Evaluation of extracellular vesicle aggregation by single vesicle analysis†

Extracellular vesicles (EVs) play pivotal roles in intercellular communication and are implicated in numerous physiological and pathological processes. Here, we introduce a quantitative technique using total internal reflection fluorescence microscopy (TIRFm)-based single vesicle analysis (SVA) to assess EV aggregation, a critical factor influencing their biological functionality. Employing two-colored fluorescent recombinant EV mixtures, this method enables precise discrimination between aggregated and non-aggregated EVs. It allows for calculating an aggregation ratio from the colocalization of fluorescence signals. We evaluate the impact of isolation methods, storage conditions, and biochemical environments on EV aggregation, including salt and pH variations and the presence of antibodies. Additionally, we quantitatively assess the efficacy of aggregation removal techniques, revealing significant variability in removal methods depending on the type of aggregates. This analytical approach is expected to enhance our understanding of EV aggregation dynamics and set a new standard for the characterization and functional analysis of EVs.