When studying extracellular vesicles (EVs), as diagnostic markers or delivery vehicles, knowing the exact concentration of the EVs is especially important. Both the quantification of the number of EVs themselves and the quantification of the EV cargo (RNA, proteins, etc.) are critical. An illustration of this is clearly stated in the Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018) guidelines, which prescribes the use of at least two different techniques for characterizing single vesicles.
Despite these guidelines however, many published experiments still rely on only one method for measuring EV concentration. The most common of these methods is Nanoparticle Tracking Analysis (NTA), and although there is a growing awareness of its critical limitations, many researchers are still surprised to find that NTA is in fact grossly misrepresenting the content of their samples. Figure 1 clearly demonstrates how NTA results can be very misleading.
Figure 1: EV sample measured by Cryo-TEM, NTA and Microfluidic Resistive Pulse Sensing (MRPS or “nCS1”). The MRPS data agrees precisely with the Cryo-TEM data, while the NTA data under-reports concentration by 10,000-fold.
Why does accurate quantification of extracellular vesicles matter?
Here’s a common example: Suppose a researcher wishes to compare the therapeutic performance of different preparations of EVs or other nanoparticles such as liposomes or gene therapy vectors. Each preparation will be tested by applying to a biological system (e.g., cultured cells or an animal model) and quantifying the biological response of that system such as viability, cell morphology, differentiation activity, or other indicator.
What scientist would consider performing such an experiment without carefully controlling for the dose of each preparation? Without such a control, important performance differences between the preparations are likely to remain hidden by variability caused by poorly controlled concentration.
No matter the downstream measurement, extracellular vesicle concentration is always a critical experimental variable and must be carefully controlled to ensure clear experimental outcomes.
A recent webinar hosted by Spectradyne discusses this subject in detail. The webinar includes a presentation by Zach Troyer, Ph.D. Candidate in Molecular Virology at Case Western Reserve University entitled, “Accurate concentration enables critical improvements in isolation of extracellular vesicles (EVs).” Troyer’s talk details how fast and reliable EV concentration measurements with MRPS enabled his group to identify and eliminate a significant source of variability in their EV isolation process, leading to quantitatively superior EV preparations.
The webinar can be viewed in full by registering here.