Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.

Enhancing Extracellular Vesicle Analysis by Integration of Large-Volume Sample Stacking in Capillary Electrophoresis with Asymmetrical Flow Field-Flow Fractionation

“…To identify the optimal injection volume of the collected AF4 fractions for LVSS/CE, we obtained the F2 fraction from the separation of 10 μL pooled human serum and injected it with various conditions: 1× F2: the nonstacking condition at 100 mbar for 10 s; 10×, 15×, and 20× LVSS: the stacking conditions at 100 mbar for 100, 150, and 200 s, respectively. This fraction was found in our previous work to elute EVs in diameters of 100–200 nm with relatively higher purity than F1 and F3 . We found that with 1× F2 EVs (Figure A) and 10× LVSS (Figure B), EVs and LDL were successfully resolved, and their migration times were consistent with the corresponding standards.…”

“…This fraction was found in our previous work to elute EVs in diameters of 100−200 nm with relatively higher purity than F1 and F3. 19 We found that with 1× F2 EVs (Figure 2A) and 10× LVSS (Figure 2B), EVs and LDL were successfully resolved, and their migration times were consistent with the corresponding standards. But more peak broadening and fronting, later elution, were observed with longer LVSS times (Figure 2B), indicating sample overloading, which also reduced the separation resolution between EV and LDL (Figure 2C).…”

“…The elution of EVs occurred at consistent migration times (Supporting Figure S2), and the EV peak area showed an excellent linear relationship (R 2 = 0.9904) with EV concentration (Figure D). As low as 2 × 10 5 particles/μL EVs can be detected by yielding an observable peak with a peak height 4× of baseline noise, much lower than what could be achieved with the normal injection mode …”

“…As low as 2 × 10 5 particles/μL EVs can be detected by yielding an observable peak with a peak height 4× of baseline noise, much lower than what could be achieved with the normal injection mode. 19 AF4-LVSS/CE for Purification and Enrichment of EVs from Serum. Offline coupling of AF4 and CE can provide effective separation of EVs from the abundant matrix components, but the protein content in serum reaches 60− 80 μg/μL.…”

“…To overcome these challenges, a gentle separation technique, asymmetrical flow field-flow fractionation (AF4), has been employed alone, , or coupled with UC for EV separation and purification . Our group has also reported the offline coupling of AF4 and capillary electrophoresis (CE), in which EVs can be separated from the smaller, abundant contaminants by AF4 (1st-dimension) and then from the impurities with comparable hydration sizes but different surface charges by CE (2nd-dimension) . This setup permits rapid monitoring of EV secretion by cells using only a few hundred microliters of the culture medium.…”