The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer‐based precipitation and size exclusion chromatography (Pre‐SEC), researchers from the Instituto Nacional de Medicina Genómica analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early‐ and late‐eluting EV fractions, the researchers performed a quantitative proteomic analysis of MDA‐MB‐468‐derived EVs. They identified 286 exclusive proteins in early‐eluting fractions and 148 proteins with a differential concentration between early‐ and late‐eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, they found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies.
Schematic overview of the EV isolation protocols and experimental workflow
After 48 h, conditioned media was collected and pooled for EV isolation by ultracentrifugation (UC), precipitation (Pre), size exclusion chromatography (SEC), and precipitation coupled to size exclusion chromatography (Pre‐SEC). For further characterization of Pre‐SEC fractions, we separated two subgroups of fractions comprised by fraction 5 to 10 (SG1) and fraction 11 to 16 (SG2). Depending on the downstream analyses the input volume for Pre‐SEC workflow was adjusted as indicated in parenthesis. WB (Western blot); NTA (Nanoparticle tracking analysis); TEM (Transmission electron microscopy); MS (Mass spectrometry); DG (Density gradient)