Systematic characterization of mammalian extracellular vesicles using nano-flow cytometry

Extracellular vesicles (EVs) are nanoscale, membrane-enclosed particles that transport bioactive cargo between cells and are increasingly studied for their potential in diagnostic and therapeutic applications. Advancing EV-based technologies for these applications depend on the ability to consistently isolate and characterize vesicle populations with defined biophysical and molecular properties. Efforts to obtain pure EV populations from cell culture systems are limited by inherent EV heterogeneity, exogenous particle contamination introduced by media supplements, and the co-isolation of non-vesicular contaminants. These challenges are further compounded by the limitations of conventional EV characterization platforms, which often lack the resolution to distinguish EVs from similarly sized non-vesicular particles or to capture molecular heterogeneity at the single-vesicle scale. Together, these limitations highlight the need for analytical approaches capable of resolving EV heterogeneity and enabling comparisons across EV production conditions and isolation strategies. In this study, we used nano-flow cytometry (nFCM) for high-resolution analysis of individual EVs, enabling simultaneous measurement of particle size, concentration, and tetraspanin expression. This approach revealed substantial amounts of exogenous particle contamination in media supplements commonly used to culture EV-producing cells, and quantified differences in EV purity and yield between methods used to isolate EVs from the media of the producing cells. Additionally, analysis of EVs derived from HEK293T, U-87 MG, and hMSC mammalian cell cultures revealed cell type-specific differences in EV production and expression of tetraspanin markers CD9, CD63, and CD81. Collectively, these results demonstrate that careful selection of media compositions and isolation strategies, combined with nFCM analytical techniques can resolve biological differences in EV populations.