Intercellular communication via extracellular vesicles (EVs) has been identified as a vital component of a steadily expanding number of physiological and pathological processes. To accommodate these roles, EVs are equipped with specific proteins, lipids, and RNA molecules by EV-secreting cells. Consequently, EVs have highly heterogeneous molecular compositions. Given that surface molecules on EVs determine their interactions with their environment, it is conceivable that EV functionality differs between subpopulations with varying surface compositions. However, it has been technically challenging to examine such functional heterogeneity due to a lack of non-destructive methods to separate EV subpopulations based on their surface markers.
Researchers at the University Medical Center Utrecht used Design-of-Experiments methodology to rapidly optimize a protocol, which we name ‘EV-Elute’, to elute intact EVs from commercially available Protein G-coated magnetic beads. The researchers captured EVs from various cell types on these beads using antibodies against CD9, CD63, CD81 and a custom-made protein binding phosphatidylserine (PS). When applying EV-Elute, over 70% of bound EVs could be recovered from the beads in a pH- and incubation time-dependent fashion. EV subpopulations were found to be devoid of co-isolated protein contaminants observed in whole EV isolates and showed intact morphology by electron microscopy. Proteinase K protection assays showed a mild and reversible decrease of EV membrane integrity during elution. Depending on the type of capturing antibody used, some antibodies remained EV-associated after elution. EV subpopulations showed uptake patterns similar to whole EV isolates in co-cultures of peripheral blood mononuclear cells and endothelial cells. However, in Cas9/sgRNA delivery assays, CD63+ EVs showed a lower capacity to functionally deliver cargo as compared to CD9+, CD81+ and PS+ EVs.
Systematic optimization of EV elution protocol from magnetic beads using DoE methodology
A: Overview of experimental setup to test elution of EVs captured on Protein G-coated magnetic beads and EV permeability after exposure to a DoE-based library of elution protocols. Elution protocols varied in incubation time, elution buffer pH and elution buffer TEA concentration. EV elution after each protocol was analyzed by onbead lipid fluorescent staining and flow cytometry. EV permeability was tested by exposing EVs to proteinase K during each elution protocol and western blotting for intraluminal EV proteins. B: Elution efficiency of HEK293T EVs captured using antibodies against CD9 (red) or CD81 (blue) after exposure to PBS as controls or each elution protocol (Run1-15). Mean ± SD of three technical replicates is shown. C: HEK293T EV permeability during each elution protocol. Bars show mean ± SD of two technical replicates and indicate average degradation of Alix, βactin, TSG-101 and syntenin, expressed as a percentage of proteinase K (PK)-treated EVs exposed to PBS. EVs exposed to SDS at physiological pH, or SDS at high pH and TEA concentrations (TEA/SDS) served as positive controls for PK activity. D, E: Prediction models of EV elution after capture with anti-CD9 (D) or anti-CD81 (E) antibodies, generated based on data in panel B. F: Prediction model of EV permeability generated based on data in panel C. G: Elution of HEK293T EVs captured on beads using antibodies against CD9, CD63, CD81 or PS, using elution buffers with pH ranging from 11.0 to 11.5 for 5 min. H: Representative western blot of HEK293T EV permeability analysis based on Alix, TSG-101, syntenin and β-actin. EVs were exposed to elution buffers with pH ranging from 11.0 to 11.5 or SDS as control in presence of PK for 5 min. Alternatively, EVs were incubated with the pH 11.2 elution buffer for 5 min, neutralized to physiological pH and subsequently incubated with PK for 5 min (11.2*). Bar chart shows average degradation of all four proteins based on band intensity analysis.
Taken together, these researchers developed a novel, easy-to-use platform to isolate and functionally compare surface marker-defined EV subpopulations. Importantly, this platform does not require specialized equipment or reagents and is universally applicable to any capturing antibody and EV source. Hence, EV-Elute can open new opportunities to study EV functionality at the subpopulation level.