A Practical Guide to Exosome Isolation and Characterization

Quick summary

• No single isolation method guarantees purity—combining approaches such as size-exclusion chromatography and density gradient centrifugation is recommended
• Researchers should remove cells from source material as early as possible to mitigate against the consequences of cell disruption, activation, or cell death.
• Characterization should use multiple orthogonal methods, including protein marker analysis, electron microscopy, and particle size/concentration analysis
• RNA profiling tools including RT-qPCR, ddPCR, and RNA-Seq can evaluate characteristic EV RNA cargo, with ddPCR offering precise absolute quantification of low-abundance transcripts
• Advances in isolation and characterization technologies are helping researchers better realize the diagnostic and therapeutic promise of exosomes

Exosomes have significant biomarker and therapeutic potential, but isolation and characterization challenges make realizing these benefits difficult. In this article, Yoon-Tae Kang and Ertan Ozymak, Staff Scientist and Senior Product Manager, respectively, from Bio-Rad Laboratories, as well as a team from Cosmo Bio USA offer guidance for isolating and characterizing exosomes and highlight some of the enabling technologies available to researchers.

What are exosomes?

Exosomes are a widely studied subtype of extracellular vesicles (EVs) that originate from the endosomal pathway and are released through the fusion of multivesicular bodies with the plasma membrane. Because exosomes carry bioactive molecules that reflect the physiological and pathological state of their cells of origin, they are of significant interest from both research and translational perspectives. Furthermore, their stability in biological fluids and intrinsic ability to mediate molecular transport support their application in diagnostics and drug delivery. According to the latest iteration of the Minimal Information for Studies of Extracellular Vesicles (MISEV2023), a field consensus document that provides recommendations and guidance for EV research, the term ‘exosome’ should be used with caution. Unless biogenesis is clearly demonstrated, operational terms like ‘small EVs (sEVs)’ are deemed more appropriate.¹

EV isolation methods

EV isolation methods include precipitation, filter concentration, size-exclusion chromatography, affinity or ion-exchange chromatography, differential ultracentrifugation, immunoprecipitation, and density gradient centrifugation. These differ considerably in terms of recovery and specificity, highlighting the need to balance yield, purity, and downstream application requirements when it comes to method selection. In general, MISEV2023 recommends using several isolation methods in combination. For example, when working with blood, both size-exclusion chromatography and density gradient centrifugation might be required to remove free, ‘soluble’ proteins such as serum albumin, immunoglobulins, and fibrinogen from the EV prep, which could otherwise co-isolate and affect downstream analysis. Notably, researchers are advised to remove cells from the source material as early as possible, since cell disruption can form particles resembling native EVs, while cellular processes like activation and death can alter EV composition and function.

• Tools for EV isolation

EV isolation products offered by Cosmo Bio USA include the EXORPTION® Extracellular Vesicles Purification Kit, which uses polymer-based precipitation to provide high purity EVs with the aid of a microcentrifuge, and OptiPrep™ Density Gradient Media, which utilizes density gradient ultracentrifugation.

Additionally, researchers can use size-exclusion chromatography (SEC) to achieve reproducible isolation of intact EVs with high yield and purity. Specifically, SEC is performed after filtration using Bio-Rad’s NGC Chromatography System, with the option to collect small volumes into 96-well plates using the NGC Fraction Collector or the BioFrac™ Fraction Collector.

EV characterization

While absolute exosome purity cannot be definitively established by any single isolation method, combining orthogonal characterization approaches provides greater confidence in the purity of your EV prep. Examples of EV characterization methods include the following:

• Protein marker characterization through western blotting², flow cytometry, ELISA, or mass spectrometry to detect EV-associated markers such as CD9, CD63, CD81, TSG101, and Alix, while also assessing the absence of common cellular contaminants like calnexin or GM130. Because marker expression is not uniform across all exosome populations, it is recommended that isolated samples be viewed as vesicle populations that are “highly enriched for exosomal characteristics.”
• Physical/morphological characterization using transmission electron microscopy (TEM) or cryo-electron microscopy (cryo-EM) to visualize vesicle structure and membrane integrity.
• Particle size and concentration analysis using techniques such as nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), or tunable resistive pulse sensing (TRPS), which help confirm the expected small EV size range (typically ~30–200 nm).
• Density-based characterization using ultracentrifugation gradients to enrich vesicles within expected buoyant density ranges.
• Functional uptake or bioactivity assays to demonstrate that isolated vesicles can interact with recipient cells and mediate biological effects consistent with EV signaling.
• RNA profiling to evaluate characteristic EV RNA cargo, including small RNAs and microRNAs associated with exosome populations. Common approaches include RT-qPCR for sensitive targeted gene expression analysis and droplet digital PCR (ddPCR) for highly precise absolute quantification of low-abundance transcripts (below twofold) with multiplexed detection. Alternatively, for comprehensive transcriptome profiling and novel transcript discovery in bulk EVs, RNA sequencing (RNA-Seq) has been utilized; products such as the SEQuoia Complete Stranded RNA Library Preparation Kit are pertinent to this application. More recently, droplet-based single cell RNA sequencing (scRNA-Seq) has been used to profile the transcriptome of individual EVs³; the ddSEQ Single-Cell 3’ RNA-Seq Kit can be utilized for similar discovery studies.

Conclusion

Exosomes are widely viewed as the most important subclass of EVs due to proposed roles in cell-to-cell communication, biomarker transport, and disease biology. While isolating and characterizing exosomes presents unique challenges, developments within the field of EV research promise to unleash the diagnostic and therapeutic potential of these critically important particles.

References

1, Welsh, JA, Goberdhan, DCI, O'Driscoll, L., et al. (2024). Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. Journal of Extracellular Vesicles, 13, e12404.

2. Kowal EJK, Ter-Ovanesyan D, Regev A, et al. (2017) Extracellular Vesicle Isolation and Analysis by Western Blotting. Methods Molecular Biology,1660:143-152.

3. Luo T, Chen S-Y, Qiu Z-X, et al. (2022) Transcriptomic Features in a Single Extracellular Vesicle via Single-Cell RNA Sequencing. Small Methods, 6, 2200881.