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.

Cit a tio n fo r fin al p u blis h e d ve r sio n: v a n Ni el, G uill a u m e , C a r t er, D avid R. F., Cl ayt o n, Ale d, L a m b e r t, D a ni el W., R a p o s o, G r a ç a a n d Vad er, Pi e t e r 2 0 2 2. C h all e n g e s a n d di r e c tio n s in s t u dyi n g c ell-c ell c o m m u ni c a tio n by ex t r a c ell ul a r v e sicl e s. N a t u r e R evi e w s M ol e c ul a r C ell Biolo gy 2 3 ,

nowledge of EV biogenesis pathways and biological activities has grown rapidly in the past decade 1 (Fig. 1a,b). EVs are membrane-enclosed structures that are released into the extracellular milieu by all organisms and cell types studied so far. EVs are a diverse family in which subtypes have been defined based on subcellular origin, size, and composition: endosome-derived vesicles (including multivesicular endosome-derived exosomes with a diameter of 50-150 nm and secretory autophagosome-derived EVs); ectosomes and other microvesicles that bud from the plasma membrane (PM) as small as exosomes or up to several µm in size; midbody remnants released by dividing cells (Box 1); migrasomes trailing behind migrating cells 2,3 ; apoptotic bodies dislodged from