Perfusable 3D human urothelial model for real-time analysis of bacterial infection dynamics and therapeutic interventions

Urinary tract infections (UTIs) remain a major health burden, yet mechanistic studies are hampered by the lack of experimental models that enable high spatiotemporal resolution tracking of infection dynamics and that faithfully recapitulate the stratified architecture of the bladder epithelium and the fluid dynamics of the urinary tract. Here, we present a modular microphysiological platform that integrates a fully stratified urine-tolerant human 3D urothelium cultured on standard transwell inserts within a custom-designed perfusion device that supports live imaging. The system maintains continuous urine flow, enabling real-time, high-resolution imaging of uropathogenic Escherichia coli (UPEC) infections under physiologically relevant clearance of planktonic bacteria, while tissue-associated bacteria are retained. Using this platform, we demonstrate that flagellar motility and type 1 fimbrial adhesin FimH provide distinct fitness advantages during urothelial colonization. Treatment with the frontline antibiotic fosfomycin confirmed the formation of L-forms and subsequent regrowth of walled bacteria upon drug withdrawal. Furthermore, the platform helped to resolve the temporal dynamics of D-mannose-mediated abrogation of FimH-dependent adhesion and revealed strain-specific bacteriophage-mediated lysis, contrasted to broad-spectrum antibiotic activity. By combining physiological tissue architecture, controlled flow, and live imaging, this integrated system offers a predictive and scalable platform for investigating UTI pathogenesis and for preclinical testing of therapeutic interventions.