An organotypic ectocervix mucosa model to study host-pathogen interaction of sexually transmitted infections.

Organotypic models closely recapitulate in vivo human tissues and organs and thus overcome limitations of animal and conventional cell cultures in studying human specific infections. Infections of the female reproductive tract by sexually transmitted pathogens typically start in the lower female reproductive tract, including the vagina and ectocervix, before ascending to the upper reproductive tract. These lower reproductive tract infections are often asymptomatic, facilitating silent transmission between partners and result in severe complications due to delayed diagnosis and treatment. However, due to the lack of relevant models and the challenge of detecting clinically asymptomatic infections, research has largely focused on symptomatic infections of the upper female reproductive tract (FRT). We present an organotypic human ectocervix mucosal (hEcCxM) model, generated by coculturing primary ectocervix epithelial cells and fibroblasts on a plastically compressed collagen scaffold. Histological analysis and single-cell RNA sequencing (scRNA-seq) revealed that the hEcCxM model developed a stratified squamous epithelium and exhibited key features essential for its physiological barrier function. Transcriptional fidelity of the hEcCxM models was validated against native ectocervix tissues. The model recapitulated several in vivo infection phenotypes of Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (Ctr). GC infection induced secretion of proinflammatory cytokines while Ctr infections did not elicit a comparable inflammatory response. Additionally, we demonstrated that neutrophils could migrate to infection sites from the basal side, but this requires the presence of an endothelial layer. Overall, the hEcCxM model represents a scalable and physiological platform for studying host-pathogen interactions in sexually transmitted infections, as well as non-infectious diseases affecting the ectocervical mucosa.