8.2. Methods for the Characterization of Exosomes

After isolation, it is necessary to evaluate whether the isolated particles are in fact exosomes, as well as the level of purity. In this sense, exosome characterization methods are mainly divided into two types: external characterization (morphology and size detection) and exosomal marker proteins (membrane and intraluminal proteins).

Electron microscopy (EM) is necessary to characterize the morphology of exosomes since particles smaller than 300 nm are too small to be observed with optical methods [313,314]. When studying biological samples, two types of EM are widely used, namely transmission electron microscopy (TEM) and scanning electron microscopy (SEM) [315]. The former is considered the gold standard for studying exosome morphology.

With TEM, a two-dimensional image is obtained. Since the wavelength of the electron beam is shorter than the wavelength of visible light by three orders of magnitude, images are recorded with a resolution of 1 nm. In addition, immuno-gold labeling opens the possibility of detecting specific proteins within the exosomes [314]. Unfortunately, the benefits of high resolution can easily be outweighed by the disadvantages related to sample preparation, as they must be fixed and dehydrated prior to their observation by TEM. In addition, image acquisition is performed under vacuum conditions which can further alter the morphology of exosomes [315].

Under TEM, exosomes appear round, making it possible to determine their diameter. This could be used as a confirmation of exosome presence in the sample, if particles are within the reported sizes for exosomes. Furthermore, exosomes have a characteristic cup shape when observed by TEM. This shape is attributed to the collapse of the structure after dehydration [316].

Although SEM resolution is lower than TEM’s, SEM has emerged as an alternative for exosome characterization [316]. Instead of using a wide beam as in TEM, SEM uses a fine-tipped beam that scans the samples line by line. As a result, instead of a two-dimensional image, SEM scans the surface of the samples to provide a three-dimensional image of the exosomes [314]. Wu et al. reported that, unlike the cup shape of exosomes observed under TEM, SEM showed rounded, bulging exosomes without a central depression. Diameter of exosomes appears to be similar when observed with either TEM or SEM. [316]. Like TEM, samples analyzed under SEM are fixed and chemically dehydrated in several steps, which often introduces artifacts that change the apparent exosome morphology [317]. Moreover, in some cases, the electron beam can also cause damage to biological samples. To circumvent these issues, cryo-electron microscopy (cryo-EM), which includes a different protocol for sample preparation, could become an alternative [314].

Cryo-EM is a type of TEM, in which samples remain in their native aqueous environments during analysis. In this method, the sample is stored and studied on vitreous ice at the temperature of liquid nitrogen, so invasive steps, such as dehydration or fixation are omitted [289]. This avoids ultrastructural changes and redistribution of elements [314]. Melyanov et al. were able to visualize with cryo-EM a broad spectrum of extracellular vesicles of various sizes and morphology with lipid bilayers and vesicular internal structures [318].

Nanoparticle tracking analysis (NTA) is a sophisticated method for measuring exosome concentration and size distribution [289]. Particles in suspension are injected into a measuring chamber where they are exposed to a laser beam. The movement of the particles over a certain period of time is then recorded by a highly sensitive camera mounted on an optical microscope. From the obtained video recording, the displacement of each particle is tracked and plotted as a function of time, which allows the calculation of the size distribution by applying the two-dimensional Stokes–Einstein equation to determine their hydrodynamic diameters [314]. The entire sample measurement process takes approximately 15 min and has better reproducibility than other methods for exosome characterization. NTA has high resolution and can detect vesicles with diameters from 30 to 1000 nm [289]. However, the lower limit can only be reached when measuring particles with a high scattering index. Exosomes range from 40 to 100 nm and may have a low scattering index [319], raising the concern that exosomes in the lower size range may not be observed as they might not scatted enough light to be detected [315].

Zhang & Lyden introduced a new method for both isolation and characterization of extracellular nanoparticles, including exosomes and exomers asymmetric-flow field-flow fractionation (AF4) [320]. With this technique, exosomes are separated based on their density and hydrodynamic properties. Exosomes flow through a forward laminar channel and, according to their Brownian motion, are classified into different populations. Smaller particles have higher diffusion rates and tend to move faster; conversely, larger particles have lower diffusion rates and tend to move slower [289,320]. This technique offers great advantages compared to other techniques, as it facilitates the successful separation of different subsets of exosomes and the identification of exomers. Moreover, the processing time is short (1 h) when compared to ultracentrifugation, and allows a number of subsequent characterizations that could provide information about the abundance of particles, particle size and purity [321,322]. However, loading capacity currently remains a drawback as large starting volumes are needed to produce sufficient material for downstream applications [321].

Tunable resistance pulse sensing (TRPS) is based on the movement of individual particles through a nanoscale pore. As the particle passes through the pore, the magnitude, duration, and frequency of the resistance pulses are detected and used to determine size, zeta potential and concentration [315]. It can detect vesicles with diameters from 50 to 1000 nm. Compared to NTA, TRPS has a higher size resolution and higher accuracy when measuring particle size distribution [289]. The main disadvantage of this technique is that TRPS measurements are susceptible to system stability problems, for instance, the pore can be blocked by particles, as well as to sensitivity problems, where particles are too small to be detected above the background noise [323].

One of the difficulties shared by the characterization methods according to the morphology and size distribution is that exosomes could be confused with other particles that may have similar characteristics, such as some lipoproteins. It is therefore important to complement these methods with the detection of exosomal protein markers to support the accurate isolation of these vesicles.

The International Society for Extracellular Vesicles (ISEV) has presented extensive recommendations for marker-based exosome characterization [324], some of which include the detection of transmembrane proteins common to all exosomes, such as tetraspanins (i.e., CD63, CD9 or CD81) as well as integrins, selectins and CD40 ligands [324]. As many of these proteins are involved in normal physiology and disease pathogenesis, they are used as important pathophysiological biomarkers of extracellular vesicles [325]. Other exosomal markers include intraluminal proteins, such as TSG101, ALIX, annexins and Rabs. It is also possible to detect any protein unique to exosomes in order to differentiate from lipoproteins that may have been co-isolated [315]. Characterization of these protein markers can be performed by Western blot, Enzyme-Linked ImmunoSorbent Assay (ELISA) or flow cytometry [315,325].

Western blot is an easy and widely known procedure. It is characterized by its accessibility and ability to detect exosomal surface and internal proteins. However, its specificity and reproducibility are limited by the quality of the antibody used, and a large amount of exosomal protein is usually needed to obtain a good signal regarding a few proteins each time [2,325]. Like Western blot, ELISA can analyze marker proteins qualitatively and quantitatively and its limit of detection is similar. However, processing is faster, making it suitable for high-throughput analysis [325]. On the other hand, flow cytometry allows for the size measurement and the observation of the structure of exosomes; while conventional flow cytometers can only measure particles larger than 300 nm, thus not allowing direct detection of exosomes [289], the new generation of flow cytometers are provided with an improved resolution, enabling the quantification and/or classification the exosomes according to the level of antigen expression detected by fluorescent antibodies or stains that will emit a signal detected by the machine [323]. Their challenge is focused on avoiding aggregation (clumping) of vesicles, which would produce inaccurate results in exosomal numbers and/or sizes [314]. In some cases, immobilization of exosomes on a bead surface is sometimes preferred, either by immunocapture or by covalent conjugation. Then, the exosomal vesicles are exposed to a fluorescently conjugated antibody against an antigen known or expected to be expressed on the exosomal surface [2,314].