Assessing Extracellular Vesicle-Host Interaction Using a Microfluidic Platform with Caco-2 Spheroides-on-Chip.

is a foodborne pathogen that adheres to and invades the epithelial cells of the human intestinal tract. The extracellular vesicles (EVs) of have an important impact during pathogenicity, but their role in invasion of host intestinal epithelial cells remains largely unknown. models lack the complexity of tissue and fail to accurately replicate the dynamic interactions between EVs and human intestinal epithelial cells, while animal infection models have species-specific differences that limit their translational relevance and are associated with ethical concerns. To bridge this gap, we propose a microfluidic platform integrated with an impedimetric sensor to monitor EV interactions with human intestinal epithelial Caco-2 cells. When cultured in this microfluidic device, Caco-2 epithelial cells underwent spontaneous 3D morphogenesis into spheroid-like structures with diameters ranging from 50 to 100 μm. Functional assays revealed that the secretome and EVs (multiplicity of infection, MOI 10) caused a 60% reduction in Caco-2 cell viability in 2D plate cultures, as measured by the MTT assay. In contrast, 3D Caco-2 spheroids showed significantly increased resistance to cytotoxic effects of secreted virulence factors of . By combining impedance spectroscopy and live microscopic observation, the platform allowed real-time monitoring of cellular spatial growth and sensitive detection of EV interactions with intestinal epithelial cells, highlighting the protective role of 3D cell organization. The physiological relevance of the model was confirmed by TEER measurements that suggested that EVs diffused paracellularly. The developed microfluidic device is a promising platform for investigating host-microbe interactions and may have a broad impact on biomedical research on gastroenteritis.