3D Oral and Cervical Tissue Models for Studying Papillomavirus Host‐Pathogen Interactions

Abstract

Human papillomavirus (HPV) infection occurs in differentiating epithelial tissues. Cancers caused by high-risk types (e.g., HPV16 and HPV18) typically occur at oropharyngeal and anogenital anatomical sites. The HPV life cycle is differentiation-dependent, requiring tissue culture methodology that is able to recapitulate the three-dimensional (3D) stratified epithelium. Here we report two distinct and complementary methods for growing differentiating epithelial tissues that mimic many critical morphological and biochemical aspects of in vivo tissue. The first approach involves growing primary human epithelial cells on top of a dermal equivalent consisting of collagen fibers and living fibroblast cells. When these cells are grown at the liquid-air interface, differentiation occurs and allows for epithelial stratification. The second approach uses a rotating wall vessel bioreactor. The low-fluid-shear microgravity environment inside the bioreactor allows the cells to use collagen-coated microbeads as a growth scaffold and self-assemble into 3D cellular aggregates. These approaches are applied to epithelial cells derived from HPV-positive and HPV-negative oral and cervical tissues. The second part of the article introduces potential downstream applications for these 3D tissue models. We describe methods that will allow readers to start successfully culturing 3D tissues from oral and cervical cells. These tissues have been used for microscopic visualization, scanning electron microscopy, and large omics-based studies to gain insights into epithelial biology, the HPV life cycle, and host-pathogen interactions.

Bacterial vaginosis and health-associated bacteria modulate the immunometabolic landscape in 3D model of human cervix

“…Here we tested the hypothesis that members of the cervicovaginal microbiota distinctly contribute to inflammation and metabolic responses in the vagina and cervix, which contribute to disease. Although BV is considered a vaginal disorder, we studied cervicovaginal microbiota–host interactions using our well-characterized 3D cervical cell model 43 , 44 for the following reasons. Sexually transmitted pathogens establish infections in the cervix 75 .…”

“…For quantification, cells were dissociated with 0.25% (v/v) trypsin (Mediatech, Manassas, VA, USA) and counted using trypan blue exclusion staining and a Countess automated cell counter (Invitrogen, Carlsbad, CA, USA). To generate 3D models, A2EN cells were grown on collagen-coated dextran microcarrier beads (Cytodex-3, Sigma-Aldrich) in rotating wall vessel (RWV) bioreactors (Synthecon, Houston, TX, USA) as described previously 43 , 44 . Briefly, A2EN cells were initially grown as monolayers in tissue culture flasks, trypsinized, and counted as described above.…”

“…Our well-characterized human three-dimensional (3D) cervical epithelial cell model 43 , 44 , based on the rotating wall vessel bioreactor technology 45 , demonstrates a remarkable resemblance to human tissue in vivo, recapitulates several hallmarks of cellular differentiation (such as microvilli, junctional complexes, mucus, and Toll-like receptor profiles), and mount physiologically and clinically relevant responses to microbial products 43 , cervicovaginal microbiota 46 , 47 , and STI pathogens 48 . When coupled with multiplexed immunoassays and metabolomics approaches and the knowledge of human and bacterial metabolism, our infection models are powerful tools for the identification of key immune mediators and metabolites in healthy and diseased microenvironments.…”

Veillonellaceae family members uniquely alter the cervical metabolic microenvironment in a human three-dimensional epithelial model

“…The 3-D cervical model is a robust system to study host-microbe interactions, as it accurately resembles ultrastructural features of the cervical tissue in vivo, such as apical and basal differentiation, tight junctions, microvilli, and more organotypic surfaces for the colonizing bacteria, and has been shown to recapitulate clinical findings 15,[44][45][46][47] . Herein, we applied a reductionist approach and infected the 3-D cervical model with single bacterial species to determine the individual contributions of these bacteria to the cervicovaginal microbiome that are currently lacking.…”

“…Herein, we applied a reductionist approach and infected the 3-D cervical model with single bacterial species to determine the individual contributions of these bacteria to the cervicovaginal microbiome that are currently lacking. This knowledge is essential to inform future experiments that involve polymicrobial infections [44][45][46][47] .…”