​Extracellular vesicles play an essential role in the human body. These tiny lipidic capsules are secreted by cells, and can transport biological information (e.g. DNA, protein, RNA) between cells. They are involved in many physiological processes, such as immune responses or tissue regeneration, and can also participate in the proliferation of metastases and in cancer progression mechanisms.

Analysis of extracellular vesicles produced by [tumor] spheroids

The analysis of extracellular vesicles can therefore help detect biomarkers of the disease, particularly since many of these cells travel through biological fluids, such as the blood or mucus. This makes them easier and less invasive to study, compared to exams such as biopsies.

“We've known about extracellular vesicles for decades, but until recently, they had only been studied in the context of two-dimensional cell culture productions," noted Vincent Agache, project manager in microfluidics at CEA-Leti. “We wanted to go further, by examining spheroids, which are three-dimensional cell clusters capable of more faithfully replicating mechanisms of the human body. The approach was inspired by research conducted at CEA-Leti on organoids-on-a-chip.“

A microfluidics platform for extracting and isolating extracellular vesicles

CEA-Leti therefore developed a microfluidics device for collecting and isolating extracellular vesicles secreted by a single spheroid, a world first. The process was broken down into successive functions, all of which were integrated within the microfluidics platform.

The first step involves trapping the spheroid in a very confined space. Using various microchannels, it is then fed nutrients that encourage growth. The technique had already been developed within the framework of previous CEA-Leti research. However, the pumping system and beads used to trap the extracellular vesicles were designed specifically for this project.

“Once the spheroid has been fed, its entire environment, including the molecules it has produced, is collected," explains Vincent Agache. “A pumping system specifically designed for this collection step was integrated. All the while, the volume obtained, in the order of 500 nanoliters, was controlled to facilitate subsequent measurements."

Co-contaminants are then removed as the fluid flows through a 200 nm filtration membrane, and through more chambers filled with magnetic beads, onto which antibodies have been grafted. Once these have latched onto the extracellular vesicles, a magnet holds back the beads as the chambers are emptied. Having been separated from their antibody bindings, the extracellular vesicles are ready for collection and analysis.

Toward personalized cancer therapies

“These analyses have revealed the presence of signatures that are specific to extracellular vesicles, which confirms a successful collection," highlighted Vincent Agache. “We are now working toward adding new features to the microfluidic device. For example, a nanometric weighing system could be integrated to measure extracellular vesicle secretion by spheroids in real time, and to observe if secretion dynamics might change in different environments or with the state of the cells."

The microfluidics platform could also be used to study the efficacy of cancer treatments. In this case, a patient's trapped spheroids would be used and exposed to a range of candidate therapeutic molecules to determine which ones might offer optimal results for that person. This would constitute a significant step toward personalized therapies.