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Page 456 Shami-shah et al. Extracell Vesicles Circ Nucleic Acids 2023;4:447-60 https://dx.doi.org/10.20517/evcna.2023.14

A PATH TOWARDS CELL-TYPE SPECIFIC EV ISOLATION
With continued advances in EV isolation techniques on many fronts, cell-type specific EV isolation is more
[65]
possible than ever. However, several challenges persist [Table 1]. Given that every cell type in the human
body secretes EVs into biofluids, relative proportions of cell-type specific EVs can depend partly on the
abundance of that cell type. Additionally, EV secretion rate has been shown to differ drastically between cell
[29]
types in cultured cells . While cell-type specific EV secretion rates have yet to be determined in vivo, both
low cell-type abundance and low basal EV secretion rates make some cell-type specific EVs difficult to
isolate using conventional technologies due to their low relative concentrations in biofluids. Furthermore, in
plasma, EVs originating from tissues have been estimated to account for less than 1% of all EVs, as opposed
[30]
to the 99.8% generated from hematopoietic cells . Because EVs act as antigen-presenting vesicles with
various membrane-bound proteins, it may be possible to capitalize on this inherent property for targeted
EV isolation . Therefore, targeted immuno-enrichment or affinity isolation-based methods are an essential
[2]
tool in using tissue-derived cell-type specific EVs for 'liquid biopsy', as the analyses of their biomolecular
contents can only be successful if their relative abundance is selectively increased.

To advance affinity-based techniques, a crucial step is to identify cell-type specific transmembrane proteins
that can be used as handles for immuno-isolation. Proteins in the human body can exist in multiple
isoforms due to alternative splicing. Consequently, in many instances, a particular transmembrane protein
can have both transmembrane as well as secreted isoforms. Hence, an antibody used to capture the
extracellular domain for EV immuno-isolation could capture both the transmembrane and secreted forms
and confound results . Therefore, selection of cell-type specific markers based solely on transcript or gene
[23]
enrichment analysis in a particular cell type is not sufficient. Thorough isoform analysis should be
performed to aid in the rational selection of both immuno-isolation targets and antibodies that ideally bind
epitopes present only in the transmembrane isoform but not in the secreted isoforms. With the growing
availability of human proteome, secretome, and single-cell transcriptomics databases, our rational approach
to select targets should improve over time .
[3]
Immunoaffinity techniques can be paired with other technologies discussed in this review to create hybrid
EV isolation procedures that increase the efficacy of immuno-enrichment [Table 1]. For example,
preliminary sample processing with SEC or DMC can help pre-purify EVs by removing secreted target
isoforms that may interfere with EV immunocapture, and/or reduce the concentration of lipoproteins that
may adhere to surfaces nonspecifically. Additionally, immunoaffinity paired with microfluidics could offer
several advantages. Using microfluidic hybrid techniques, EVs could be selectively filtered based on their
size, shape, and surface biomarkers, which may allow for the isolation of even rarer subtypes. Regardless of
the technique chosen, these complementary purification steps must be carefully optimized to maximize EV
yield, given the low abundance of some cell-type specific EVs. Moreover, even with effective preliminary
sample processing, the results of cell-type specific EV immunocapture can be extremely misleading if
rigorous validation of the immuno-enrichment protocol is not performed to account for nonspecific
binding. This is a crucial step in any cell-type specific EV isolation process that relies on immunoaffinity or
utilizes surfaces to which lipid particles may adhere. However, even with careful optimization, techniques
such as ultracentrifugation that can compromise the integrity of the EV membrane are not well suited for
cell-type specific EV isolation, as EV fusion and aggregation could interfere with the analysis of specific
internal cargo. Overall, rigorous target analysis paired with emerging isolation techniques should enable
cell-type specific isolation and pave the path towards a new era of EV research and clinical applications.

OUTLOOK
The growing understanding of extracellular vesicle biology, paired with advances in EV isolation tools and