An optimized method for the isolation of extracellular vesicles (EVs) including exosomes is crucial to increase the knowledge on the immunomodulatory effects of tumor derived exosomes (TEX). Hence, this study compares Sepharose and ExoSpin midi columns (Cell Guidance Systems) with classical ultracentrifugation to obtain TEX from blood of HNSCC patients. The blood of healthy donors served as a control. Protein concentration of the isolated TEX was determined by using BCA Colorimetric and Bradford assays. The quality of the isolated TEX was validated by Dynamic Light Scattering (DLS) for measuring size, Transmission Electron Microscopy (TEM) for morphology, and flow cytometry for the expression level of typical exosomal markers.

Our results indicate that all methods enrich particles with sizes in the standard range for exosomes (30-150 nm). The evaluation of the expression of tetraspanins CD9, CD63, CD81 and Hsp70 by flow cytometry showed variable expression patterns with the different isolation methods. The highest protein concentrations were obtained after ultracentrifugation, followed by ExoSpin midi and Sepharose-based isolation which resulted in the highest purity and most homogeneous TEX enrichment as demonstrated by TEM.

Sepharose is a reliable method for small-scale applications along with pre-selecting small extracellular vesicle subpopulations. In ultracentrifugation, while EVs contain impurities, the isolated particles might more closely reflect the whole range of vesicles present in the blood. ExoSpin results in higher protein concentrations and achieves a more homogenous vesicle population than ultracentrifugation. Future functional studies with EVs will tell which isolation method most likely reflects the in vivo function of exosomes in HNSCC patients.

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