatus, a critical bacterium found in a healthy vaginal microbiome. After 24 hours of culture in the mimetic mucus, L. crispatus abundance was measured via PCR and normalized to the initial abundance (Figure 3). The qPCR data showed that mimetic mucus from the menstrual and ovulation phases of the menstrual cycle had differentially higher L. crispatus abundance compared to HBSS++ and MC only, supporting the ability of the mimetic mucus to support L. crispatus culture.

Collectively, this study established a novel mimetic mucus that enables the incorporation of mucus into FRT in vitro models to study mucosal biology across menstruation, pregnancy, menopause, and disease. This critical tool enables the incorporation of vaginal microbiota to investigate epithelial barrier function, host-bacteria interaction, infection and inflammatory immune responses. Whereas this study focused on the development of a cervical mucus mimetic, these methods can be applied to generate flowable mucus compositions for other mucosal in vitro models including the gut and lung.

Acknowledgements (Optional): Thanks Xinqiao Jia's group for letting us use rheometer for material charaterization.