8/6/2023 0 Comments Capto core 700 manualProteins that are often considered “classical” exosome markers 10 can be present at variable levels or can even be absent in some cases 11 and have been found to be expressed in other subcellular compartments or by other types of EVs 12, 13. This is challenging due to shared characteristics with ectosomes, such as size, membrane architecture, density and many marker proteins. Despite increasing interest in exosomes for their natural role in cell-to-cell communication and potential as diagnostic and therapeutic tools 8, it remains difficult to selectively isolate them 9. Our studies reveal new possibilities for the isolation of defined subpopulations of EVs with preserved biological function that can easily be upscaled for production of larger amounts of EVs.Įxtracellular vesicles (EVs) refer to a group of naturally occurring small lipid bilayer particles that are derived from cells and cannot replicate 1, 2 including exosomes (EVs of endosome-origin 3, 4, 5) and ectosomes (microparticles/microvesicles, plasma membrane-derived vesicles) 6, 7. Characterisation and comparison of the EVs obtained by this method to EVs purified by differential centrifugation, currently the most common method to isolate EVs, demonstrated higher purity and more selective enrichment of exosomes in EV preparations using our FPLC method, as assessed by comparison of marker proteins and density distribution. The method comprises size exclusion chromatography followed by immobilized metal affinity chromatography, which is enabled by expression of poly-histidine tagged folate receptor α in the parental cells. For this reason, we developed a new two-step fast performance liquid chromatography (FPLC) protocol for purification of large numbers of EVs. Most EV purification methods include a precipitation step that results in aggregation of vesicles and most available techniques do not efficiently separate the various types of EVs such as exosomes and ectosomes, which are involved in distinct biological processes. Despite this, few protocols have been reported for the isolation of EVs with preserved biological function. Extracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential.
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