Dissertação de Mestrado Investigação Biomédica 2023 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Abstract Small extracellular vesicles (EVs) are key components of intercellular communication in multiple physiological and pathological processes. In biomedical research, EVs are gaining significant attention as disease biomarkers and therapeutic targets, particularly in cancer development and metastasis. Given the small size of EVs of interest (30 to 150 nm) and the high risk of contamination with non-small EVs, reliable isolation techniques are required. Ultracentrifugation (UC) is the gold standard for EV isolation despite being time-consuming and exhibiting poor reproducibility. Recent methods, such as size-exclusion chromatography (SEC), offer promising results, but comparative studies are necessary. In this study, we compared the yield, small EV specificity, and reproducibility of EV isolation between SEC, combined with ultrafiltration, and sucrose cushion ultracentrifugation (sUC). EVs were isolated from conditioned culture medium (CCM) using three myeloma cell lines: MM.1S, ANBL-6, and ALMC-1. The concentration and size of particles were assessed by nanoparticle tracking analysis, the protein concentration was determined by bicinchoninic acid assay, and the presence of specific EV markers and non-specific contaminants was evaluated by western blot. Our findings indicate that SEC outperforms sUC in yield, recovering more than 8 times more EVs per volume of CCM used. We also observed that the variability using sUC was 3 times higher than SEC, highlighting better reproducibility when using SEC. The western blot analysis of non-small EV contaminants revealed that HSP90B1 can be detected in some sUC samples, whereas it was consistently absent from SEC samples. In summary, SEC emerges as a more practical, efficient, and consistent method for EV isolation than sUC. In terms of EV characterization, our work unveiled disparities in vesicle size and biomarker expression among the different cell lines, underscoring the importance of cell line selection when studying EVs in multiple myeloma. Our study of different SEC fractions also revealed distinct particle and protein concentrations, sizes, and expression of markers within each fraction. This highlights the influence of the fraction window’s choice in downstream research outcomes. Additionally, our results established HBS with 0.005% of Tween 20 as an optimized buffer for isolating and preserving EVs at -80°C. Standardization of small EV isolation methods is lacking, and comparative assessments are essential to ensure efficient and reproducible EV recovery, regardless of following applications. Our study shows that SEC outperforms sUC in terms of EV isolation quality, offering a quicker and potentially more reliable method