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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.

Cyclical electrical field flow fractionation (CyElFFF) is a technique for characterizing and separating nanoparticles based on their size and charge using cyclical electric fields. The high diffusion rate of nanoparticles has prevented CyElFFF from being applicable to particles smaller than 100 nm. In this work, the diffusion challenges associated with nanoparticles was resolved using biased cyclical electric fields. This new method, biased cyclical electrical field flow fractionation (BCyElFFF), achieves baseline separation of 15 and 40 nm gold nanoparticles. Theoretical considerations show that the optimal resolution is achieved when the applied bias yields electrical transport that counteracts the diffusive transport of nanoparticles. BCyElFFF greatly extends separation capabilities of the cyclical electrical field flow fractionation to sub 50 nm nanoparticles and provides a powerful alternative to other separation and characterization techniques capable of separating nanoparticles smaller than 50 nm.

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