Table 4 Methods for exosome characterization

Ultracentrifugation [42]

400×g (to remove cells and large cell debris)

10,000–20,000×g (to remove large debris and intact organelles)

100,000–150,000×g (to pellet exosomes)

Golden standard, obtain highly pure exosomal fraction

1. Only valid for exosomes purified from cell conditioned medium, but not the body fluids with complex mixture of many components

2. It sediments exosomes as well as other vesicles, proteins, and/or protein-RNA aggregates

3. Time-consuming, labor-intensive, and requires expensive equipment

Size exclusion (filtration [75] or chromatography [137])

Filtration through a series of filters down to 100 nm pore size followed by centrifugation (100,000×g) to concentrate

Collect exosomes away from smaller protein contaminants

Risk of impurity or fragmentation of larger vesicles under filtration pressure

CCM or biofluid is dissolved in the mobile phase followed by passing through the stationary phase, wherein the various constituents of the mixture travel at different speeds so as to separate

Preserves the integrity and biological activity of exosomes

1. Deformation and breaking-up of larger vesicles, which may potentially skew results

2. Requiring a long run time, limiting its scalability for high-throughput applications

Immune affinity capture [138]

Incubate CCM with specific microbeads to bind exosomes, separate exosome-bound microbeads from CCM using solid support magnet or flow cytometry

Collect exosomes with specificity

Yields are often quite low

ExoQuick precipitation methods [139]

This precipitation solution is combined with biofluid containing exosomes and is incubated overnight at 4 °C. The mixture is then centrifuged at low speed to form a pellet containing exosomes

1. Enable high-throughput, quantitative isolation of exosomes from low sample volumes

2. No need specialized equipment and shorten the operation time just in less than 2 h

3. Be efficient, reliable and reproducible

Co-precipitating non-vesicular contaminants, such as lipoproteins and polymer materials

Microfluidic technologies (ExoChip) [140]

Immunoaffinity, sieving, and trapping exosomes on porous structures

Quantitative and high-throughput analysis of exosome contents with high sensitivity

Inadequate quality control and normalization across study groups, not yet in clinical use

Dynamic light scattering [141]

Determine the fluctuations in the intensity of light scattered from nanoscale particles in solution, thereby calculating the sizes and the diffusion coefficients of the particles under Brownian motion

1. Accurate, reliable, and repeatable particle size analysis in 1 or 2 min

2. Size measurement of molecules with MW < 1000 Da

3. Low volume requirement (as little as 2 µL)

Low refractive index of vesicles and a bias towards detection of larger particles renders it problematic to distinguish MVs from exosomes in mixtures

Nanoparticle Tracking Analysis [81]

Quantification of particles in the range of 50–1000 nm in liquid suspension through real-time image of light scattered from individual nanoscale particles moving under Brownian motion

Detection of single vesicles with a diameter as low as 50 nm

1. Only semi-quantification of the exosomes/microvesicles preparations

2. Risk of no detection of nanoparticles with a certain size or optical density

3. Considerable intra-assay count variability exists

Surface plasmon resonance [142]

Sensitively detects molecular interactions occurring on the metal/dielectric interface through monitoring minute changes in the refractive index resulting from adsorption of molecules onto the metal surface, which is then converted into the surface bound mass and related to the concentration of exosomes in solution

1. Competence for the total mass of exosomes, including proteins, lipids, and nucleotides

2. Compatibility with selective surface immobilized capture molecules for the binding partner; under investigation

3. Small sample volumes

Inadequate quality control and normalization across study groups; not yet in clinical use