High-grade extracellular vesicles preparation by combined size-exclusion and affinity chromatography

Abstract Extracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols been reported the isolation of EVs with preserved biological function. Most EV purification methods include a precipitation step that results in aggregation most available techniques do not efficiently separate various types such as exosomes ectosomes, which are involved distinct processes. For this reason, we developed new two-step fast performance liquid chromatography (FPLC) protocol large numbers EVs. The method comprises size exclusion followed by immobilized metal affinity chromatography, is enabled expression poly-histidine tagged folate receptor α parental cells. Characterisation comparison obtained to purified differential centrifugation, currently common isolate EVs, demonstrated higher purity more selective enrichment preparations using our FPLC method, assessed marker proteins density distribution. Our studies reveal possibilities defined subpopulations function can easily be upscaled production larger amounts