Automated Microfluidic Platform for Single Spheroid Culture and Extracellular Vesicle Isolation: Application to...

Extracellular vesicles (EVs) are key mediators of intercellular communication and carry molecular information that reflects the state of their cell of origin. 3D cell cultures more accurately reflect the in vivo microenvironment and the biogenesis of extracellular vesicles compared to 2D cultures. Despite these advantages, studying EVs in 3D systems such as spheroids remains technically challenging. Conventional EV isolation and characterization methods often require pooling multiple spheroids to obtain sufficient material, which masks the intrinsic heterogeneity between individual spheroids and limits applications in precision medicine. To overcome these challenges, this work develops an automated microfluidic platform capable of single-spheroid culture, continuous secretion collection, and high-efficiency EV isolation. The platform incorporates 200 nm filtration and immunomagnetic capture targeting CD63/CD81-positive EVs, achieving a 60% recovery yield. Using adrenocortical carcinoma spheroids as a model, this work demonstrates that inhibiting β-catenin signaling selectively reduces the levels of EV-derived miR-139-5p and miR-483-5p, consistent with prior findings from 2D culture studies. This platform represents a groundbreaking approach to EV profiling at the single-spheroid level, unlocking new opportunities for personalized medicine, drug discovery, and targeted therapies by enabling the analysis of cellular heterogeneity and scarce biological samples such as patient-derived organoids.