Generation of a Mouse Prostate Organoid-Based Model for Studying Host-Pathogen Interactions.

Aging men are highly affected by prostate diseases, such as benign prostatic hyperplasia, prostatitis, and prostate cancer. Together, these conditions contribute significantly to the global health burden and are a leading cause of illness in men. Organoid models have transformed disease modeling by allowing the replication of key features of epithelial structure and function in vitro. However, the closed lumen structure of organoids limits experimental access to the apical surface, hindering studies of host-pathogen interactions. To overcome these challenges, we generated a physiologically relevant culture system in which cells derived from mouse prostate organoids are seeded as a two-dimensional (2D) model. This approach maintains essential epithelial characteristics while providing direct access to the apical surface. This step-by-step protocol describes the dissociation of mature mouse prostate organoids and optimal seeding conditions to support cell attachment, proliferation, and differentiation. Immunofluorescence microscopy confirmed the expression of prostate-specific markers in the organoid-based model and demonstrated an accurate representation of the prostate tissue's cellular organization. TEER measurements, along with F-actin and tight junction staining, further confirmed cell differentiation and barrier formation. Infection of the model with uropathogenic Escherichia coli (UPEC) demonstrated its utility in investigating host-pathogen interactions. This model provides a valuable platform to investigate prostate epithelial biology and disease mechanisms and is particularly well-suited for studying host-pathogen interactions and evaluating potential therapeutic strategies.