Comparison of Exosome Isolation and Analysis Methods

Your body makes heavy use of the versatile lipid-membrane-enclosed nanoparticles known as exosomes. In humans, they have direct roles in immunity, intercellular communication, and cancer (Wang et al., 2023). Especially given that exosomes are in common, easy-to-sample biological fluids such as blood and urine (Martins et al., 2023), it’s no surprise that hundreds of exosome-focused clinical trials have been conducted or are in progress (Chavda et al., 2023). In fact, the global exosome research market was $144 million in 2021, and is predicted to reach $661 million by 2026 (MarketsandMarkets, 2022).

There are established guidelines for documenting the functional activities that are associated with exosomes and other extracellular vesicles (Thére et al., 2018). Nevertheless, as of yet there is no single established procedure for isolating exosomes, and a comprehensive means of analyzing them might require multiple technologies (Eisenstein, 2022). Such capabilities are foundational to minimizing toxicity and other off-target outcomes of their use.

This lack of standards in isolation and analysis is hindering exosome research (Li et al., 2019). In the meantime, researchers need to identify the procedures and tools that are most appropriate for their application. Here, we provide an overview of exosome isolation and analysis, and discuss pertinent issues with representative academic and industry experts.

Exosome isolation: Come out wherever you are

Researchers commonly isolate exosomes by some combination of the following (Xu et al., 2023). Ultracentrifugation is a longstanding workhorse of cell and molecular biology, but is slow and the resulting exosome purity can be low. Size-exclusion chromatography and precipitation can be fast, cheap, and gentle; but can also have contamination issues. Ultrafiltration minimizes the necessary sample quantity but its use of force can damage exosomes. Immunoaffinity chromatography can isolate exosomes, but eluting them from antibody-coated beads, for example, can be difficult. Microfluidics-based exosome separations and other recent developments, including commercial isolation and purification kits, can require further validation (Chen et al., 2022).

Three main challenges have hindered researchers’ development of standard procedures for isolating exosomes. One, an isolation procedure that at first glance seems to be successful can nevertheless select for non-representative sub-populations of exosomes (such as in terms of exosome size and tetraspanin content; Bettio et al., 2023). Two, storage conditions after isolation (such as freeze–thaw cycles and the storage temperature) can affect exosome properties (Gelibter et al., 2022; Kusuma et al., 2018). Three, many custom laboratory procedures are on an insufficient scale for clinical validation or commercial utility (Ahn et al., 2022).

Exosome analysis: Interrogate me

Many of the myriad tools available for cell analysis are also applicable to exosome analysis. Here, we focus on electrochemistry and mass spectrometry.

Electrochemistry is a straightforward means of exosome analysis. For example, Keke Hu (Xiamen University), Andrew Ewing (University of Gothenburg), and coworkers have measured the catecholamine neurotransmitter content of single exosomes, released in real time from single neuroendocrine cells (Hu et al., 2023). Ewing says: “Exosomes are known to contain proteins and RNA, for example, but containing neurotransmitters was a surprise. This is potentially a unique way for chemical messaging between cells—not yet proven but possible.” Hashkavayi et al. (2020) reports biomolecular targets, signaling elements, linear response ranges, and detection limits for various means of electrochemical analysis of cancer-derived exosomes.

Mass spectrometry is central to ongoing single-cell multiomics efforts—interrogating multiple layers of physiology within an individual cell—and thus is widely used in exosome analysis (Jajaludin et al., 2023). For example, a recent study of cutaneous melanoma exosomes identified numerous proteomic biomarkers, classified in accordance with angiogenesis and other cancer-related categories (Surman et al., 2023). By mass spectrometry, thousands of proteins can be quantified within a single exosome (Vlachakis et al., 2023). Such information is pertinent to the mechanisms by which various metabolites enter exosomes, and thus to understanding disease progression.

Industry perspectives

ZenBio is a contract research organization for preclinical screening, drug discovery, and other needs; and has a track record of procuring SBIR grants for extracellular vesicle research. They primarily isolate exosomes by ultracentrifugation and size-exclusion chromatography, and subject their exosomes to rigorous quality control—such as by any of 37 surface proteins. John Ludlow, Vice President for Regenerative Medicine, says: “We have ten years of experience in exosome isolation and analysis, and are further opening the door to using exosomes in clinical settings. We are advancing exosome research in diseases, tissue engineering, regenerative medicine, and biopsies for diagnosis as well as prognosis.”

Proteintech is an expert resource for monoclonal antibodies that are ideal for isolating and characterizing exosomes, and determining their functions. Dr. Sreethu Sankar, Product Manager, says that their antibodies “can be used in techniques like flow cytometry, western blotting, ELISAs, or immunoprecipitation to study exosome dynamics. These methods allow you to analyze the expression levels or interactions of specific proteins or cargo molecules in exosomes. Monoclonal antibodies can also be used for neutralizing certain exosome surface proteins to assess their role in uptake, targeting, or signaling in recipient cells.” Dr. Sankar further highlights that “Proteintech offers several highly validated primary conjugated and unconjugated monoclonal antibodies against popular exosome markers like Alix, TSG101, CD9, and many more.

Exosomes are no longer considered cellular junk or artifacts of sample preparation (Eisenstein, 2022). Perhaps with ongoing research, exosome isolation and analysis will become more streamlined than they are today.

References

Ahn S-H, et al. (2022). Manufacturing therapeutic exosomes: From bench to industry. Mol. Cells 45(5):284–290.

Bettio V, et al. (2023). Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques.  PLoS ONE 18(5):e0285440.

Chavda VP, et al. (2023). Exosome nanovesicles: A potential carrier for therapeutic delivery. Nano Today 49:101771.

Chen J, et al. (2022). Review on strategies and technologies for exosome isolation and purification. Front. Bioeng. Biotechnol. 9:811971.

Eisenstein M (2022). Eavesdropping on extracellular vesicles. Nat. Meth. 19:1518–1523.

Gelibter S, et al. (2022). The impact of storage on extracellular vesicles: A systematic study. J. Extracell. Vesicles 11(2):e12162.

Hashkavayi AB, et al. (2020). Advances in exosome analysis methods with an emphasis on electrochemistry. Anal. Chem. 92(19):12733–12740.

Hu K, et al. (2023). Single exosome amperometric measurements reveal encapsulation of chemical messengers for intercellular communication. J. Am. Chem. Soc. 145(21):11499–11503.

Jalaludin I, et al. (2023). A guide to mass spectrometric analysis of extracellular vesicle proteins for biomarker discovery. Mass Spectrom. Rev. 42(2):844–872.

Kusuma GD, et al. (2018). To protect and to preserve: Novel preservation strategies for extracellular vesicles. Front. Pharmacol. 9:1199.

Li X, et al. (2019). Challenges and opportunities in exosome research—Perspectives from biology, engineering, and cancer therapy. APL Bioeng. 3(1):011503.

MarketsandMarkets (2022). Exosome research market size, growth by product and services (kits, reagents, instrument), indication (cancer, infectious diseases), application (biomarkers, vaccines), manufacturing services (stem cell, dendritic cell-derived), end user & region - global forecast to 2026. Rep. BT 6939, March. (last accessed Jun. 5, 2023)

Martins TS, et al. (2023). A review on comparative studies addressing exosome isolation methods from body fluids. Anal. Bioanal. Chem. 415:1239–1263.

Surman M, et al. (2023). Similarities and differences in the protein composition of cutaneous melanoma cells and their exosomes identified by mass spectrometry. Cancers 15(4):1097.

Thére C, et al. (2018). Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7(1):1535750.

Vlachakis DP, et al. (2023). Future of exosome bioinformatics. EMBnet J. 28:e1015.

Wang X, et al. (2023). Recent progress in exosome research: Isolation, characterization and clinical applications. Cancer Gene Ther. 1–15.

Xu W-M, et al. (2023). Research development on exosome separation technology. J. Membr. Biol. 256:25–34.