A Rapid and High-Recovery Extracellular Vesicle (EVs) Isolation Technique from Blood Samples.

Abstract

Extracellular vesicles (EVs) circulating in blood serve as non-invasive "liquid biopsies," carrying molecular cargo that reflects the physiological and pathological state of distant cells. Their analysis is crucial for understanding disease mechanisms and discovering novel biomarkers. Clinically, blood EVs hold significant promise for early disease diagnosis, prognostic assessment, and monitoring treatment response in diverse areas such as organ transplantation, cancer, and neurological disorders. Current EV isolation techniques, beyond ultracentrifugation, include size exclusion chromatography (separation by size for high purity) and immunoaffinity capture (using antibodies for high specificity). Here, we present a simplified, rapid, and reproducible method for isolating EVs from small-volume blood samples. This protocol consistently yields a concentrated EV pellet covering 50-300 nm EVs, amenable to direct downstream analysis. Developed and validated in our laboratory using human, porcine, and murine blood samples, this method has proven instrumental in identifying EV-based biomarkers for predicting outcomes related to organ transplantation. The protocol's adaptability and reliance on readily prepared, cost-effective reagents further enhance its utility. This scalable approach can be further integrated with subsequent purification or enrichment steps to optimize sample preparation for protein and nucleic acid assays. Key features • This method uses Ficoll-isolated plasma or centrifuged serum from any source, fresh to frozen. • Protocol is easy to handle, fast, and has fewer technical details. • 50-200 μL of plasma can be used to isolate enough EVs to run 5-8 western blot gels. • Protocol acquires 50-300 nm EVs enriched with markers and nucleic acids. • EVs isolated by this method can be further purified.