Exosome Isolation and Analysis Methods

Exosomes are small, secreted organelles formed by budding at both plasma and endosome membranes. In recent years, they have received growing interest for their diagnostic and therapeutic potential, spawning the development of novel tools and technologies for exosome characterization. This article provides a recap of why exosomes are so appealing to researchers and discusses methods for exosome isolation and analysis.

What’s in a name?

The term ‘exosome’ is a source of some controversy. First used in the literature to describe transforming DNA fragments that transferred between Drosophila cells, it was subsequently adopted as a generic alternative  to ‘extracellular vesicle’. However, the recently updated guidelines of the International Society for Extracellular Vesicles (ISEV) on minimal information for studies of extracellular vesicles (MISEV) propose exosomes to be a subtype of extracellular vesicles (EVs), along with other cell-released, membranous structures such as microvesicles, oncosomes, and apoptotic bodies. “The term exosome is now generally understood to reference EVs characterized by a diameter of 40–150 nm and a density of 1.09–1.18 g/mL,” clarifies John W. Ludlow, Ph.D., Vice President, Regenerative Medicine, at ZenBio. “Exosomes participate in a wide variety of cellular activities and can be isolated from multiple body fluids as well as from the conditioned media of any cell type that can be grown in culture.”

Diagnostic and therapeutic potential

Exosomes have been implicated as critical messengers in cell-to-cell communication since they selectively transport cargo including proteins and RNA. Such intercellular vesicle trafficking underpins many aspects of human health and disease and also suggests a role for exosomes as drug delivery systems. “EV research focused on human disease can be broadly categorized as either diagnostic or therapeutic,” reports Emily Cartwright, Ph.D., Product Marketing Specialist, Antibodies, at Bio-Techne. “Within the diagnostic field, cancer and neurodegenerative disease researchers are working to identify EV-associated biomarkers from readily accessible sites like blood, serum, and cerebrospinal fluid, which would reduce the need for invasive tissue biopsies. For therapeutic purposes, researchers are interested in creating synthetic EVs and using them as carriers for small molecule drugs, vaccine antigens, and other water-soluble therapeutics, as they can travel to distal sites. Additionally, using EVs harvested from mesenchymal stem cells to promote tissue repair and regeneration is under investigation for conditions including liver, cardiac, and neuronal disease, as well as for repairing cartilage.”

Exosome isolation

A major challenge for researchers studying exosomes lies in isolating a consistent population from the total pool of EVs. Ultracentrifugation remains the gold standard approach, although equipment costs can limit its utility. “Alternative techniques for exosome isolation include size exclusion chromatography, precipitation, and immunoaffinity,” notes Ludlow. “However, the latter two methods often result in exosome preps being contaminated with ancillary proteins and nucleic acids that can impede downstream analysis. At ZenBio, we use ultracentrifugation followed by size exclusion chromatography, which provides opportunities for scale-up.”

Exosome analysis

Exosome analysis is based on either size or phenotype, each requiring different technologies for characterization. “Nanoparticle Tracking Analysis is the most prevalent method for determining EV size distribution and concentration,” explains Ludlow. “By simultaneously measuring diffusion events and providing direct observation of individual particles, NTA yields high-resolution results that are supported by visual validation. Moreover, where NTA employs a fluorescence mode, it enables differentiation of intrinsic or fluorescently labeled nanoparticles.” Another approach, Tunable Resistive Pulse Sensing technology, provides accurate measurements of nanoparticles suspended in electrolytes, as opposed to the estimates provided by light scattering techniques. This is complemented by an emerging technology for nanoparticle sorting, Deterministic Lateral Displacement (DLD) pillar array, which has previously been used to sort, separate, and enrich micrometer-scale particles including parasites, bacteria, and circulating tumor cells in blood.

“While the methods just described are highly precise for determining the presence of intact vesicles, they do not provide a wealth of information about phenotype or the messages the EVs are carrying,” comments Cartwright. “For this type of analysis, researchers commonly use western blot, ELISA, and increasingly, flow cytometry.” To streamline western blot-based exosome analysis, Bio-Techne has developed exosome marker antibody sampler packs containing antibodies from each of the categories suggested by the MISEV 2018 guidelines for exosome characterization; these are validated both for traditional western blotting and for use on the automated Simple Western platform.

Another company investing in dedicated reagents for exosome analysis is Biotium, which recently launched the ExoBrite™ EV Membrane Staining Kits for flow cytometry. “Analyzing exosomes by flow cytometry is complicated by the small size of these particles,” reports Alexis Madrid, Ph.D., Biotium’s Assistant Director of Bioscience. “Not only must researchers take care when determining the size detection limit of the instrument, but they must also implement measures aimed at minimizing debris and small particles that can confound results, as well as making certain to include appropriate controls.”

Madrid adds that a further, rarely discussed, challenge for fluorescence-based detection of exosomes by flow cytometry is that longer wavelengths of light are not as efficient at exciting small particles compared to shorter wavelengths, which limits the available selection of dyes and may also impact data accuracy. “ExoBrite™ EV Membrane Stains were designed to circumvent common problems such as aggregation or dim signal and are formulated to give clean, bright exosome staining,” she says. “Currently, they are available with four color options, compatible with antibody co-staining, and can be used to stain purified or bead-bound exosomes for analysis via flow cytometry or other fluorescence-based detection methods.”