Exosomes, a type of extracellular vesicle, are membrane-bound vesicles released by cells into the extracellular milieu. Exosomes are thought to have multiple and varied functions, from disposing of unwanted molecules to intercellular communication. Formed when multivesicular bodies fuse with the cell membrane, exosomes are of diagnostic and therapeutic interest because they can carry proteins throughout the bloodstream. The young and quickly evolving field of exosome research still burgeons with technological and methodological developments. Here’s a glimpse at the main methods used today for isolating exosomes, along with some expert advice.
Alexander Vlassov, senior manager of R&D at Thermo Fisher Scientific, is impressed by the evolution of the exosome field since its explosion in 2012–13, driven by academic research. But the protocols continue to evolve, because the small size (about 30–150 nm in diameter) and complex and variable composition of exosomes make them challenging to purify and characterize. “They’re in between individual molecules and cells in terms of size,” he says. “They have a very complex composition, different cargo for different nanovesicle sub-populations, different paths of circulation in the body, and extremely diverse functions.” With characterization ongoing, there is still no “rock solid” definition of exosomes and other extracellular vesicles, despite classifying based on size, density, or surface markers.
Ultracentrifugation
Originally, ultracentrifugation was considered the gold standard for isolating exosomes, but Vlassov believes this is no longer true. “When you pellet exosomes with ultracentrifugation, you often recover contaminating entities such as large protein complexes, so it’s okay for some studies but overall preparations are not very clean,” he says. Instead, density gradient ultracentrifugation can return a higher degree of purity, is a well-established method for exosome isolation, and is suitable for samples of large volumes. Unfortunately, its drawbacks include a time-consuming, complex protocol and special equipment. Furthermore, the result may contain other unwanted cellular components that have the same density as the desired exosomes, or exosomes of the same density but different sizes.
Immunoaffinity capture
In immunoaffinity capture, exosomes are captured by binding to monoclonal antibodies, which are attached to magnetic particles or beads for easy isolation. “For processing multiple samples, or for small volumes of body fluids like serum and plasma, precipitation with different reagents can be a better approach,” says Vlassov, for instance the use of Thermo Fisher Scientific’s Total Exosome Isolation reagents.“You can obtain pretty high purity and yield without advanced training or an ultracentrifuge, and this is sufficient for most needs.”
Miltenyi Biotec’s Exosome Isolation Kits use magnetic MicroBeads with antibodies to the tetraspanin proteins CD9, CD63, and CD81, which are known to reside on exosome surfaces. “The isolation protocol is based on the well-established MACS®Technology, which enables fast isolation of high purity and high yield exosomes in less than two hours,” says Ariadna Pascual, product manager at Miltenyi Biotec. Subsequent use of the MACSPlex Exosome Kit facilitates the detection of 37 exosome surface proteins by flow cytometry.
Vlassov notes that the great diversity observed among exosomes means that, so far, there is no universal marker for their isolation and purification. Antibodies to surface markers, such as CD63, can be used to isolate subsets of exosomes, but not to recover a total population. “The most important thing is to document exactly all the details of the vesicle isolation and characterization methods in each publication,” he says. “I don't think there will be a consensus regarding the best isolation protocol in the near future.”
Size exclusion chromatography
Size exclusion chromatography (SEC) is another widely used method that separates extracellular vesicles according to size using a resin inside a chromatography column. SEC has the advantages of being relatively simple, repeatable, and scalable, according to Hans van der Voorn, CEO of Izon Science. Izon’s qEV platform isolates extracellular vesicles/exosomes using SEC columns, augmented by “precisely tuned automatic fraction collectors,” says van der Voorn. “This offers the precision, repeatability, and scalability of isolation that the extracellular vesicle field has been missing.” Different column sizes accommodate different sample volumes, and are available in in two size ranges for exosome size selectivity (35nm+ and 70nm+).
Best isolation method depends on application
Ultimately, applications may dictate the best exosome isolation method. “For example, for protein analysis by western blot, RNA sequencing, and electron microscopy, the isolation method based on immunolabeling would be the best choice due to its high specificity,” says Pascual. “However, if you want to analyze isolated exosomes by mass spectrometry, a non-labeling method like density gradient ultracentrifugation would be a better choice.” Pascual believes that the lack of standardized protocols especially impacts downstream applications.“It is very important to choose a method that gives you a high purity population of exosomes with the minimum amount of contaminants and that it is highly reproducible,” she says.
Van der Voorn believes that subsequent analytical steps should also be considered. For example, the successful use of exosomes or extracellular vesicles containing RNA (EV-RNA) in biomarker analysis for various diseases or conditions will require repeatability and standardization. “Accurate extracellular vesicle concentration measurement is also expected to become a standard requirement for quantification, as the EV-RNA quantities will need to be normalized back to the number of EVs that generated the EV-RNA,” he says. “That will likely mean an automated splitting of the isolated EVs.”
The surge of developments in exosome research continues with a fervent energy that may spill over into other areas of research. “This field was born in a fantastic collaboration of academic researchers, biotech and pharma industries,” says Vlassov. “It’s still early days, but I already see exciting developments in exosome applications such as liquid biopsy, diagnostics, and development of vaccines and therapies.”