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

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.

Size and shape characterization of hydrated and desiccated exosomes

“…It contradicts common claims that exosomes have a Bcup shape^ [23] (as illustrated in Fig. 5d) reported on the basis of electron microscopy imaging of desiccated samples [12,[45][46][47] and AFM of both hydrated and desiccated exosomes [48][49][50]. Our finding is supported by prior cryo-TEM results that report round morphology [51] of exosomes released by hepatocytes, spheroid shape [52] of human mesenchymal stem cell exosomes, and naturally spherical [53] exosomes secreted by prion-infected cells.…”

Extracellular Vesicles Isolated from the Leaf Apoplast Carry Stress-Response Proteins

“…PATL1 and PATL2 both bind phosphoinositides and are thought to mediate vesicle transport and/or fusion (Petersen et al, 2014). PATL1 has been localized to the cell plate in dividing cells but also associates with PLASMODESMATA-LOCATED PROTEIN1 (PDLP1) during downy mildew infection and may colocalize with PDLP1 at the extrahaustorial membrane (Caillaud et al, 2014).…”

Post Isolation Modification of Exosomes for Nanomedicine Applications

“…For example, Momen-Heravi et al reported that culturing B lymphocytes in the presence of IL-4 and CD40 increased exosome production by over 200-fold [25]. Large numbers of exosomes might then be isolated from culture or biological fluids using advanced microfluidic isolation strategies [39]. Other potential options to scale up exosomal nanocarrier production include the use of bovine milk-derived [40] or plant-derived [14,15] exosomes following biocompatibility testing.…”