Salivary Extracellular Vesicles Separation: Analysis of Ultracentrifugation-Based Protocols

• Published in: Oral diseases
• Main Authors: Itzel, Castillejos-García, Eduardo, Martínez-Martínez, Velia, Ramírez-Amador, Mireya, Cisneros-Villanueva, Alfredo, Hidalgo-Miranda, Pilar, Ramos-Godínez María Del, Gabriela, Anaya-Saavedra
• Format: Journal Article
• Language: English
• Published: Denmark 27-10-2024
• Subjects: saliva; PEG; extracellular vesicles; miRNAs; ultracentrifugation
• ISSN: 1354-523X; 1601-0825; 1601-0825
• DOI: 10.1111/odi.15171

The clinical potential of extracellular vesicles (EVs) is widely acknowledged, yet the standardization and reproducibility of its separation remain challenging. This study compares three protocols: ultracentrifugation (UC), UC with purification step (UC + PS), and a combined protocol using polymer-based precipitation and UC (PBP + UC).
Salivary samples were collected from healthy donors. EVs were separated (UC, UC + PS, and PBP + UC) and characterized using transmission electron microscopy, nanoparticle tracking analysis, EV purity, RNA concentration, and Western blotting. miRNA expression was evaluated by quantitative RT-PCR. Statistical analyses comparing groups were performed using ANOVA.
All methods successfully separated CD9+ and CD63+ EVs from saliva. The UC + PS and PBP + UC protocols yielded the highest concentrations of EVs, enriched in < 200 nm vesicles. EV purity and RNA recovery were comparable among all methods. Expression of miR-16, miR-27a, and miR-99a was successfully detected using all methods.
The UC + PS and PBP + UC protocols demonstrate comparable efficiency in separating salivary EVs. However, the combined PBP + UC protocol, with its simplified processing capability, offers a significant advantage, particularly in the initial phase of EV separation. This finding suggests its potential application in clinical settings where time-sensitive simple processing is critical. Further validation is needed to confirm its effectiveness for transcriptomic and proteomic analyses.