Dataset Information

Ultracentrifugation versus kit exosome isolation: nanoLC-MS and other tools reveal similar performance biomarkers, but also contaminations.

ABSTRACT:

Aim

For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared.

Materials & methods

'Case study' exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed.

Results

Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLC-MS. Western blotting and nanoLC-MS also revealed negative exosome markers regarding both isolation approaches.

Conclusion

The two isolation methods had an overall similar performance, but we hesitate to use the term 'exosome isolation' as impurities may be present with both isolation methods. NanoLC-MS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures.

SUBMITTER: Skottvoll FS

PROVIDER: S-EPMC6331754 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Ultracentrifugation versus kit exosome isolation: nanoLC-MS and other tools reveal similar performance biomarkers, but also contaminations.

Skottvoll Frøydis Sved FS Berg Henriette Engen HE Bjørseth Kamilla K Lund Kaja K Roos Norbert N Bekhradnia Sara S Thiede Bernd B Sandberg Cecilie C Vik-Mo Einar Osland EO Roberg-Larsen Hanne H Nyström Bo B Lundanes Elsa E Wilson Steven Ray SR

Future science OA 20181109 1

<h4>Aim</h4>For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared.<h4>Materials & methods</h4>'Case study' exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed.<h4>Results</h4>Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in th ...[more]

PMID: 30652024

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Project description:BackgroundExosomes are nanovesicles (30-120 nm) of endosomal origin. These exosomes contain various functional proteins and RNAs that could be used for therapeutic purposes. Currently, having a standard method for exosome isolation retaining its biological properties with increased yield and purity is a major challenge. The most commonly used method is differential ultracentrifugation but it has its own disadvantages, which include high time consumption, low yield due to disruption of exosome integrity, and high protein contaminants. In this study, we have identified an improved method addressing these problems for exosome isolation using ultracentrifugation since it is cost-effective and used worldwide.MethodWe have compared differential ultracentrifugation with the modified method called one-step sucrose cushion ultracentrifugation for exosome isolation. The conditioned serum-free media from human mesenchymal stem cells cultured for 48 h was collected for exosome isolation. The cellular debris was removed by centrifugation at 300g for 10 min, followed by centrifugation at 10,000g for 30 min to remove microvesicles. Equal volumes of pre-processed conditioned media were used for exosome isolation by direct ultracentrifugation and one-step sucrose cushion ultracentrifugation. The exosomes isolated using these methods were characterized for their size, morphology, concentration, and surface marker protein expression.ResultIt was observed that the recovery of exosomes with cup-shaped morphology from one-step sucrose cushion ultracentrifugation was comparatively high as estimated by nanoparticle tracking analysis and electron microscopy. These results were confirmed by Western blotting and flow cytometry.ConclusionWe conclude that this one-step sucrose cushion ultracentrifugation method provides an effective and reproducible potential standard method which could be used for various starting materials for isolating exosomes. We believe that this method will have a wide application in the field of extracellular vesicle research where exosome isolation with high yield and purity is an imperative step. Schematic representation of comparison of UC and SUC exosome isolation methods for tissue-specific human mesenchymal stem cells. The SUC isolation method yields a greater number of cup-shaped exosomes with a relatively homogenous population for mass-scale production of exosomes for downstream analysis.AbbreviationsSUC One-step sucrose cushion ultracentrifugation, UC Direct ultracentrifugation.

Dataset Information

Ultracentrifugation versus kit exosome isolation: nanoLC-MS and other tools reveal similar performance biomarkers, but also contaminations.

Aim

Materials & methods

Results

Conclusion

Publications

Ultracentrifugation versus kit exosome isolation: nanoLC-MS and other tools reveal similar performance biomarkers, but also contaminations.

Similar Datasets

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