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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 455

get trapped [56,57] . The requirement for frequent filter and channel de-clogging, even with small sample
volumes, is a major drawback of this technique. In microfluidic affinity isolation, the sample flows through
polydimethylsiloxane (PDMS) microfluidic chips that are functionalized with affinity reagents, such as
antibodies. ExoChip™ is such a microfluidic affinity system that exploits both the affinity and filtration
capabilities of microfluidics. It consists of an array of microfluidic channels coated with a specific binding
[60]
agent, such as an antibody against the EV surface marker CD63 . When a sample containing EVs is passed
through the chip, the EVs are captured by the binding agent and retained, while other molecules pass
through. ExoChip™ has been shown to be highly efficient at isolating EVs, with a reported recovery rate of
over 90% for tetraspanins . It is also fast, with a processing time of less than 30 min. In addition, it is
[60]
relatively low-cost and easy to use, making it a promising tool for EV isolation and analysis. In microfluidic
DEP, EVs are separated from a sample using an alternating current electrokinetic (ACE) microarray
chip [58,59] . The sample is subjected to an alternating current, and the EVs are concentrated into high-field
regions within a short time scale while other components of the sample are separated based on their
dielectric properties.

There are several advantages to using microfluidics-based methods for EV isolation. They have the potential
to be automated and miniaturized, making them a high-throughput and portable method for EV
isolation [20,47] . Additionally, sample volumes as low as 10 µL can be used for isolation. Some limitations of
this technique include the requirement for specialized equipment, low sampling efficiency, frequent channel
clogging, and the need for antibodies with high affinity and specificity. Due to such limitations,
microfluidics-based methods are not yet considered standard methods for EV isolation [20,47] .

Reduced solubility approaches
EV precipitation by attenuating solubility is one of the earliest conventional approaches used to isolate EVs
from a variety of complex biofluids. One such method includes using hydrophilic polymers, such as dextran
[61]
or polyethylene glycol (PEG) . These polymers, when added to a sample biofluid containing EVs, will form
a complex with EVs due to their negative charge. The complex can then be precipitated by adding a
neutralizing agent, such as cationic beads or an excess of cationic protein. The EVs can then be recovered
from the precipitate by centrifugation at a low speed (< 1500 xg) or by filtration [20,47] . There are several
commercial PEG-based EV precipitation kits available, including ExoQuick®, ExoQuick® ULTRA (System
Biosciences), ExoPrep™ (HansaBioMed), Total Exosome Isolation Kit (Invitrogen), and miRCURY™
(Qiagen) that can be used based on the application, type, and volume of biofluid being used as a starting
[20]
material . Any approach should be thoroughly optimized, as the properties of the hydrophilic polymer and
the neutralizing agent can affect the efficiency of EV isolation.

While bulk sample yields may be higher relative to other isolation techniques, purities are often
compromised when utilizing these reduced solubility approaches. For instance, in a study conducted by
Garcia-Romero et al., a PEG-based EV precipitation method led to the highest yield for both precipitated
EVs along with protein contents from the sample . While another study by Gamez-Valero reported similar
[62]
results with high protein and EV yields using PEG-based precipitation methods, downstream cryo-EM
analysis of the precipitants did not detect any EVs, and molecular profiling of the precipitants showed EVs
that were only CD9 + /CD63-/CD81-suggesting the presence of other bulk contaminants confounding EV
[63]
isolates . Thus, PEG-based polymers are nonspecific reagents for EV precipitation that is often
accompanied by other non-EV precipitants such as albumin, lipoproteins, and immunoglobulins [47,64] . These
limitations make reduced solubility approaches ineffective for cell-type specific EV isolation. Importantly,
efficient processing time, simplicity, and low cost make this method particularly attractive for crude and
rapid EV isolation. Researchers may want to use this method for preliminary analysis before pursuing other
isolation methods that could help improve yield and purity.