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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 451

[39]
sucrose or iodixanol (OptiPrep) . The process starts by filtering or centrifuging the samples to remove
debris and large particles that could interfere with the EV isolation. This process also dilutes the samples
allowing them to pass through the thick gradient during the prolonged centrifugation times required. The
sample is then layered on top of the density gradient and centrifuged at high speed for many hours,
depending on the type of biofluid, to separate the different components of the samples [23,38] . Exosomes have a
[40]
characteristic buoyant density of 1.10-1.19 g/mL on sucrose density gradients . Post centrifugation,
samples are collected from each density fraction of the gradient and EVs are found in the denser fractions.
[23]
Typically, a refractometer is used to measure the density using the refractive index of each fraction .
DGC has been a useful technique for many cell-biological applications, including isolating organelles and
[41]
[42]
determining the oligomeric states of a protein . While it has been a widely applied technique in the field of
EV research, it has some limitations for EV isolation . Depending on the medium being used, DGC
[20]
requires specialized equipment and can be expensive, low throughput, and time intensive (more than
18-hour spin to reach density equilibrium) [23,37,43,44] . Because of the long time required, DGC is not highly
applicable to use in a clinical setting or efficient enough for biomarker discovery . While DGC yields EVs
[24]
with a relatively high purity compared to ultracentrifugation, higher-order protein aggregates and
lipoproteins with similar densities to EVs persist as contaminants [20,45] . For example, while KBr-density
gradient ultracentrifugation on plasma successfully removes VLDLs from EV fractions, HDLs persist at a
ratio of EVs to HDLs estimated to be as high as 1:100 by TEM . Impurities can also arise due to the
[45]
improper setting up of the gradient, causing intermixing of the gradient interfaces and fraction mixing. All
these issues can contribute to an overall low yield and EV contamination, which should be considered when
optimizing DGC isolation . Even with these limitations, DGC has been an important technique in EV
[20]
research, providing utility both as an orthogonal validation tool for evaluating subtype EV markers and as a
method for disease-state bulk EV analysis in biofluids such as urine [23,46] .

Size Exclusion Chromatography
Size Exclusion Chromatography (SEC), conventionally referred to as gel filtration, is a method for isolating
biological species such as proteins or vesicles, based on their hydrodynamic radius, which is equivalent to
the apparent size of the solvated species [20,47] . EVs can be separated from soluble proteins based on their
ability to be excluded or pass through pores of different sizes in a chromatography column [Figure 1]. To
isolate EVs using SEC, sample is passed through the stationary phase of a chromatography column that is
packed with beads of a porous material, such as agarose (Sepharose) [24,48] , allyl dextran (Sephacryl) , or
[48]
[48]
cross-linked dextran (Sephadex) . The beads have pores of different sizes, and as the sample passes
through the column with the flow of the mobile phase, the EVs are separated based on their size. Smaller
EVs get trapped in the pores of the resin for longer times, so they are eluted from the column later, while
larger EVs avoid the pores of the resin and mostly pass through the larger channels, so they are eluted
earlier. The separated EVs are collected in fractions and can then be analyzed or used for further
experimentation [20,24,47,48] .

The separation of EVs from various biological fluids, including CSF and plasma, can be optimized based on
the column length, size, type of resin, and flow rate of the mobile phase . These parameters can further
[24]
influence the yield and purity of the isolated EVs; therefore, the protocol must be thoroughly optimized
based on the application. SEC has been adapted as a single-step isolation system and modified over the
years for greater purity and yield . This technique aids in separating small from large EVs and from
[47]
non-EV soluble protein contaminants in samples, with an average 20 min processing time, resulting in time
and cost-effective, pure, intact, and functional EV retrieval l [47,49] . SEC can also be scaled up and automated,
making it a high-throughput method for EV isolation. It can be performed with varying sample volumes,
making it useful for isolating EVs from samples with limited volume availability (e.g., CSF). Additionally,