Evaluation of exosome separation from human serum by frit-inlet asymmetrical flow field-flow fractionation and multiangle light scattering

Exosomes are extracellular vesicles that mediate intercellular communication, immune response, and tumour metastasis. However, exosome isolation from the blood is complicated because their size and density are similar to those of blood lipoproteins. Here, we employed field programming frit-inlet asymmetrical flow field-flow fractionation (FIAF4) coupled with multiangle light scattering (MALS) for the effective separation of exosomes from free unbound proteins and lipoproteins present in serum samples using different pre-treatment methods, namely, a commercial exosome isolation kit, ultracentrifugation (UC), and a simple centrifugation followed by ultrafiltration (UF). Sizes of the eluted exosomes, as calculated by MALS signals, approximated well with the results of batch dynamic light scattering of the collected fractions and with the sizes of polystyrene particles. Exosome separation from lipoproteins was validated by western blotting with several markers of exosomes and lipoproteins, followed by proteomic analysis using nanoflow ultrahigh-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry. UC requires relatively large amounts of serum samples (at least 2 mL) but is more efficient at removing lipoproteins. The UF method with a centrifugal concentrator (300 kDa) was found to be more effective in retrieving exosomes with low serum volumes (50 μL). Altogether, this study demonstrates the application of field programming FIAF4 for the isolation/purification of exosomes from proteins and lipoproteins in the serum.