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Page 2 Ribovski et al. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305 https://dx.doi.org/10.20517/evcna.2023.26

INTRODUCTION
Extracellular vesicles (EVs) are lipid bilayer vesicles that are secreted by cells and contain a diverse array of
cargo, including proteins, lipids, DNA, and RNA. They play a role in cell-cell communication through the
delivery of their cargo to recipient cells and thereby influencing cellular processes such as proliferation,
[1-3]
differentiation, and immune responses . EVs can be divided into three subpopulations according to their
biogenesis route and size: exosomes are formed through the inward budding of the late endosome and have
a size between 30-150 nm, microvesicles or ectosomes (150-2,000 nm) are created through outward budding
of the plasma membrane, and apoptotic bodies (500-4,000 nm) are the result of outward blebbing of the
plasma membrane during apoptosis. The different populations of vesicles are highly heterogeneous, and
their methods of isolation and classification are under constant scrutiny. The MISEV 2018 guidelines
suggested EV-enriched markers to identify the subcellular origin of secreted vesicles and if those could not
be validated to refer to small EVs (sEVs) or large EVs (LEVs) purely on the basis of their size, i.e., being
smaller or larger than 200 nm, respectively . Thanks to the development of innovative methods to study EV
[4]
biogenesis, endocytosis and cargo release, researchers have started to unveil EV structure-function
relationships, leading to a vast increase in knowledge on EV behavior and function.

With regard to the formation of EVs, a few main regulators and machineries can be pointed out. Exosomes
originate from late endosomes upon the inward budding of the endosomal membrane, forming
intraluminal vesicles (ILVs). When the multivesicular body (MVB) fuses with the plasma membrane, the
[5,6]
ILVs are released into the extracellular space and are now called exosomes . Besides small GTPase activity,
both endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent
mechanisms control the formation and release of ILVs . Microvesicles are plasma-derived vesicles
[7-9]
originating from the outward budding of the plasma membrane. The vesicles are released directly after
formation and fission, which involves the ESCRT machinery together with small GTPases [10,11] . Apoptotic
bodies are a product of programmed cell death, i.e., apoptosis. Cells start to bleb and disassemble into
membrane-enclosed fragments, named apoptotic bodies, which are typically heterogeneous in size and
[12]
content .

Size and composition are highly heterogenous both between and within EV populations. Besides size and
surface composition heterogeneity, cargo heterogeneity is also observed among subpopulations of EVs, e.g.,
in exosomes [13,14] . Differential centrifugation and size exclusion chromatography are two largely employed
methods to isolate EVs and separate subpopulations based on size. Immunoaffinity-based methods are
effective options for highly selective populations, but are not suitable for large-scale production and limited
to the use of validated marker proteins for which antibodies exist with high affinity and specificity. Polymer-
based precipitation of EVs is simple and provides a high yield, but at the cost of heterogenous
populations [15-17] . Combinations of the above-mentioned methods may be beneficial for isolating specific
populations or enhancing purity and yield [18-21] .

Because the content of EVs reflects the physiological state of the cells they are derived from, understanding
the fundamental biological processes that control extracellular vesicle biogenesis and trafficking holds great
potential to develop EVs as drug delivery systems or signaling pathway modulators within disease contexts.
This review discusses the current knowledge on cellular interactions with EVs, their uptake and intracellular
trafficking, and cargo delivery, highlighting the reported techniques for studying endocytosis and cargo
release.