Automated Optofluidic Analysis of Single Extracellular Vesicles for Organoid Culture Monitoring.

Advances in organoid-on-chip technology are transforming culturing methods by enabling precise control over biochemical environments and sustained nutrient delivery. Conventional methods for assessing organoid health are invasive, requiring skilled manual handling and sacrificing the tissue for analysis. We present a fully automated, integrated system for noninvasive extraction, labeling, and optical analysis of extracellular vesicles (EVs) secreted by 3D organoid cultures. EVs contain important molecular biomarker cargo, such as nucleic acids and proteins, enabling real-time insight on the health of their cell of origin. Using a polydimethylsiloxane (PDMS) optofluidic biosensor, we demonstrate: (1) optical detection of single EVs derived from organoid culture; (2) on-chip sample processing and counting of single EVs; and (3) automating conditioned media delivery, on-chip sample preparation, and optical detection of EVs in a single integrated system. Together, these results showcase a noninvasive approach to monitoring organoid health through EV signatures present in conditioned media. The integrated platform based on a modular optofluidic analysis chip further offers a foundation for integrating a variety of downstream analysis techniques, advancing continuous organoid-on-a-chip monitoring.