Systematic characterization of mammalian extracellular vesicles using nano-flow cytometry

Authors: VYZOUREK, BENJAMIN - University Of Nebraska; ANDERSON, DIRK - University Of Nebraska; SKRABAL, LUKE - University Of Nebraska; HUMPHREY, CHRISTINE - University Of Nebraska; ROMERO, EDUARDO - University Of Nebraska; SCHWEIGER, BRITTANY - University Of Nebraska; KIEVIT, FORREST - University Of Nebraska; Miles, Jeremy; PANNIER, ANGELA - University Of Nebraska

Submitted to: Extracellular Vesicle
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/10/2025
Publication Date: 12/1/2025
Citation: Vyzourek, B.T., Anderson, D., Skrabal, L., Humphrey, C.E., Romero, E., Schweiger, B., Kievit, F., Miles, J.R., Pannier, A.K. 2025. Systematic characterization of mammalian extracellular vesicles using nano-flow cytometry. Extracellular Vesicle. 6. Article 100098. https://doi.org/10.1016/j.vesic.2025.100098.
DOI: https://doi.org/10.1016/j.vesic.2025.100098

Interpretive Summary: Extracellular vesicles (EVs) are cell-derived, non-replicating nanoparticles comprised of lipid bilayer and include exosomes (30-100 nm diameter), microvesicles (50-1000 nm diameter), and apoptotic bodies (100 nm – 5 µm diameter). Research interest in EVs has substantially expanded in recent years due to their ability to transport a wide array of biomolecules and facilitate communication and function of nearby cells. However, there is a need for standardized isolation and characterization protocols to isolate and characterize EVs with higher purity and greater resolution. In addition, cell culture media components, particularly fetal bovine serum (FBS), introduces substantial contaminating particles that influence downstream applications. As a result, the objective of the current study was to develop an adjustable workflow for EV isolation from serum-free cultures in a variety of human cell lines, establish quantifiable metrics for comparing isolation methods, and optimize protocols for comprehensive particle characterization using the Flow NanoAnalyzer. This study demonstrates that serum-free culture conditions significantly reduce exogenous EV contamination from FBS while maintaining cell viability and EV production for limited incubation periods. Nano-flow cytometry effectively distinguishes EV populations, quantifies yield and purity differences between isolation methods, and resolves molecular heterogeneity at the single-vesicle level. Ultracentrifugation yields purer EV populations but at lower concentrations compared to ultrafiltration, which provides higher yields with more contaminants. Finally, distinct tetraspanin expression profiles among EVs from a variety of human cell lines reflect biological heterogeneity relevant for diagnostic and therapeutic applications. In conclusion, the approach outlined in this study provides a framework for standardized EV production, isolation, and phenotyping, enhancing reproducibility and utility in downstream applications.

Technical Abstract: 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.