Commentary for the incorporation of extracellular vesicle markers varies among vesicles with distinct surface charges

Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication, transporting diverse molecular cargoes such as proteins, lipids and nucleic acids. Despite their growing importance in both basic biology and clinical applications, the remarkable heterogeneity of EVs remains a major obstacle to their functional characterization. In a recent study, Maeda and colleagues developed a highly sensitive and quantitative method for monitoring EV release using high-affinity binary technology (HiBiT)-tagged marker proteins, combined with a novel chromatographic approach that fractionates EVs based on surface charge properties. This strategy enabled the identification of distinct EV subpopulations harbouring specific protein markers and differing in their biogenesis and intracellular origin. By integrating CRISPR-mediated tagging, live-cell luminescence assays and ion-exchange chromatography, the study establishes surface charge as a new physicochemical parameter for EV classification. These findings offer a powerful framework for dissecting EV heterogeneity and lay the foundation for the development of more precise EV-based diagnostic strategies.