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Page 16 Asao et al. Extracell Vesicles Circ Nucleic Acids 2023;4:461-85 https://dx.doi.org/10.20517/evcna.2023.37

Particularly, EVPs derived from MSCs may be effective in immune modulation and cell regeneration, and
their application in neurological and cardiovascular diseases is being investigated.

Research is also being conducted to evaluate the inhibition of EVP production or uptake to improve
[163]
pathological conditions propagated by EVPs themselves . Cancer-derived EVPs expressing PD-L1 block
antitumor immunity by interacting with programmed cell death-1 (PD-1) on immune cells, reducing the
efficacy of immune checkpoint inhibitors (ICBs) [53,55] . Hence, a combination of a peptide that destroys
tumor-derived EVPs expressing PD-L1 and ICB has been shown to improve antitumor immunity in the
[138]
mouse model . This demonstrates the success of strategies that target EVPs themselves for treatment.
In summary, as our understanding of the pathophysiology of EVPs advances, research on applying EVPs for
therapeutic purposes is rapidly progressing. Notably, ongoing research aims to enhance the functionality of
EVPs, not only by incorporating various biomolecules and compounds as cargoes, but also by modifying
the EVPs themselves. Nevertheless, the clinical application of engineered EVPs presents several challenges,
including assessing their efficacy and safety in preclinical trials, selecting appropriate source cells and
isolation methods, establishing clinical-grade manufacturing, and standardizing dosing and administration
routes. Despite the aforementioned challenges, therapies involving EVPs have advanced to the stage of
clinical trials, and it is expected that their applications will continue to expand in the future.

TECHNOLOGICAL ADVANCES ENABLING EVP RESEARCH
The progress in EVP research cannot be discussed without considering technological advancements,
including the isolation and purification of EVPs and analysis of single EVPs. In this section, we highlight
the technological progress made in EVP analysis over the last decade.

Isolation and purification of EVPs
A variety of EVP isolation methods exist based on various EVP properties: traditional differential
ultracentrifugation and ultracentrifugation using density gradient, for example, rely on size and density
differences [172,173] . More recently, size exclusion chromatography (SEC) and ultrafiltration have also emerged
as size-based separation methods [174,175] . Other methods based on surface markers, such as immunoaffinity
capture (IAC), and polymer precipitation using commercially available kits, have also been developed.
Recently, asymmetric flow field flow fractionation (AF4) emerged as a method to separate small-size EVPs
that contain various populations [12,14] . AF4 controls two streams flowing through a thin channel with a semi-
permissive bottom wall membrane, allowing separation of EVPs ranging from a few nanometers to 100 nm
in size. AF4 has enabled the separation of ENPs smaller than 50 nm that were difficult to separate using
traditional methods, facilitating the characterization of ENP cargo and function. Microfluidic-based
technologies are also gaining attention as a method to separate and analyze EVPs from small sample
volumes [176,177] . By flowing the sample through microchannels on the micron scale and detecting EVPs based
on surface markers or size, it is possible to detect and analyze EVPs from limited samples at high
throughput, rendering this method highly sought-after. Each separation method has its own advantages and
disadvantages, and a standard method has not been established [16,178] . The main challenges are how to
effectively separate heterogeneous EVPs, prevent co-isolation of non-EVP material (such as aggregate
proteins, viruses, and lipoproteins), prevent the loss of EVPs during separation, and minimize time and
cost. Furthermore, the optimal separation method should be chosen according to the sample source. For
instance, plasma samples contain a variety of impurities and require more advanced separation methods.
For biomarker discovery and detection, it is desirable to use methods that can separate EVPs from small
amounts of samples, while for therapeutic purposes, it is necessary to consider methods that can efficiently
separate large amounts of EVPs.