Almost all cells secret extracellular vesicles (EVs) which play important roles in cell-cell communications. They are present in circulation, and carry molecules that could signal pathological development in remote tissues. Thus, tremendous research efforts have been devoted to study the functions of EVs and identify the EVs carrying high potential in disease diagnosis, prognosis, and even therapeutics. One obstacle for EV study is that EVs in biological samples are highly heterogeneous. Multiple rounds of separation and purification are often needed to obtain pure EVs for downstream characterization or function assessment. Our group applies two open-channel separation methods for EV analysis. One is asymmetrical flow field flow fractionation (AF4) that provides gentle and fast EV separation based on their size difference, while preserve their fragile structures. However, size-based separation is insufficient to resolve EVs from potential contaminants like proteins and lipoprotein complexes. Thus, the EVs having similar sizes and potentially co-eluted with contaminants of comparable dimensions can be further resolved by Capillary Electrophoresis (CE) by size-to-charge ratios. To demonstrate the effectiveness of AF4 coupled with CE for EV separation, we optimized the AF4 method to mainly separate EVs from free proteins and high-density lipoprotein complexes, although the low and very-low density lipoprotein complexes with comparable sizes as EVs could be co-eluted. The collection window of AF4 was determined by testing the EV marker proteins with Western Blot (WB). Integrity of the EV after AF4 was confirmed by NTA and TEM. We also examined the proper CE conditions for EV separation, and found that the CE voltage and buffer ionic strength strongly affected the separation profile.  With the proper voltage and buffer ionic strength, CE does not change the morphology of the collected EV and the EV peak can be used to quantify the total amount of eluted EVs. The system is being employed to analyze EVs in cell culture media for monitoring EV secretion by cells under various stimulations. We believe that, these two open-channel separation methods and their off-line coupling can provide rapid analysis of EVs and enhance EV resolution from other contaminants. This system will be helpful for study of EV functions by rapidly revealing alteration in EV abundance induced by different triggers.

Analysis of extracellular vesicles by asymmetrical flow field flow fractionation (AF4) and capillary electrophoresis (CE)

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