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.
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.
P-03-Free-flow electrophoresis provides a new and effective way of isolating extracellular vesicles from plasma and ascites
The combination of Free Flow Electrophoresis (FFE) as a separation technique and AMNIS as an analytical technique can display differences in charge density of extracellular vesicles (subfractions), as we have already shown previously. Here in addition we use Nano Flow Cytometry (NanoFCM) to further characterize EV derived from various bio fluids (plasma, ascites) isolated using FFE. FFE can offer various separation protocols, differing in the distance of electrophoresis or time of electrophoresis. The separation protocol with a prolonged distance of electromigration was elected for these experiments.
P-04-Free Flow Electrophoresis (FFE) enables subfractionation of human plasma-derived exosomes (EV) from blood samples of healthy donors.
P-05 Free flow electrophoresis setup and experimental workflow for the separation of extracellular vesicles from human plasma
The complete workflow of start-up of FFE-instrument prior the experiment for the enrichment of exosomes from melanoma plasma sample