Establishment of a simplified dichotomic size‐exclusion chromatography for isolating extracellular vesicles toward clinical applications

Size‐exclusion chromatography (SEC) is a widely adopted method for the isolation of extracellular vesicles (EVs) from complex samples. SEC can efficiently remove high‐abundant proteins, while often requires multiple fractionation operation using diversified column settings. In this study, we aim to establish a simplified SEC method to acquire high quality EVs. In comparison of all three cross‐linked Sepharose resins with the sample types of FBS and human serum (HS), CL‐6B and CL‐4B showed superior performance in regular SEC to CL‐2B in terms of significantly narrower EV and protein peaks, higher resolutions and EV purity. By increasing their bed volumes to 20 ml, the resolutions of CL‐6B and CL‐4B columns could be significantly improved, while the CL‐6B column had the best performance with higher particle yields and tighter EV peaks. With the CL‐6B 20 ml column, we further established a simplified dichotomic SEC method that only requires two bulk elutions to acquire EVs in the Eluate 1 and proteins in the Eluate 2. We further justified that such CL‐6B columns were reusable for at least 10 consecutive times, and the dichotomic SEC was applicable to EV isolations from HS and FBS‐free supernatants of fluorescently labelled and unlabelled SW620 cells. The proteomics analysis implicated that although the two methods had dissimilar abilities in removing different co‐isolating contaminant proteins from EVs, the dichotomic SEC and ultracentrifugation could isolate EVs from human plasma with comparable purity. This dichotomic SEC has its intriguing potential to be used for EV preparation toward clinical testing and/or basic research.