Exosome Characterization: Key to Emerging Rx, Dx

Among the identifiable and distinct extracellular vesicles (EVs), exosomes have captured the most interest due to their potential in both diagnostic and therapeutic medicine. Exosomes, which form during organelle fusions, shuttle a wide variety of molecular cargo between cells. Those contents, which may be genes, proteins, macromolecular fragments, metabolites, or other small molecules, provide clues on an organism's health status. Although the "natural" application would appear to be Dx, more than 50 exosome-based therapies are under development.

Characterizing exosomes for their origins, dimensions, and contents is now a distinct branch of subcellular research, but one that borrows extensively from cell biology. At the level of enumeration and gross characterization, researchers turn to cell counting or other physical/mechanical methods like centrifugation; for exosome content analysis, they borrow methods from single-cell and rare-cell proteomics, genomics, and metabolomics. The twist or challenge is their size: exosomes are found roughly in the 30–150 nm diameter range, which is about the size of a lipoprotein. A typical mammalian cell is a thousand times larger.

Of counting and contents

"Exosomes are produced by all cell types," explains John Ludlow, Ph.D., VP for regenerative medicine at Zen-Bio. "In cultured cells, we collect conditioned medium and isolate exosomes either by ultracentrifugation, size exclusion chromatography, or immunoaffinity. We also isolate exosomes from bodily fluids, most often from blood serum or plasma, by the same methods. We program exosomes to contain either the protein or nucleic acid of interest by electroporation of the exosomes themselves, or transfection of cultured cells to overexpress the protein or nucleic acid of interest, and screen to see that exosomes carry this cargo."

Zen-Bio characterizes exosomes using a Thermo Fisher NanoDrop spectrophotometer, which quantifies protein and estimates RNA content through direct absorbance. "Exosomes are not lysed, stained, or RNA-extracted before these measurements," Ludlow adds. "We assess particle diameter and concentration through nanoparticle tracking analysis (NTA) using the ZetaView^® nanoparticle tracking video microscope from Particle Metrix. We also isolate miRNA from exosomes and perform sequencing to assess their nucleic acid cargo."

Investigators then perform surface protein analysis for the exosome-specific markers CD-9, CD-63, and CD-81, to confirm that the isolated particles are indeed exosomes. For these studies, Zen-Bio uses the MACSPlex Exosome Kit from Miltenyi Biotec to isolate and analyze exosome surface proteins. From there, they can analyze for proteomic content via LC/MS, and conduct biological function studies for cell proliferation, migration, angiogenesis, cytokine inhibition, etc.

Prep-free preps?

"To analyze exosomes accurately, the nanoparticles must be purified from complex biofluids, which requires laborious purification, followed by confirmation that the biomarkers of interest are associated with the collected exosomes," says George Daaboul, Ph.D., Chief Scientific Officer at NanoView. Another approach involves single vesicle analysis methods that allow the colocalization of exosome and biomarker of interest on single exosomes. "This allows accurate identification and analysis of exosome-associated biomarkers in the presence of other biomolecules present in the matrix of biological fluids. This is the benefit or our ExoView^® platform, which requires no purification."

Daaboul notes that biomolecules associated with exosomes can also be present, in other forms, in biofluids. "To ensure that what you are analyzing is associated with exosomes, researchers rely on purification methods to ensure that only exosomes and molecules associated with them are evaluated. Since exosomes are nanoparticles, they are typically purified using size-based separation techniques like ultracentrifugation and size exclusion chromatography." Simpler physical separation techniques like precipitation also enrich for exosomes, but contaminants tend to co-precipitate. "It is possible to bypass these issues by using single vesicle analysis, where co-localization of multiple markers provides orthogonal verification that the detected biomarker is associated with exosomes."

Exosomes are powerful biomarkers because they participate actively in the course of many diseases, and that activity sheds light into disease pathology. "Unlike other circulating biomarkers, the lipid vesicle nature of the exosome allows enriching for subpopulations that reflects different tissues or cell types, and then exploring their nucleic acid and protein contents," Daaboul says. "When analyzing exosomes, special attention needs to be taken in matching the appropriate sample-preparation technique with the specific downstream analysis. For example, mass spectrometry requires very pure samples and the total exclusion of any non-exosome associated proteins, requirements that are less critical for immunoassays."

Complex milieu

A major challenge in studying extracellular vesicles is their small size, so instruments must be up to the task of reliably detecting them in a complex milieu. "High sensitivity flow cytometry is a great approach since it is quantitative, high-throughput, with a high detection rate and high signal to noise ratio, all of which facilitate separating exosomes from background debris," explains Stephanie Brunelle, Ph.D., Product Manager for flow cytometry at Luminex. "Orthogonal approaches are possible as well, for example electron microscopy, nanoparticle tracking, and microfluidic resistive pulse sensing."

Controls are just as important as samples when studying extracellular vesicles, she adds, and should include buffer only, antibody/dye only, and detergent plus labeled exosome samples; analysts should also perform a dilution series.

"These controls can help determine whether the 'swarming' effect is present. This occurs when multiple extracellular vesicles form a swarm and are detected as a single object, which is undesirable," Brunelle tells Biocompare.

Exosome analysis is full of potential but adopting and commercializing these techniques, at scale and in routine medical settings, presents several challenges. Operational hurdles include generating clean samples for clinical trials. Additional challenges in commercializing, or even industrializing, exosome analysis in mainstream medicine involve consistency in assay design, "so that all research is consistent from lab to lab and country to country," Brunelle says. "The field is relatively new and we are constantly learning."