The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at ∼1.3 V AC. A375 melanoma exosome recovery was approximately 70–80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from ∼−1.6 to −0.1 μm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from ∼−5.5 to −0.1 μm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.

No abstract

Although many properties for small extracellular vesicles (sEVs, formerly termed “exosomes”) isolated at ∼100 000g are known, a wide range of values are reported for their electrophoretic mobility (EM) measurements. This paper reports for the first time the effect of dilution on the EM of U87 glioblastoma cell-derived and plasma-derived sEVs and medium size EVs (mEVs, commonly termed “oncosomes”) preisolated by differential centrifugation. Furthermore, the effect of resalting on the EM of sEVs and mEVs was evaluated. The EM of U87 sEVs and U87 mEVs showed an increase as the salt concentration decreased to 0.005% of the initial salt concentration. However, for the plasma sEVs and plasma mEVs, the electrophoretic mobility increased as the salt concentration decreased to 0.01% of the initial salt concentration and then increased to its initial value when the salt concentration decreased to 0.005% of the initial salt concentration. For both U87 and plasma sEVs and mEVs, the EM remained almost constant when the concentration of the particles changed and the salt concentration was kept the same as its initial value. This indicates that the EM of EVs is only a function of the salt concentration of the buffer and is independent of the concentration of the particles. The sEVs and mEVs were separated with cyclical ElFFF for the first time. The results indicate that ElFFF was able to fractionate the EVs, and a crescent-shaped trend was found for the retention time when the applied AC voltage was altered (increased).

Nanoscale and microscale cell-derived extracellular vesicle types and subtypes are of significant interest to researchers in biology and medicine. Extracellular vesicles (EVs) have diagnostic and therapeutic potential in terms of biomarker and nanomedicine applications. To enable such applications, EVs must be isolated from biological fluids or separated from other EV types. Developing methods to fractionate EVs is of great importance to EV researchers. Our goal was to begin to develop a device that would separate medium EVs (mEVs, traditionally termed microvesicles or shedding vesicles) and small EVs (sEVs, traditionally termed exosomes) by elasto-inertial effect. We sought to develop a miniaturized technology that works similar to and provides the benefits of differential ultracentrifugation but is more suitable for EV-based microfluidic applications. The aim of this study was to determine whether we could use elasto-inertial focusing to re-isolate and recover U87 mEVs and sEVs from a mixture of mEVs and sEVs isolated initially by one round of differential ultracentrifugation. The studied spiral channel device can continuously process 5 ml of sample fluid per hour. Using the channel, sEVs and mEVs were recovered and re-isolated from a mixture of U87 glioma cell-derived mEVs and sEVs pre-isolated by one round of differential ultracentrifugation. Following two passes through the spiral channel, approximately 55% of sEVs were recovered with 6% contamination by mEVs (the recovered sEVs contained 6% of the total mEVs). In contrast, recovery of U87 mEVs and sEVs re-isolated using a typical second centrifugation wash step was only 8% and 53%, respectively. The spiral channel also performed similar to differential ultracentrifugation in reisolating sEVs while significantly improving mEV reisolation from a mixture of U87 sEVs and mEVs. Ultimately this technology can also be coupled to other microfluidic EV isolation methods in series and/or parallel to improve isolation and minimize loss of EV subtypes.

Electrical field flow fractionation (EFFF) is a particle separation method that employs an electrical field to cause particles in the carrier flow transferring in the vertical direction. The electrical characteristics of EFFF are one of the important aspects to explore electrical field how to work on the particle separation in EFFF channel. The equivalent electrical circuit model of EFFF channel was built. The experiments of chronoamperometry and electrochemical impedance spectroscopy (EIS) were conducted on the EFFF channel. The electrical circuit was utilised to fit the data of EIS experiments on EFFF channel. The fitting data were compared with the experimental data of EIS. The result indicates that the experimental data is highly quantified with the fitting data. It manifested that the equivalent electrical circuit reflected the electrical characteristics of the EFFF channel.