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Raposo et al. Extracell Vesicles Circ Nucleic Acids 2023;4:240-54 https://dx.doi.org/10.20517/evcna.2023.18 Page 244

[44]
and midbody remnants that can also be considered EVs. A new particle, the Exomere, possibly not of
[45]
endosome or plasma membrane origin, has been recently described and reported to transfer cargo ,
although no biogenetic pathway(s) has been assigned to it [Table 1]. Lastly, the molecules surrounding EVs,
[46]
so-called the corona , may influence EV function. They are not merely contaminants as EVs from blood
plasma surrounded by such a “corona” were functional by inducing an increased expression of TNF-α, IL-6,
[46]
CD83, CD86 and HLA-DR in human monocyte-derived dendritic cells ; other proteins associated with the
outer leaflet of EVs, such as tetherin, allow EVs to cluster to potentially increase their affinity for a target .
[47]
In short, massive heterogeneity requires the development of optimized isolation and quantification
procedures to standardize reporting of results and to better assign the observed functions to different types
of vesicles and vesicle subpopulations . Currently, several methods are being used depending on the
[39]
starting samples (e.g., conditioned media from cell cultures, biological fluids, tissues, model organisms), the
paucity of material that can often be limiting depending on the applications. When handling conditioned
media from cultured cells, differential ultracentrifugation is generally used as a first approach followed by a
thorough characterization using western blotting, nanoparticle tracking, and importantly, electron
microscopy, the only method with enough resolution to visualize small membrane-bound vesicles and even
their origin (namely, budding from the plasma membrane or endosome fusion with the plasma
membrane) [48,16,49] . Floatation gradients (sucrose, Percoll) are also generally performed to show the vesicular
nature of the isolates, including their density . Moreover, possible heterogeneity can be appreciated by
[48]
monitoring the distribution of marker proteins using western blot. Interestingly, mass spectrometry reveals
several protein components that can be common to exosomes and ectosomes from different cell types in
addition to cell type-specific proteins . These methods are certainly lengthy and difficult to adapt to small
[50]
[51]
amounts of material. Other methods include Size Exclusion Chromatography (SEC) and Asymmetrical-
flow field-flow fractionation that optimally maintain EV function. Immunoaffinity-based methods,
[52]
ultrafiltration, anion exchange chromatography, and microfluidics have promising possibilities . All these
[53]
methods have advantages and disadvantages and allow the isolation of EVs with different features such as
density, size, and charge. The characterization of recovered EV fractions and description of the methods
used is essential in the reporting of results (see MISEV Guidelines) .
[39]
ELUCIDATING THE MOLECULAR MECHANISMS THAT OVERSEE EV BIOGENESIS AND
SECRETION-A MAJOR CHALLENGE FOR THE BIOMEDICAL RESEARCH COMMUNITY
The intracellular mechanisms that govern the biogenesis and secretion of EVs are clearly complex and
overlapping. Moreover, our understanding of their depth and scope is clearly incomplete, even at this stage
in the work. An important goal in unraveling the molecular mechanisms of EV biogenesis, secretion and
targeting is to eventually understand EV function and whether one can selectively interfere with EV
generation, release, and targeting. Over 20 years ago, the discovery of the ESCRT machinery in yeast and in
mammalian cells revealed a first hint that led ultimately to a general understanding of how cargo is sorted
within endosomes and incorporated into newly formed intraluminal vesicles that could either be released as
exosomes or transferred to lysosomes for degradation [54,55] . Although these initial observations offered great
progress, the overall picture of EV biogenesis remains a sketch and appears much more complex than
[6]
initially conceived . The biogenesis of extracellular vesicle subpopulations relies on several steps that are
common to exosomes and ectosomes, the latter referring to vesicles emanating from the plasma membrane.
A membrane microdomain is formed engaging specific sets of cargoes, including a portion of cytosol, after
which the domain buds and forms a neck that fissions to release a vesicle either directly in the extracellular
milieu, if happening at the plasma membrane, or into the lumen of a multivesicular body. If ESCRT
subunits can modulate the sorting of certain cargoes leading to the formation of a subpopulation of
intraluminal vesicles within endosomes, then it seems reasonable and perhaps likely that selected ESCRT
[56]
subunits may also operate at the plasma membrane . There are other protein components and lipid-based