Human salivary extracellular vesicles: a comparative study of isolation techniques

Saliva offers a valuable source of analyte for extracellular vesicle (EV) -based biomarker research, however, due to the complex composition of saliva, the isolation of salivary EVs remains technically challenging. The aim of our study is to systematically compare three salivary EV isolation techniques by assessing the obtained EV sample quality and quantity, and to highlight differences between the three methods with respect to costs, isolation time, and potential for parallel sample handling (i.e. sample throughput). For the enrichment of EVs we used three EV isolation techniques with saliva samples of four healthy donors: polymer precipitation (PP), membrane affinity (MA), and size exclusion chromatography (SEC). The vesicles obtained were characterized via transmission electron microscopy (TEM), SDS-PAGE and Western blot (WB), and conventional nanoparticle tracking analysis (NTA) and interferometric nanoparticle tracking analysis (iNTA). Our results indicate that all isolation methods resulted in successful enrichment of EVs of similar size distribution. However, we observed differences with respect to vesicle yield and detectability of positive EV markers, potentially reflecting differences in vesicle integrity and sample purity. Assessment of particle number via NTA and iNTA approaches revealed prominent differences in EV number of MA samples but not in the case of samples prepared with other enrichment methods, highlighting the importance of the choice of analysis method. Our study highlights advantages and potential shortcomings of three salivary EV isolation approaches, and calls attention to the importance of multimodal analysis of EV samples, combining approaches for EV marker detection, imaging, NTA and iNTA.