The beginning of the 21st century marked a turning point in understanding the role of extracellular vesicles (EVs) in both physiological processes and diseases. Scientists began to investigate the content of EVs using different approaches, including proteomics, lipidomics, genomics, and biochemistry. These studies shed light on the potential roles of EVs in different contexts. This research focused on immune cells, inspired researchers in the field of immune therapies against cancer, as demonstrated by the work of Chaput (2003) and Zitvogel (2005). Ratajczak (2006) made a breakthrough when demonstrating that cell-derived microvesicles could reprogram other cells through the horizontal transfer of mRNA and protein delivery, a discovery that highlighted the significant impact EVs could have on cellular behavior. Today, there is now compelling evidence of EVs having multiple roles in regulating the immune response (Figure 1. Also refer to Buzas, Nature Reviews Immunology, volume 23, pages 236–250 (2023).

Figure 1: Regulatory Functions of EVs. The central EVs express specific markers on their surface and interact with various immune cells, including T cells, Natural Killer cells (NK), and dendritic cells (DC). Regulatory T cells (Treg), cytotoxic T lymphocytes (CTL), adenosine triphosphate (ATP), and adenosine monophosphate (AMP) are involved in these interactions. Please note that the size of the extracellular vesicle in the figure has been intentionally exaggerated for illustrative purposes.

Currently, there is a faction of scientists within the scientific community who use the term “exosome,” while originally these structures were referred to as “microparticles.” This discrepancy in nomenclature can lead to confusion, particularly when arbitrary size ranges are assigned to different types of vesicles. To address this issue, it has been suggested that the umbrella term “extracellular vesicles” (EVs) should be used to describe non-replicating structures that are bounded by a lipid bilayer. The establishment of the International Society for Extracellular Vesicles (ISEV) in 2011 has played a crucial role in fostering consensus on this terminology. The definition of EVs as non-replicating structures delimited by a lipid bilayer has been formalized in the current recommendations outlined in the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines (2018). The official journal of the society, the Journal of Extracellular Vesicles (JEV), further reinforces the accepted terminology and provides a platform for research in the field.

A major topic of debate in the field of extracellular vesicles (EVs) revolves around the selection of methods for their isolation and enrichment. Various techniques are available, including ultracentrifugation, ultrafiltration, size-exclusion chromatography, precipitation methods, immunoaffinity-based assays, magneto-immunocapture, microfluidic immunochips, and lipid nanoprobes. The choice of method depends largely on the specific objectives of the study.

In terms of EV characterization, several biophysical approaches have been developed, such as nanoparticle tracking analysis and reverse pulse sensing, which enable the counting and sizing of EVs. Further characterization involves methodologies like proteomics, genomics, lipidomics, and more. While these techniques provide insights into EV populations as a whole, flow cytometry allows for the individual characterization of EVs. Single- EV information is particularly valuable as it unravels the heterogeneity within EV populations. However, ensuring the accuracy and reproducibility of studies is crucial. To address this, various working groups came together in 2011 to establish the International Society for Extracellular Vesicles (www.isev.org), which has played a pivotal role in advancing the EV field.

In 2018, the society published updated guidelines for EV analysis known as MISEV (Minimal Information for Studies of Extracellular Vesicles). These guidelines aim to address controversies and questions surrounding EV research. As stated by Ramirez et al. (2018), “a persistent concern in flow cytometry is the reliable distinction between EVs carrying specific protein markers and those that do not, in order to accurately measure the proportion of EVs of a particular type.” Quantitative and qualitative analyses of EV heterogeneity within samples are crucial for a comprehensive understanding of the biology. To improve this process, a new resource on the use of flow cytometry for EV research has been made available recently (Welsh JA, Arkesteijn GJA, Bremer M, et al. A compendium of single extracellular vesicle flow cytometry. J Extracell Vesicles. 2023;12(2):e12299. doi:10.1002/jev2.12299).

The research on extracellular vesicles (EVs) has evolved into a distinct field, complete with its own dedicated society and scientific conferences. The discovery of EVs has illuminated a previously unknown realm that plays a crucial role in regulating various physiological processes. Further studies in this field hold the potential to unravel the heterogeneity of EVs and elucidate their diverse functions. Moreover, they could pave the way for identifying novel biomarkers of diseases. By monitoring cell-specific EVs, researchers can search for specific biomarkers that are present on or within EVs. This approach offers a non-invasive means of defining disease biomarkers, as it only requires a simple blood draw. Promising results have already been demonstrated in the context of Alzheimer’s disease (Tao-Ran (2019).

Furthermore, EVs are currently the subject of intense investigation for their potential use as carriers to transport therapeutic compounds to target cells or organs. By engineering EVs loaded with drugs that can specifically target certain cells, such as tumors, researchers hope to overcome challenges associated with conventional therapies. This targeted delivery approach could improve the efficacy of treatments and minimize unwanted side effects. This has important implications, as current therapies are often systemically administered, resulting in suboptimal functionality and potential to cause harm to health tissues.

References

• Asleh et al. Extracellular vesicle-based liquid biopsy biomarkers and their application in precision immuno-oncology. Biomark Res 11, 99, 2023.
• Ramirez et al. Technical challenges of working with extracellular vesicles. Nanoscale 10, 881-906, 2018.
• Théry et al. 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, 2018.
• Couch et al. A brief history of nearly EV-erything – The rise and rise of extracellular vesicles. J. Extracell Vesicles. 10(14):e12144, 2021.
• Tao-Ran et al. Extracellular vesicles as an emerging tool for the early detection of Alzheimer’s disease. Mech Aging and Dev. 184:111175, 2019.
• Welsh JA, Arkesteijn GJA, Bremer M, et al. A compendium of single extracellular vesicle flow cytometry. J Extracell Vesicles. 2023;12(2):e12299. doi:10.1002/jev2.12299.

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