Three-dimensional models of the cervicovaginal epithelia to study host-microbiome interactions and sexually transmitted infections

Abstract

2D cell culture systems have historically provided controlled, reproducible means to analyze host-pathogen interactions observed in the human reproductive tract. Although inexpensive, straightforward, and requiring a very short time commitment, these models recapitulate neither the functionality of multilayered cell types nor the associated microbiome that occurs in a human. Animal models have commonly been used to recreate the complexity of human infections. However, extensive modifications of animal models are required to recreate interactions that resemble those in the human reproductive tract. 3D cell culture models have emerged as alternative means of reproducing vital elements of human infections at a fraction of the cost of animal models and on a scale that allows for replicative experiments. Here, we describe a new 3D model that utilizes transwells with epithelial cells seeded apically and a basolateral extracellular matrix (ECM)-like layer. The model produced tissues with morphologic and physiological resemblance to human cervical and vaginal epithelia, including mucus levels produced by cervical cells. Infection by Chlamydia trachomatis and Neisseria gonorrhoeae was demonstrated, as well as the growth of bacterial species observed in the human vaginal microbiota. This enabled controlled mechanistic analyses of the interactions between host cells, the vaginal microbiota, and STI pathogens. Affordable and semi high-throughput 3D models of the cervicovaginal epithelia that are physiologically relevant by sustaining vaginal bacterial colonization, and facilitate studies of chlamydial and gonococcal infections.

Keywords: 3D model; cervicovaginal epithelium; microbiome; sexually transmitted infections.

Figure 1.

Model set-up. A volume of 70 µl of collagen was added to the basal portion of the inverted transwell (A) and stored at 4°C. BJ’s were added to the basal membrane 2–3 days later at 3 × 10⁴ in a volume of 80–100 µl and incubated at 37°C, 5% CO₂(B). Epithelial cells (A2EN or VK2) were apically added at 1 × 10⁵ in a volume of 50–200 µl (C). After 6–9 days of incubation at 37°C, 5% CO₂ cells were ready to be used in experiments.

Figure 2.

Characterization of the 3D cervical epithelium model (A2EN). Transepithelial resistance values over the course of A2EN epithelial cell transwell 3D model set up (A). Histology (H&E) imaging (B) and electron microscopy (TEM) imaging (C) of the epithelial cells of the model 6 days post set-up. Confocal imaging of mucin gel formation (MUC-5B) on the model 6 days post set-up (D). Error bars represent SD. Image of invivo tissue purchased from shutterstock.com.

Figure 3.

Characterization of the 3D vaginal epithelium model (VK2). Transepithelial resistance values over the course of VK2 epithelial cell transwell 3D model set up (A). Histology (H&E) imaging (B) and electron microscopy (TEM) imaging (C) of the epithelial cells of the model 8 days post set-up. Error bars represent SD. Image of invivo tissue purchased from shutterstock.com.

Figure 4.

Infection of the 3D cervical model (A2EN) by C. trachomatis. Analysis of chlamydial infectivity on the conventional 2D (coverslip) model by fluorescent imaging (A) compared to the 3D (transwell) model (B), and resultant enumeration of infected cells (C). TEM image of infected cells on the 3D model (D). Cytokine profile of uninfected as compared to infected 3D cervical cells (E).

Figure 5.

Infection of the 3D cervical cells (A2EN) by N. gonorrhoeae (Gc). Transmigration of Gc across the cervical epithelium model is similar to that obtained with a commonly used cell line (HEC-1-B) (A). TEM image of Gc attached to 3D cervical epithelial cells (B).

Figure 6.

The 3D vaginal (VK2) epithelium model supports the growth of vaginal bacteria. pH (A) and D(-) lactate concentrations (B) of apical media after 48 h of anaerobic bacterial growth on 3D VK2 cells. TEM, FISH, and viability images of bacteria and host cells after 48 h of growth (C).