An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 31 INTRODUCTION Extracellular vesicles (EVs) are lipid bilayer membrane structures (30-8000 nm in diameter 1) released by cells. They are involved in cellular communication through transfer of surface receptors and/or a variety of macromolecules carried as cargo (e.g., lipids, proteins, nucleic acids, protein-coding mRNAs and regulatory microRNAs) 2,3. As EVs are excreted by virtually all cell types in the human body, they can be found in most body fluids, such as the blood 1, saliva 4 and urine 5,6. Often regarded as a “snapshot” of the status of the cell of origin, EVs are examined for their biochemical signatures to assess the presence of various diseases, e.g., cancer or viral infections 7,8, and are considered excellent minimally invasive biomarkers in so-called liquid biopsies 9-11. While no unique antigens representative for specific EV classes and subpopulations have been reported to date, tetraspanins (CD9/ CD63/CD81) are recognized as common antigens. These proteins are enriched on EVs and are involved in EV biogenesis, cargo selection, and cell targeting 12,13. Despite the increased interest in EVs as biomarker, their quantification and characterization is hampered by physical characteristics such as their small size and low epitope copy number 14, the variety of their protein markers depending on the cell source, and the confinement of some markers to the luminal side of the EVs 3,15. The identification of EVs in blood plasma is further hindered by the molecular complexity of the plasma, which contains multiple elements (e.g., lipoproteins, cell debris and soluble proteins), that interfere with EV analysis 3,16. Moreover, a lack of robust methods and ambiguities in how data should be interpreted for EV analysis makes data interpretation between studies challenging 17,18. Currently, the gold standard approach for EV analysis is based on the isolation or concentration of EVs. Ultracentrifugation, density-gradient, and size exclusion chromatography are the most widely used EV isolation techniques 19, despite yielding low-purity EV samples due to the co-isolation of non-desired molecules such as lipoproteins 3,16. Additionally, a variety of analytical platforms are available. Nanoparticle tracking analysis (NTA) allows the determination of the size distribution and a rough indication of the concentration 20 of individual nanoparticles in suspension, but provides limited phenotyping capabilities. In turn, transmission electron microscopy (TEM) is able to image particles <1 nm, but is time consuming. Other methods, such as ELISA and Western blot analysis, offer bulk phenotyping abilities but lack quantification 5,21-23. Thus, a tool for the accurate determination of the concentration and phenotyping of single EV in complex samples such as plasma represents an unmet need.
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