Single-step isolation of extracellular vesicles by size-exclusion chromatography

By A. Böing, E. van der Pol, Anita E. Grootemaat, F. Coumans, Auguste Sturk, R. Nieuwland. Journal of Extracellular Vesicles, 2014

898 citations recorded by the source corpus.

Background Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim To develop a single-step protocol to isolate vesicles from human body fluids. Methods Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18–20 (32%±2 of total), and protein in fractions 19–21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles.

Subfields: Spectroscopy

View Paper: https://www.semanticscholar.org/paper/187427fae272b89690e2b05a12f2eb5ab21f227b

Citations in the corpus

• Isolation of Extracellular Vesicles: General Methodologies and Latest Trends (2018, neutral): “As has been shown, this method is also applicable to separation of EVs from the blood plasma and urine protein complexes and lipoproteins [42, 63, 64, 120], which is challenging and many other methods fail [120].”
• Exosome: A Review of Its Classification, Isolation Techniques, Storage, Diagnostic and Targeted Therapy Applications (2020, neutral): “51 The method of ultrafiltration usually uses ultrafiltration membranes with different molecular weight cutoffs (MWCO) to selectively separate samples.”
• A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option? (2020, neutral): “This technique was adapted into a single-step isolation system for exosomes from biological fluids in 2014 [83].”
• Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. (2015, neutral): “Here, we present a systematic comparison study between a robust EV purification method, ultrafiltration with size-exclusion liquid chromatography (UF-LC), adapted from previous studies, 14 – 16 and UC.”
• Exosomes as a new frontier of cancer liquid biopsy (2022, neutral): “developed an SEC-based protocol by cross-linked sepharose CL-2B column, which could efficiently isolate exosomes with a diameter larger than 70 nm from platelet-free supernatant [50].”
• Extracellular Vesicle Heterogeneity: Subpopulations, Isolation Techniques, and Diverse Functions in Cancer Progression (2018, neutral): “, vesicles disruption, aggregation, and co-purification of non-EV material (89, 90)].”
• Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods (2015, neutral): “SEC was performed as described previously with minor modifications [16].”
• Optimized exosome isolation protocol for cell culture supernatant and human plasma (2015, neutral): “Recently, a single-step SEC isolation of exosomes from human plasma has been described (23).”
• Extracellular vesicle isolation and characterization: toward clinical application. (2016, neutral): “…the notion that size-exclusion chromatography (SEC) coupled with membrane filtration is the preferred procedure for isolating EVs from plasma/serum (71, 90, 94, 95); the preferred method for harvesting EVs after SEC, i.e., precipitation versus centrifugation, requires further evaluation (95).”