Comparison of light scattering-based detection methods for the sizing and number density characterization of extracellular vesicles (EV) isolated from human embryonic kidney (HEK) cell cultures

Extracellular vesicles (EVs) are membrane-bound nanosized particles excreted by all cells and are of significant interest for biomedical applications such as diagnostic testing and as vectors for therapeutic delivery. EVs are abundant in biofluids, including urine, saliva, blood, and cell culture media, but must be isolated from their complex matrix for use. Once isolated, a primary challenge is determining the EV size distribution and the total number of particles recovered. Multiple detection methods are currently used to characterize EV recoveries in terms of sizing and number density determinations, the most common being nanoparticle tracking analysis (NTA), flow cytometry, and electron microscopy (transmission (TEM) or scanning (SEM)). Addressed here is a practical assessment of three common light scattering-based methods for the determination of EV population sizing and number densities. NTA, multi-angle light scattering (MALS), and nano-flow cytometry (nFCM) are directly compared. Specifically, the baseline practical advantages and disadvantages of each technique are evaluated via analysis of silica nanoparticle standards. Subsequently, EVs isolated from human embryonic kidney (HEK) cell culture supernatant using a hydrophobic interaction chromatography-based separation on the previously developed polyester (PET) capillary-channeled polymer (C-CP) fiber column platform were characterized. This isolation of high-purity EVs from HEK cell culture matrix components was validated using UV chromatograms, Bradford protein assays, SEM, TEM, and fluorescence nFCM analysis. The relative attributes of these important light scattering methods are presented in terms of their fitness for specific applications and overall effectiveness in EV size and concentration analysis.