Developments in Extracellular Vesicle Research Present a Promising Future

Extracellular vesicle (EV) populations and their low-abundance cargo have long offered promising possibilities for research and treatment, but the actual art of studying them has been a cumbersome challenge, as many workflows and systems have not been able to provide the required sensitivity, precision or reproducibility. Recent advances in nanoscale technology ensure researchers more data with less hands-on time, vastly increasing what they can study and discover about diseases.

The importance of EV research

EVs and other microvesicles are the result of a variety of cellular processes, and the history of EVs can be traced back to E.J. Chargaff in the mid-1940s with his discovery of cell structure in relation to blood coagulation using centrifugation. The abundance, size and cargo of EVs offer researchers a lens into cellular physiology by presenting a snapshot in time of a particular cellular state, which can be used to measure disease state or progression.

It is particularly exciting to see the progress that nanoscale technologies now offer researchers. Alfonso Blanco, director of the Flow Cytometry Core at University College Dublin, explains the favorable alternative that analyzing these tiny particles can offer and how this impacts patients: “You can see a lot of potential biomarkers coming into clarity, and you are getting the samples from the blood or even urine, rather than invasive tissue or surgery. That’s crucial in situations where the doctor doesn't want to intervene more than once because the patient is suffering. Getting the blood sample is easier, so liquid biopsies are coming as a big alternative to predict a disease, follow the disease, treat that disease and then follow up that the treatment is effective, all in an easy way by looking at these biomarkers.”

Traditional challenges to EV research

If you were to draw a Venn diagram representing critical needs for the measurement of EVs, you would find that no one technology sits comfortably in the middle. Where we’ve had sensitivity and precision, it’s come at a cost of throughput, technical acumen and expense. Where we’ve been able to access affordable, high-throughput methods, we’ve given up sensitivity and precision or settled for answers that can only be applied to populations as an average instead of individual particles.

“Labs have often had to combine two orthogonal technologies to get one picture – two platforms would have to work in harmony to deliver the full portfolio of different particles. This would be frustrating for a variety of reasons, including the largely manual workflow, variability, extra equipment, cost, space constraints and operator sensitivity,” Blanco explained.

New technology finally emerges

Technologies for measuring EVs, in bulk or individually, have evolved. We now have methods to correlate and compare results across some orthogonal technologies, giving us the ability to have more expansive data sets that can answer a variety of questions. Competition in the space and the need for powerful but practical user experiences have given researchers access to more affordable and more capable tools.

Nanoscale flow cytometry technology finally answers the problem with existing analyzer platforms by offering a discernibly lower level of detection while increasing resolution to characterize lower abundance targets in EV populations between 1 μm and 40 nm, utilizing polystyrene when triggering on violet side scatter. This enables 30–50% more data creation compared to previous methods – and all in one instrument that can detect, count and characterize these biological nanoparticles.

The importance of automation

Researchers need a sensitive, consistent and flexible solution to advance their nanoparticle research, which can be beneficial in finding a cure for a myriad of diseases. Automation is the easiest way to achieve this, by cutting down on manual steps that can often lead to errors, delays or a lack of consistency and reproducibility.

Automation could be capable of sample preparation, sample delivery and macro or process automation during an analytical process. The task of sample preparation in this space is highly dependent upon the source material, so I expect that as we move closer and closer to a diagnostic space wherein an assay is regulated, we’ll see automation applied as well.

Sizing, counting and characterizing are also hallmarks of automation in the EV space. To determine particle sizes using flow cytometry, it is necessary to calibrate the instrument using a well-characterized reference particle and a calibration method. Latex polystyrene particles are commonly used for this purpose due to their NIST-traceable nature and consistent manufacturing process. By calibrating the flow cytometer and providing training to laboratory staff, the resulting data can be reported in absolute units, allowing for accurate and precise method comparison and correlation.

Quality control processes assist statistical analyses

Quality control has historically been focused on consistency. While existing technologies have become more powerful, the lower limits of detection have historically not been sensitive enough in addressing our target particles. Quality control’s purpose can now be expanded through our new nanoscale technology to define the edges of dynamic range and give user-suggested settings that are both robust and consistent.

In order to achieve accurate and reliable counting in flow cytometry, it is important to have precise mechanical control over the volume of samples being delivered, rather than introducing variability by loading samples by hand. Users look for the ability to customize their sample handling so that they can ensure the appropriate amount of sample is used to obtain the desired results. This customization allows for more precise and tailored analysis in flow cytometry.

The future of EV research and discoveries

With more clarity in results, EVs offer advanced diagnostic, prognostic, theranostic and therapeutic procedures, while simultaneously reducing the invasive nature of sample collection. Monitoring disease progression or creating custom therapeutic models is incredibly powerful but it requires specificity in analytical methods. This is finally available for a variety of researchers.

“Updating this workflow is really an exciting moment in research,” said Blanco. “Biomarkers for many different diseases, treatments and more – all from EVs that we can introduce in their cargo, and the possibility that it can lead to a drug being created that will go directly to a target and kill that particular cell type – that’s where the future lies.”

Accessing previously undetectable nanoparticles with more abundance extends our statistically significant data set, allowing us to make important observations. One day this might include advanced disease detection with less invasive sample collection or precise characterization of modalities for cell and gene therapy. This is all possible with the advent of advanced technology that powers both sensitivity and precision using novel tools with focused delivery and low-complexity user experiences. Merging all of these advances into one method is really powerful.

Nanoscale technology is accelerating EV research, the ability of scientists to investigate novel disease biomarkers and individuals’ responses to therapeutic options. This is a paradigm shift in the pursuit and delivery of precision medicine. Size determination is nothing without precision, and precision is nothing without ease of use.

About the interviewee:

Alfonso Blanco is the director of the Flow Cytometry Core Technologies at UCD Conway Institute in the University College Dublin. He is the co-organizer of several national and international courses, workshops and conferences. Alfonso is the president and founder of different societies (e.g., Cytometry Society of Ireland, Spanish Research Society in Ireland,) and he is highly involved as a member and chair of different committees of the International Society for the Advancement of Cytometry (ISAC).