Monitoring Chlamydia trachomatis Development in Polarized Endocervical Epithelial Cells.

Understanding Chlamydia trachomatis infection requires physiologically relevant models tailored to various research questions. One of the main sites of C. trachomatis infection is the columnar epithelium of the endocervix; therefore, a model that accurately mimics the environment of endocervical epithelium is essential. Polarized cell models provide a valuable solution, offering a cost-effective yet biologically relevant alternative to conventional 2D culture systems and more complex and costly 3D or animal models. Immortalized human endocervical epithelial cells (A2EN cells) form a polarized monolayer on transwell membranes, reproducing key in vivo features, including tight junctions, mucus secretion, proinflammatory cytokine production, and hormone receptor expression, when maintained at low passage. This chapter outlines protocols for generating a polarized A2EN culture and assessing polarization through morphological, biochemical, and functional assays. Furthermore, we describe how C. trachomatis development can be monitored in polarized cells through microscopy, flow cytometry, immunofluorescence, and reinfection assays.