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

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.

Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival

“…22 For immunoblotting, total protein (10/40 µg) isolated in NP40 buffer supplemented with protease 1X PPI cocktail was loaded on to SDS-polyacrylamide gel and resolved by electrophoresis. 22 For immunoblotting, total protein (10/40 µg) isolated in NP40 buffer supplemented with protease 1X PPI cocktail was loaded on to SDS-polyacrylamide gel and resolved by electrophoresis.…”

“…Protein-based quantitation of isolated EV or cell lysates was done using the protein DC assay kit as described earlier. 22 For immunoblotting, total protein (10/40 µg) isolated in NP40 buffer supplemented with protease 1X PPI cocktail was loaded on to SDS-polyacrylamide gel and resolved by electrophoresis. Subsequently, resolved protein bands were transferred on to polyvinylidene difluoride membrane and probed with specific antibodies against thrombospondin (THBS1), ARF6, CD9, CD63, HIF-1α, and β-actin.…”

Optimized method for extraction of exosomes from human primary muscle cells

“…Previous publications showed lower exosomal protein detection (e.g. CD63) by Western blot using the polymer-based precipitation compared to ultracentrifugation, despite observing a greater number of vesicles by NanoSight using polymer-based precipitation [28,38]. Based on these publications, we suspected that the polymer matrix was hiding epitopes.…”

“…Looking at the literature, we noticed that the polymer kit consistently led to a greater number of vesicles detected by NanoSight [49,52,56], and yet led to a reduced detection of exosomal markers by Western blot [28,38,49,52,56,57]. Interestingly, Rider et al, while optimizing a polymer to extract extracellular vesicles, showed that rinsing of exosomes that had been precipitated using the polymer resulted in an increase in exosome markers detected by Western blot [28].…”

Engineering Extracellular Vesicles as Nanotherapeutics for Regenerative Medicine

“…This is a relatively simple process and does not require the use of specialized equipment. However, the use of foreign polymers can introduce impurities into the sample that is difficult to remove, and may adversely impact downstream applications [30,31]. A third method, immunoaffinity, relies on the interactions between exosome membrane proteins and their respective antibodies [29] This is a more precise method of capturing specifically exosomes, but is limited in that it cannot be used for large sample volumes.…”