Despite the increasing interest in extracellular vesicles (EVs), obtaining EVs in high purity remains a tedious process. Neither fractionation by density nor by size alone is sufficient to separate EVs from most contaminants including lipoproteins. For now, a time-consuming combination of two methods (density and size separation) is required to enrich EVs to high purity at the expense of time. In recent years, we have qualified Free Flow Electrophoresis (FFE) as an efficient method for EV separation.
P-02-Fractioning by Free Flow Electrophoresis (FFE) followed by Imaging Flow Cytometry analysis displays EV charge density heterogeneities within human plasma and cell culture supernatants
FFE allows quick separation and preparation of biological analytes including cellular organelles from various biological liquids according to their outer electric charges. Performing subsequent imaging flow cytometry (IFCM) analyses we demonstrate that also extracellular vesicles (EVs) can be effectively and quickly fractioned by FFE. Interestingly, EVs prepared from cell culture supernatants on average reveal higher negative charges than EVs from human plasma of healthy donors or melanoma patients, respectively.
Overall, applying the established and optimized EV separation protocols FFE provides a fast and feasible method for EV fractioning for appropriate down-stream analysis including IFCM. In its current form FFE allows fractioning of approx. 100 samples per working day.
Free flow electrophoresis allows quick preparation of extracellular vesicles from cell culture supernatants and human plasma
Upon applying imaging flow cytometry analyses to identify EV containing fractions, we have optimized FFE protocols for the preparation of bona fide EVs from conditioned cell culture media and demonstrated the reproducibility of the method.
Applying a comparable strategy, we now have improved FFE protocols for the preparation of EVs from human plasma samples. Notably, EVs from plasma show much higher EV complexity than those from cell culture supernatants. Specifically, plasma EVs are recovered in more than three subfractions. Several of these fractions also have relatively high protein contents, which could effectively be reduced by subsequent ultra-filtration (UF).
Separation via FFE can produce up to 20 samples per hour, with each sample containing 100 µl which is needed for AMNIS analysis. The entire procedure, including UF (300 KD), can yield approximately 100 samples per day and can be described as relatively fast compared to other technologies.
Free Flow Electrophoresis can display the degree of depletion of proteins from EVs from supernatant of cell culture of Mesenchymal stem cells via various standard techniques, like Ultracentrifugation (UC), precipitation with PEG + UC and tangential ultrafiltration (TFF)
A Free Flow Zone Electrophoresis (FF-ZE-pH) protocol has been developed for the purification and isolation of EVs from supernatant of cell culture, using a set of buffer media of different pH-values ranging from pH 4.6 to pH 7.
According imaging flow cytometry analyses with EV specific antibodies on an AMNIS ISXII platform, EVs subtypes were detected with different charge densities across the area of electrophoretic migration. Only some subtypes of EVs were free of proteins, others were still associated with proteins. Upon combining FFE with subsequent ultrafiltration (300 KD-UF-membranes) even high amounts of contaminating protein can be removed from obtained samples.
FFE demonstrates that EV samples obtained with different preparation methods vary regarding the complexity of EVs and contaminating proteins. Furthermore, the results demonstrates that none of the methods removes non-EV associated proteins appropriately. Notably, if required, the amount of contaminating protein can significantly be reduced obtained FFE-EV samples are additionally cleaned by ultrafiltration.