Identification of Extracellular Vesicle Subpopulations Using Single Extracellular Vesicle Analysis

Abstract

Extracellular vesicles (EVs) are cell-secreted membranous vesicles containing cellular proteins, lipids and nucleic acids. EVs are often classified into a few subtypes called exosomes, microvesicles and apoptotics bodies according to their biogenesis. EVs are known to consist of heterogeneous population having various shapes, sizes and densities, and thus they are also expected to be heterogeneous in biological functions. However, current approaches for EV analysis are mostly ensemble methods that inevitably average out the information from heterogeneous EV populations. Although there are some single EV analysis methods such as nanoparticle tracking analysis and electron microscopies, they are mostly inefficient for analyzing biological properties of EVs. Therefore, establishing an efficient and reliable single EV analysis method for studying biological properties of EVs is urgent. In this dissertation, a single EV analysis based on total-internal reflection fluorescence microscopy is developed and demonstrated. This method can directly visualize individual EVs immobilized on a passivated surface and successfully revealed heterogeneity presented in EVs. The single EV analysis discovered unique EV subpopulations that are expected to be exosomes and microvesicles. Co-localization analysis of tetraspnin and endogenous organelle markers also provided evidences that could support origins of the two subpopulations. In addition, the tetraspanin analysis using different purification methods identified that density gradient and buoyant density gradient successfully isolated these two populations whereas early fractions of size exclusion chromatography cannot. The single EV analysis discovered many unknown properties of single EVs and it is further expected to improve our understanding of EV heterogeneity and subpopulation.