Methodological Guidelines to Study Extracellular Vesicles

All body fluids contain cell-derived membrane-enclosed vesicles. Such vesicles are shed by prokaryotes and eukaryotic cells and contain messages to the environment. Cell-derived vesicles are thought to contribute to homeostasis, disease development, and progression, may provide novel biomarkers, and may be suitable for use as therapeutic drug carriers.

Owing to the relationship between these extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. Researchers from the University of Amsterdam provide an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, they focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.

Working principle of common methods to isolate extracellular vesicles (EVs)

exosomes

Separation is based on size, density, and immunophenotype. Straight brackets: isolated EVs; yellow: soluble components; and blue: buffer. A, In differential centrifugation, separation is based on size, and large EVs (gray) collect earlier at the bottom of the tube and at lower g forces than small EVs (green). The soluble components are not affected by centrifugation, but non-EV particles such as lipoproteins and protein aggregates may copellet with EVs. B, In density gradient centrifugation, separation is based on density, and EVs will travel to their equilibrium density. Non-EV particles such as lipoproteins may coelute with EVs because of similar density or interaction. The soluble components with a high density relative to the gradient will collect at the bottom of the tube. C, Size exclusion chromatography uses a porous matrix (dotted circles) that separates on size. Soluble components and particles smaller than the size cutoff enter the porous matrix temporarily, whereas EVs and particles larger than the size cutoff do not enter the porous matrix. As a result, EVs and particles larger than the size cutoff elute before the soluble components and particles smaller than the size cut-off. D, In ultrafiltration, soluble proteins and particles smaller than the size cutoff (≈105 kDa) are pushed through the filter, and the EVs are collected at the filter. E, In immunocapture assays, EVs are captured based on their immunophenotype. EVs are captured using an monoclonal antibody (mAb) directed against an antigen exposed on the targeted (green) EVs only. F, In precipitation, addition of a precipitating agent induces clumping of EVs, non-EV particles, and soluble proteins. The clumps will sediment, and sedimentation can be accelerated by centrifugation.

Coumans FAW, Brisson AR, Buzas EI, Dignat-George F, Drees EEE, El-Andaloussi S, Emanueli C, Gasecka A, Hendrix A, Hill AF, Lacroix R, Lee Y, van Leeuwen TG, Mackman N, Mäger I, Nolan JP, van der Pol E, Pegtel DM, Sahoo S, Siljander PRM, Sturk G, de Wever O, Nieuwland R. (2017) Methodological Guidelines to Study Extracellular Vesicles. Circ Res 120(10):1632-1648. [article]

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