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Shami-shah et al. Extracell Vesicles Circ Nucleic Acids 2023;4:447-60 https://dx.doi.org/10.20517/evcna.2023.14 Page 449

discusses key EV isolation techniques focusing on their advantages, limitations, and potential areas of future
development and provides a perspective on a path forward to achieving cell- or tissue-type EV isolation.

CHALLENGES IN EXTRACELLULAR VESICLES ISOLATION
Depending on the biogenesis pathway, EVs can be of many different subtypes such as exosomes,
microvesicles, and apoptotic blebs. Many of these EV subtypes tend to overlap in size, surface proteome,
[1,2]
and cargo, making EVs broadly heterogeneous in nature . Additionally, biofluids of interest for EV
isolation contain varying compositions of proteins and non-EV lipid particles that contribute to the
heterogeneity, complexity, and viscosity of the biofluid. For instance, analysis of blood serum and plasma
estimates that there are roughly 10 EVs and up to 10 lipoproteins (LDLs/VLDLs/HDLs) per mL of blood
11
16
plasma [21,22] . Furthermore, many biofluids, including blood plasma, serum, and cerebrospinal fluid (CSF),
have a high abundance of albumin and matrix proteins. The lower relative abundance of EVs in these
complex matrices can make high-purity EV isolation difficult, as both proteins and lipoproteins can co-
isolate with the EVs [21,23,24] .

Apart from the problem of biological heterogeneity in EV isolation, many technical challenges persist that
can also impact the overall yield and purity of EVs. Lipids have a high propensity to adhere to the walls of
plastic tubes . Additionally, researchers have shown that plastics can destabilize membranes by mechanical
[25]
[26]
stretching . Evtushenko et al. have shown that EV losses can reach 51% ± 3% when cell-culture-derived
EVs are stored for 48 h at +4 °C in polypropylene Eppendorf tubes. Such significant EV loss could be
[27]
reduced to 18%-21% by using Eppendorf Protein LoBind tubes or surface blocking with protein blockers .
Other critical challenges in EV isolation can be attributed to the methods employed for sample collection.
Although EV counts seem to remain consistent over time in undisturbed samples, mild agitation designed
to simulate blood handling during transportation results in a notable and artificial discharge of EVs derived
[28]
from platelets . Moreover, EVs tend to form EV-blood cell clusters over time, which can further impact
EV isolation. Overall, the broad heterogeneity of EVs and their low relative abundance in biofluids, paired
with technical inconsistencies associated with sample collection, pose a major challenge for EV isolation.

CHALLENGES AND OPPORTUNITIES IN CELL-TYPE SPECIFIC EV ISOLATION
EV subpopulations that originate from specific types of tissue in the body carry the biomolecular signature
of their cells of origin. EVs are rich sources for a new form of 'liquid biopsy' because they have a lipid shell
comprising transmembrane proteins on their surface and cargo proteins, nucleotides, and metabolites
inside of them . They may provide detailed molecular profiling of cells from hard-to-access organs (e,g.,
[2-4]
brain), which would otherwise require an invasive biopsy. This can open diagnostic opportunities for
diseases that cannot be detected presently with blood tests. Additionally, cell-type specific EVs could pave a
pathway to study disease etiology by monitoring progressive changes at the cellular and molecular levels in a
minimal to non-invasive way.

One of the major barriers to cell-type specific EV isolation is the rare and very low abundance of cell- and
tissue-type specific EVs in human biofluids. For instance, blood plasma is a repository of EVs from many
different cells and tissues. Auber et al. used EV RNA cargo profiling to estimate that, of the total EVs in
healthy blood plasma, around 99% originate from platelets, erythrocytes, and white blood cells. Less than 1%
originate from solid tissues [29,30] . Furthermore, when the researchers examined the EV-to-parental cell type
ratio on a cell-specific level, they observed a they observed a significant range spanning four orders of
magnitude. That range extended from 0.13 ± 0.1 erythrocyte-derived EVs per erythrocyte to 1.9 ± 1.3 × 10
3
monocyte-derived EVs per monocyte . Therefore, this broad range in the number of EVs released per cell
[29]
between different cell types, along with existing contaminants in the biofluids, make isolating cell-type