Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics

Abstract

Nano-sized extracelullar vesicles (EVs) released by various cell types play important roles in a plethora of (patho)physiological processes and are increasingly recognized as biomarkers for disease. In addition, engineered EV and EV-inspired liposomes hold great potential as drug delivery systems. Major technologies developed for high-throughput analysis of individual EV include nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (tRPS) and high-resolution flow cytometry (hFC). Currently, there is a need for comparative studies on the available technologies to improve standardization of vesicle analysis in diagnostic or therapeutic settings.We investigated the possibilities, limitations and comparability of NTA, tRPS and hFC for analysis of tumor cell-derived EVs and synthetic mimics (i.e. differently sized liposomes). NTA and tRPS instrument settings were identified that significantly affected the quantification of these particles. Furthermore, we detailed the differences in absolute quantification of EVs and liposomes using the three technologies. This study increases our understanding of possibilities and pitfalls of NTA, tRPS and hFC, which will benefit standardized and large-scale clinical application of (engineered) EVs and EV-mimics in the future.

Systematic review of factors influencing extracellular vesicle yield from cell cultures

“…Currently, the qNano (Izon Science) is the most widely used TRPS system in the EV field and commercial tunable nanopores suited to measure polydispersed EV samples are available. The smallest tunable nanopores (NP100) can measure particles in a size range of 50-70 to 200 nm (Maas et al 2014(Maas et al , 2015. However, this does not cover the full size spectrum of EVs, which stretches down to 30 nm (Vlassov et al 2012).…”

“…It has been suggested that the variability in the lower detection limit could be due to pore-to-pore variability (Coumans et al 2014). If a sample contains particles with larger diameters than the pore, clogging can occur (Maas et al 2015). The qNano sample flow is controlled by a variable pressure module, allowing for a more sensitive concentration estimation (Willmott et al 2010).…”

Extracellular Vesicles in Renal Diseases

“…Different techniques, including NTA/RPS and FCM, have been now thoroughly compared in two studies. 17,21 It is very clear that absolute quantification of EVs substantially differs among the methods described above. A gold standard for quantification of such particles has not yet been established but is being addressed by 43,55 In addition, these techniques lack the capacity for visual differential confirmation between small cells, debris, and MPs or exosomes but can be used as confirmatory test, such as to show an exosome-specific marker in an EV sample preparation.…”

“…18,19 Currently there is no single method which can phenotype, size and enumerate the whole range of EVs, therefore posing a challenge to truly understand the biology of EVs. 17,21 In this review, we will summarize the current knowledge about the characterization of EVs and the evolving more accurate detection techniques, and provide an overview of the role of EVs in various renal diseases.…”

Tunable Resistive Pulse Sensing: Potential Applications in Nanomedicine

“…The researchers concluded that the differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The importance of correct settings to obtain accurate and reproducible results were highlighted in the study undertaken by Mass et al [62]. They undertook a study to compare nanoparticle tracking analysis (NTA), high resolution flow cytometry and TRPS in quantifying EVs and 212nm sized synthetic liposomes.…”