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TECHNIQUES FOR EXOSOME CHARACTERIZATION
There are various isolation procedures of exosomes from body fluids and cell culture supernatants,
including immunoaffinity capture, polyethylene glycol-mediated precipitation, ultracentrifugation, and
microfluidics. To be able to use exosomes as a tumor biomarker, it is crucial to preserve their origin,
structural integrity, molecular and genetic substance, and their functional properties as well to be suitable
for DNA, RNA, and protein analysis.

Nevertheless, the simultaneous purification of exosomes is essential since plasma-derived exosomes have
[33]
a high abundance of proteins . From current data, the mini-SEC method so far seems to be a favorable
method compared to other isolation methods due to its better recovery and consistency of vesicles, because
there is a risk of protein complex aggregation and loss of vesicles and biological functions as a result of
ultracentrifugation.

In the following section, we give an overview of commonly used exosome characterization procedures.

Electron microscopy
Electron microscopy (EM) is the gold standard for exosome identification and verification of exosome
preparation quality since it was one of the first methods used. It is also the only technique for direct
exosome visualization. However, this method is difficult to use on an everyday basis. One of the reasons is
[34]
that EM results in exosome loss during dehydration and embedding . Other variations of this technique
may improve the results such as scanning EM and cryoEM, but further quantification of exosomes is a
bigger issue.

Nanoparticle tracking analysis
This method provides an assembled analysis of Brownian motion via light dispersion to rank and size
nanoparticles in a liquid suspension [35-38] . Based on Brownian motion, analyzing over time light scatter
[39]
produced by a laser beam that hits the particles yields a size distribution and concentration . Although
good and regular calibration is essential, it is one of the more favorable methods used because it does not
depend on detecting a definite marker and also due to the suspension of exosomes in a broad range of
[39]
solutions .

Tunable resistive pulse sensing
Tunable resistive pulse sensingdiscovers the passage of distinct particles through a membrane’s pores.
This happens when temporary electrical resistance is measured. It correlates with the amount and size of
particles present. Pore size can be modified to improve the measurement. The analysis gives a concentration
[39]
and particle distribution similar to nanoparticle tracking analysis .

Developing a concordance between techniques for quantification of exosomes plays an important role in
[39]
establishing the future of exosome biomarkers in clinical use .

Immunodetection methods
These are analyses that based on the detection of mono- or polyclonal antibody bound to the antigen in the
sample.

Western blots are used for the study of exosome cargo, and specifically, the semi-quantitative densitometry
[12]
analysis of protein bands is insightful for disease progression . It also reveals that PD-L1 or TGF-β/LAP
[12]
are significantly linked with disease progression .