Size-exclusion chromatography for the characterization of urinary extracellular vesicles

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1228; p. 123828
• Main Authors: Park, Sanghwi, Jalaludin, Iqbal, Hwang, Hyojin, Ko, Minjeong, Adelipour, Maryam, Hwan, Myung, Cho, Namjoon, Kim, Kee K., Lubman, David M., Kim, Jeongkwon
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-08-2023
• Subjects: Centrifugation; Chromatography, Gel; EVs; Extracellular Vesicles; Humans; Size-exclusion chromatography; Urine; Urine; Extracellular vesicles; Size-exclusion chromatography; EVs; Centrifugation
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2023.123828

•Size-exclusion chromatography (SEC) was used to evaluate EV isolation from human urine.•SEC was able to confirm the presence of EVs and non-EV proteins.•Repeated centrifugations increased EVs and reduced non-EV proteins.•From 40 mL of human urine, EVs were successfully enriched.
In recent years, extracellular vesicles (EVs) have gained attention for their potential as biomarkers for the early diagnosis and treatment of various diseases. Traditionally, EV isolation has relied exclusively on ultracentrifugation. However, alternative enrichment methods such as size-exclusion chromatography (SEC) and polyethylene glycol-based precipitation have been introduced. This study utilized SEC as a characterization tool to assess the efficiency of EV isolation. Urinary EVs isolated from human urine using centrifugation (40,000 × g) were analyzed using an SEC column with a pore size of 1000 Å, an inner diameter of 7.8 mm, and a length of 300 mm. The EVs were detected sequentially using UV (280 nm) and fluorescence (λex/em = 550 nm/565 nm); the EVs were observed at approximately 6 min, while the proteins were observed at approximately 12 min. The repeated centrifugation enrichment steps resulted in an increase in EV peaks and a decrease in protein peaks. SEC analysis of the enriched EV samples confirmed that a four-cycle repetition of centrifugation is necessary for successful EV enrichment and removal of non-EV proteins from 40 mL of human urine.