EVerZom provides services to evaluate the size and biophysical properties of exosomes.
Measuring size of exosomes
Extracellular vesicles (EV) are a heterogeneous population of biological particles. In the perspective of therapeutical production, EV size is one the simplest measurement to follow batch-to-batch reproduction, as a surrogate marker of EV identity and with major consequences on EV loading or biodistribution for instance. EV size is generally provided as a size distribution covering the heterogeneity of the EV populations, associated to statistical values such as the diameter mean, median or mode. Depending on the measurement method, the provided size is often quite different. This is due to several phenomena: 1) some technics do not detect smaller particles, which automatically increase mean and median diameters; 2) some technics measure the hydrodynamic diameter Dh, related to its diffusion properties while others measure the true particle diameter D. Dh>D, as it also considers the corona of particles diffusing together with the vesicle. The main technics available and recommended to measure EV size are: 1) nanoparticle tracking analysis; 2) electronic microscopy; 3) interferometry; 4) atomic force microscopy; and 5) resistive pulse sensing. Dynamic light scattering is not recommended as it under-estimate the size heterogeneity of the sample.
Nanoparticle tracking analysis (NTA)
NTA follows single particles movement into a solution and calculate their hydrodynamic diameter based on Brownian motion equations. Its advantage are its association with concentration information to provide distribution per volume, and its simplicity once the instrument is available. However, optical limitation reduces its sensitivity to vesicles bigger than 50-70 nm in scatter mode. Fluorescent staining may help to detect smaller particles. EVerZom offers NTA measurements of any kind of samples using the ZetaView® QUATT (Particle Metrix, Germany) or the NS300 (Malvern, UK) with the cheapest price compared to other analysis.
Electronic microscopy (EM)
Different EM technics exist, but cryo-transmission EM is the gold standard to determine extracellular vesicles size. Its advantage is the direct visualization of EV, providing information on morphology and the true particle diameter of any size. However, it is quite complex, cryo-TEM services are very expansive instruments, and the size distribution is independent of concentration. EVerZom offers cryo-TEM services.
The instrument can provide extracellular vesicles size measurement using interferometric images. The advantages are the selection of particles for positive EV markers (CD81, CD63, CD9), and their sensitivity up to 50 nm, better than NTA. EVerZom is using ExoView® R100.
Atomic force microscopy
AFM allows surfaces to be imaged and forces to be measured from an atomic point of view, either in a gaseous or liquid environment. It can measure EV size with a very small sample, and offers structural information. However, its resolution can be limited to 100 nm, and is not adapted to routine measurements.
Resistive pulse sensing (RPS)
Tunable RPS is a recent method using an elastic diaphragm with adjustable holes adapted to a particular size range. Particles crossing the hole cause a transient change in the resistance between the inner and outer electrodes of the small orifice tube in the circuit of the constant current design to generate a potential pulse1.
1. Sivakumaran, M. & Platt, M. Tunable resistive pulse sensing: potential applications in nanomedicine. Nanomed. 11, 2197–2214 (2016).
To summary, each technique has its own pro and cons, and will not give similar results. For this reason, different samples should always be compared based on the same instrument measurement.