Size-Exclusion Chromatography: A Path to Higher Yield and Reproducibility Compared to Sucrose Cushion Ultracentrifugation for Extracellular Vesicle Isolation in Multiple Myeloma

• Published in: International journal of molecular sciences Vol. 25; no. 15; p. 8496
• Main Authors: Grenhas, Madalena, Lopes, Raquel, Ferreira, Bruna Velosa, Barahona, Filipa, João, Cristina, Carneiro, Emilie Arnault
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.08.2024
• Subjects: Analysis; Cell culture; Cell Line, Tumor; Chromatography; Chromatography, Gel - methods; Culture Media, Conditioned - chemistry; extracellular vesicle isolation methods; Extracellular vesicles; Extracellular Vesicles - chemistry; Extracellular Vesicles - metabolism; Health aspects; Humans; Medical research; Medicine, Experimental; Multiple myeloma; Multiple Myeloma - pathology; Physiological aspects; Plasma; Proteins; Ratios; Reproducibility; Reproducibility of Results; size-exclusion chromatography; Sucrose; sucrose cushion ultracentrifugation; Ultracentrifugation - methods; ultrafiltration; Portugal; extracellular vesicle isolation methods; multiple myeloma; myeloma cell lines; extracellular vesicles; sucrose cushion ultracentrifugation; newly diagnosed multiple myeloma; size-exclusion chromatography; plasma; ultrafiltration
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms25158496

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.