Abstract
Exosomes have received significant attention for their role in pathobiological processes and are being explored as a tool for disease diagnosis and management. Consequently, various isolation methods based on different principles have been developed for exosome isolation. Here we compared the efficacy of four kits from Invitrogen, 101Bio, Wako and iZON along with conventional ultracentrifugation-based method for exosome yield, purity and quality. Cell culture supernatant was used as an abundant source of exosomes, and exosome quantity, size-distribution, zeta-potential, marker-expression and RNA/protein quality were determined. The Invitrogen kit gave the highest yield but the preparation showed broader size-distribution likely due to microvesicle co-precipitation and had the least dispersion stability. Other preparations showed <150 nm size range and good stability. Preparation from iZON column; however, had a broader size-distribution in the lower size range suggestive of some impurities of non-vesicular aggregates. RNA quality from all preparations was comparable; however, proteins from Invitrogen method-based exosomal preparation showed polyethylene glycol (PEG) contamination in mass spectrometry. Chemical impurities from the precipitant could also be the cause of toxicity of Invitrogen method-based exosomal preparation in biological growth measurement assay. Together, these findings should serve as a guide to choose and further optimize exosome isolation methods for their desired downstream applications.
Highlights
Exosomes are small membrane vesicles (30–150 nm) of endocytic origin, which are shed by all cell types under normal- and patho-physiological conditions
We observed that the precipitation-based Total Exosomes Isolation kit (Invitrogen) had the maximum yield followed by gel-filtration chromatography, PureExo kit (101Bio), and differential ultracentrifugation
As we observed that the size distribution of exosomal preparations from, different isolation methods varied, we examined their purity by immunoblotting for specific marker proteins
Summary
Exosomes are small membrane vesicles (30–150 nm) of endocytic origin, which are shed by all cell types under normal- and patho-physiological conditions. Exosomes in patient’s body-fluids have emerged as a promising source for biomarker development[5] They can be isolated from the small amount of biological fluids and clinical samples and their cargo, which represents tissue-specific molecules with higher stability, can serve as disease-specific biomarkers[16,17]. Since their release and composition can be modulated by environmental factors, they can serve as markers for disease status and treatment outcomes[18,19]. Presented data from this study will serve as a guide to choose and further optimize exosome isolation methods for their desired applications in biomarker development and/ or biological assays
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