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A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM

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Abstract

Exosomes participate in cancer metastasis, but studying them presents unique challenges as a result of their small size and purification difficulties. Asymmetrical field flow fractionation with in-line ultraviolet absorbance, dynamic light scattering, and multi-angle light scattering was applied to the size separation and characterization of non-labeled B16-F10 exosomes from an aggressive mouse melanoma cell culture line. Fractions were collected and further analyzed using batch mode dynamic light scattering, transmission electron microscopy and compared with known size standards. Fractogram peak positions and computed radii show good agreement between samples and across fractions. Ultraviolet absorbance fractograms in combination with transmission electron micrographs were able to resolve subtle heterogeneity of vesicle retention times between separate batches of B16-F10 exosomes collected several weeks apart. Further, asymmetrical field flow fractionation also effectively separated B16-F10 exosomes into vesicle subpopulations by size. Overall, the flow field flow fractionation instrument combined with multiple detectors was able to rapidly characterize and separate exosomes to a degree not previously demonstrated. These approaches have the potential to facilitate a greater understanding of exosome function by subtype, as well as ultimately allow for “label-free” isolation of large scale clinical exosomes for the purpose of developing future exosome-based diagnostics and therapeutics.

Flow path of exosome sample through the asymmetrical field flow fractionation instrument, detectors, and transmission electron microscope.

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Acknowledgments

This research was supported in part by the US National Science Foundation Division of Graduate Education under grant DGE-0903715 and in part by the National Institutes of Health under grant 1R21GM107894-01. We would also like to thank Michael J. Scott at C-TRAIN for assisting in exosome collections and shipments between C-TRAIN at Washington University and the University of Utah laboratories. Additional grant support for C-TRAIN investigators included the Elsa U. Pardee Foundation (J. L. Hood) and the National Institutes of Health R01HL073646-08 (S. A. Wickline).

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Authors and Affiliations

  1. Department of Mechanical Engineering, University of Utah, Salt Lake City, UT, 84112, USA

    Kevin E. Petersen & Bruce K. Gale

  2. Department of Geology and Geophysics, University of Utah, Salt Lake City, UT, 84112, USA

    Eliana Manangon, Diego P. Fernandez & William P. Johnson

  3. Consortium for Translational Research in Advanced Imaging and Nanomedicine, School of Medicine, Washington University, St. Louis, MO, 63110, USA

    Joshua L. Hood & Samuel A. Wickline

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  1. Kevin E. Petersen
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  2. Eliana Manangon
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  3. Joshua L. Hood
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  4. Samuel A. Wickline
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  5. Diego P. Fernandez
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  6. William P. Johnson
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  7. Bruce K. Gale
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Correspondence to Kevin E. Petersen.

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Published in the topical collection Field- and Flow-based Separations with guest editors Gaetane Lespes, Catia Contado, and Bruce Gale.

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Petersen, K.E., Manangon, E., Hood, J.L. et al. A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM. Anal Bioanal Chem 406, 7855–7866 (2014). https://doi.org/10.1007/s00216-014-8040-0

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