Exosome enrichment from human serum using polyethylene glycol precipitation

Abstract

Exosomes, nanosized extracellular vesicles that play key roles in intercellular communication, have great potential in diagnostic and therapeutic applications. However, isolating exosomes from complex biological matrices such as human serum remains challenging due to limitations in scalability, cost, and purity of current methods. In this study, we developed and optimized a polyethylene glycol (PEG) precipitation method as a cost-effective alternative to the commercial ExoQuick kit for efficient exosome enrichment from pretreated human serum. The efficiency and reproducibility of PEG precipitation were systematically evaluated and compared with ExoQuick using various analytical techniques, including size exclusion chromatography (SEC), scanning electron microscopy (SEM), nanoparticle tracking analysis (NTA), Western blotting, and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). SEC confirmed comparable exosome recovery between PEG and ExoQuick, while SEM and NTA analyses verified intact morphology and consistent size distribution of exosomes. Western blotting confirmed the presence of characteristic exosomal markers (CD63 and CD9) and a reduction of contaminating proteins such as albumin and apolipoprotein A1. Moreover, proteomic analysis by nano-LC-MS/MS identified diverse exosomal proteins, confirming the isolation of intact exosomes. Repeated PEG precipitation up to four cycles further improved exosome purity without compromising yield. These results demonstrate that PEG precipitation is a scalable, accessible, and cost-effective method that provides comparable exosome enrichment efficiency to ExoQuick. The optimized protocol offers a practical solution for analytical laboratories to enable cost-effective exosome-based studies in diagnostics and therapeutics. Supplementary Information: The online version contains supplementary material available at 10.1186/s40543-025-00521-0.

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