To obtain the defining characteristics of a given group of electric vehicles, researchers need ways to selectively isolate particular subsets of these nanoparticles. Many methods are now available, but each involves tradeoffs in ease of use, throughput, and reproducibility.
Witwer describes the options in terms of the “specificity/recovery matrix,” where high performance on one axis means sacrifices on the other. “You have methods that are very efficient at recovering everything in the sample – and that might be fine,” he says. One of these methods consists in using reagents like polyethylene glycol to precipitate the nanoparticles and microparticles in suspension from a given sample. But it also reduces non-vesicular proteins, RNAs and other debris, and while this is not a problem in the context of biomarker discovery or analysis, it is poorly suited for the selective characterization of subsets of electric vehicles.
The most common starting point for EV purification today is differential ultracentrifugation, in which highly heterogeneous preparations of cell culture medium or other biological samples are centrifuged at increasing speeds to selectively precipitate particles. of a particular size. For exosomes, it is usually a multi-step process in which the EVs themselves are ultimately pelletized at forces greater than 100,000 times the force of gravity for many hours. “It’s very reproducible,” says Juan Manuel Falcón, a researcher at CIC bioGUNE in Derio, Spain. “The problem is that it’s too laborious.”
Electron micrograph of small exosomes purified from cultured cells by differential ultracentrifugation and AF4.
Credit: David Lyden and Haiying Zhang, Weill Cornell Medicine
And if the goal is to obtain truly pure preparations of a particular vesicle subtype, this is often only a first step in the process, since the products of differential ultracentrifugation inevitably remain contaminated with d other biomolecules. This is particularly problematic with blood products like plasma and serum, which are teeming with proteins that become enriched together with exosomes in differential ultracentrifugation – notably low and high density lipoproteins, which are similar in size and density. to many EV subpopulations. Buzás also notes that they are several orders of magnitude more abundant than vesicles in blood-derived samples. Complementary methods can help achieve superior purification, including density gradient centrifugation and size exclusion chromatography – strategies that allow more precise separation of EVs from other contaminating molecules.
New alternatives are also emerging. For example, Lyden’s group worked with a technique called asymmetric flux field flux fractionation (AF4), in which a liquid sample containing ultracentrifugally purified EVs is passed through a channel while also being subjected to a perpendicular fluid flow.3. This results in highly efficient particle size separation as they pass through the channel while preserving much of the sample’s inherent heterogeneity. “It’s a great analytical tool…but it’s still a little labor intensive,” says Haiying Zhang, a Weill Cornell researcher who regularly collaborates with Lyden. Another promising platform, developed by Luke Lee at Harvard and Fei Liu at Wenzhou Medical University in China, uses a sophisticated ultrafiltration method to purify EVs directly from clinical samples such as urine, saliva or plasma. In a 2021 publication, they showed that their EXODUS platform could extract EVs from a urine sample in ten minutes while achieving higher yield and purity than a range of other isolation methods. well established.4.
Super-resolution microscopy imaging of EVs of purified fibroblasts (left and top right) with the ONI Nanoimager, with individual tetraspanin distribution at a single EV (bottom right).
Credit: Olesia Gololobova (Witwer laboratory) and Laura Hüser (Weeraratna laboratory), Johns Hopkins
Finally, there are opportunities to selectively isolate vesicle subpopulations via affinity purification with antibodies that specifically recognize known EV-specific surface markers. The most popular targets here are three transmembrane proteins belonging to the tetraspanin family: CD63, CD81 and CD9. “This is an opportunity to permanently purify the vesicles,” says Buzás. But even here, the complexity is considerable. For example, Koen Breyne, a researcher in Xandra Breakefield’s lab at Harvard Medical School, notes that although tetraspanins are a defining characteristic of electric vehicles, not all electric vehicles produce them at equivalent levels. As such, even affinity capture experiments will inevitably give an incomplete picture of the vesicle population. “I think a statistic can’t identify a certain class or type of electric vehicle,” Breyne says.