The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium‐sized nascent EVs of THP1 cells as well as of Optiprep‐purified platelets, and incubated them in EV‐depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein‐coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α‐chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF‐α, IL‐6, CD83, CD86 and HLA‐DR of human monocyte‐derived dendritic cells, EV‐free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein ‘contamination’ of EV preparations and may add a new perspective to EV research.

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Mast cells have been shown to release extracellular vesicles (EVs) in vitro. However, EV‐mediated mast cell communication in vivo remains unexplored. Primary mast cells from GFP‐transgenic and wild type mice, were grown in the presence or absence of lipopolysaccharide (LPS), and the secreted EVs were separated from the conditioned media. Mast cell‐derived EVs were next cultured with LPS‐naïve mast cells, and the induction of TNF‐α expression was monitored. In addition, primary mast cells were seeded in diffusion chambers that were implanted into the peritoneal cavities of mice. Diffusion chambers enabled the release of GFP+ mast cell‐derived EVs in vivo into the peritoneal cavity. Peritoneal lavage cells were assessed for the uptake of GFP+ EVs and for TNF‐α production. In vitro, LPS‐stimulated mast cell‐derived EVs were efficiently taken up by non‐stimulated mast cells, and induced TNF‐α expression in a TLR4, JNK and P38 MAPK dependent manner. In vivo, using implanted diffusion chambers, we confirmed the release and transmission of mast cell‐derived EVs to other mast cells with subsequent induction of TNF‐α expression. These data show an EV‐mediated spreading of pro‐inflammatory response between mast cells, and provide the first in vivo evidence for the biological role of mast cell‐derived EVs.