Author(s):
Grenhas, Madalena ; Lopes, Raquel ; Ferreira, Bruna Velosa ; Barahona, Filipa ; João, Cristina ; Carneiro, Emilie Arnault
Date: 2024
Persistent ID: http://hdl.handle.net/10362/171170
Origin: Repositório Institucional da UNL
Subject(s): extracellular vesicle isolation methods; extracellular vesicles; multiple myeloma; myeloma cell lines; newly diagnosed multiple myeloma; plasma; size-exclusion chromatography; sucrose cushion ultracentrifugation; ultrafiltration; Catalysis; Molecular Biology; Spectroscopy; Computer Science Applications; Physical and Theoretical Chemistry; Organic Chemistry; Inorganic Chemistry
Description
Funding Information: The authors express their gratitude to the Electron Microscopy Unit of Instituto Gulbenkian de Ci\u00EAncia (IGC) for the acquisition of the EV images. Additionally, we extend our appreciation to Carolina Pestana MSc for her contribution to the statistical analysis of this study, to the Systems Oncology Research Group (Champalimaud Foundation) for granting access to the required equipment for this study, and to the Champalimaud Foundation for funding. Publisher Copyright: © 2024 by the authors.
Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal.
