Exosome Isolation: Cyclical Electrical Field Flow Fractionation in Low-Ionic-Strength Fluids

Abstract

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.

Detection of Inflammation-Related Melanoma Small Extracellular Vesicle (sEV) mRNA Content Using Primary Melanocyte sEVs as a Reference

“…In general, primary melanocyte sEVs were smaller and possessed lower negative, closer to neutral, zeta potentials. Such differences support the feasibility of assessing means to distinguish normal sEVs from melanoma sEVs or other tumor sEV types based on innate sEV biophysical properties [19,20].…”

“…Small EVs were sized in 1× PBS using dynamic light scattering set to the lognormal number mode, and zeta potentials determined using a NanoBrook 90Plus (Brookhaven Instruments, Holtsville, NY, USA) nanoparticle tracking analyzer according to established methods [7,15,16,18,19]. Small EV protein concentrations were measured using a Pierce BCA protein assay (Thermo Fisher Scientific Inc., Waltham, MA, USA) as described previously [9,18,19,20,49].…”

An efficient method to isolate lemon derived extracellular vesicles for gastric cancer therapy

“…According to the size of EVs, several methods, such as ultrafiltration [ 23 ], size exclusion chromatography [ 14 ], asymmetric flow field-flow fractionation [ 24 – 26 ], and dialysis [ 19 , 27 ] have been used for EVs isolation. However, with the inherent drawbacks, these methods for EVs isolation are unsatisfactory, e.g.…”

Multifunctional microfluidic chip for cancer diagnosis and treatment

“…The most commonly used exosome separation method is to use ultracentrifugation (UC) [68], which is time-and reagentsconsuming. Although various new methods emerged in recent years, there is not much change in exosome separation and detection [69][70][71]. Microfluidic chip can not only efficiently complete the separation and detection of exosomes, but also integrate these two techniques on a single chip, which greatly simplify the procedure.…”