ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles

TLDR

Extracellular vesicles, once thought to be waste disposal, are now known to mediate many physiological and pathological processes, yet their purification is difficult due to overlapping properties with other secreted products. The study introduces ExtraPEG, a polyethylene glycol–based approach to purify exosomes and other extracellular vesicles. ExtraPEG rapidly enriches vesicles from large volumes using low‑speed centrifugation followed by a single small‑volume ultracentrifugation step, offering a quick and inexpensive protocol. The method yields sufficient protein and RNA for proteomics and sequencing, preserves vesicle biological activity, and can be adapted to isolate various vesicle populations from diverse fluids.

Abstract

Abstract Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes.