Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications

TLDR

Exosomes play key roles in disease pathobiology and are increasingly explored for diagnostic and therapeutic purposes, prompting the development of diverse isolation techniques. The study compared the efficacy of four commercial kits (Invitrogen, 101Bio, Wako, iZON) and conventional ultracentrifugation for exosome yield, purity, and quality. Using cell‑culture supernatant, the authors quantified exosome number, size distribution, zeta potential, marker expression, and assessed RNA and protein quality. The Invitrogen kit yielded the most exosomes but exhibited broader size distribution, PEG contamination, and toxicity, while the other kits produced <150 nm, stable exosomes with comparable RNA quality, informing optimal method selection for downstream applications.

Abstract

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 &lt;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.