The Role of Flow Cytometry in Cutting-Edge Extracellular Vesicle Research

Researchers are always seeking innovative tools to unlock the mysteries of cell-to-cell communication, biomarker discovery, and clinical diagnostics. Extracellular vesicles have emerged as intriguing players in these arenas, offering insights into intercellular signaling and potential clinical applications. In this blog post, we will talk about the pivotal role of extracellular vesicles in biology, the clinical implications, and how advanced flow cytometry is enhancing our understanding of these tiny but potent entities.

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Extracellular vesicles: nature's tiny messengers

Extracellular vesicles are small, membrane-bound vesicles released by cells into the extracellular space. These nano-sized particles carry a cargo of proteins, lipids, nucleic acids, and metabolites, serving as critical mediators of intercellular communication in physiological processes, including immune regulation, tissue repair, and cellular waste disposal.

These vesicles are also involved in the pathophysiology of diseases such as cancer, neurodegenerative disorders, and infectious diseases. Understanding their composition, biogenesis, and functions has become crucial for helping us understand these diseases and develop therapeutic interventions.

Clinical implications of extracellular vesicles

Extracellular vesicles hold great promise as biomarkers for early disease detection, disease monitoring, and as vehicles for targeted drug delivery.

For instance, specific markers on a type of extracellular vesicle called exosome can provide valuable insights into tumor progression and metastasis. By analyzing the protein and nucleic acid cargo of exosomes, researchers can identify signature molecules associated with specific cancer types, paving the way for more precise and non-invasive diagnostic methods.

Extracellular vesicles also have notable advantages as drug carriers owing to their innate capacity for molecular transport. They can travel in the body without attracting undue attention from the immune system, while also maintaining qualities like low toxicity, high stability, efficient delivery, biocompatibility, and the ability to cross the blood-brain barrier. Exosome-based carriers have produced promising results in this area, with demonstrated safety data in animal models of Parkinson’s disease.

Role of flow cytometry in extracellular vesicle research

Flow cytometry is a powerful tool for the analysis of exosomes and other extracellular vesicles. This technique enables researchers to profile and quantify extracellular vesicles to better understand their size, surface markers, cargo, and biological impacts on other cells.

Still, detecting and characterizing extracellular vesicles by flow cytometry presents some challenges. Flow cytometers are designed for cells, which are much larger than extracellular vesicles. Exosomes for example, are 30–150 nm in diameter; that is roughly 1,000 to 10,000 times smaller than a typical eukaryotic cell. Extracellular vesicle preparations are also highly heterogeneous and prone to aggregation, further complicating data analysis. 

For these and other reasons, researchers need to optimize their cytometry protocols based on the properties of the particles to ensure the best results.

Application highlights using flow cytometry

While there are flow cytometry protocols for direct analysis of extracellular vesicles, the technology is most ideally suited for functional studies of extracellular vesicles. Here are a few examples.
 

• Surface marker expression: Flow cytometry can assess changes in surface marker expression on recipient cells after exposure to extracellular vesicles. By labeling recipient cells with fluorescent antibodies against specific markers, researchers can monitor alterations in cell surface proteins, which may indicate cellular activation or signaling events triggered by exosome interactions.
• Functional assays: When extracellular vesicles release their miRNA, protein, or other cargo, there is a functional response on the recipient cells. Flow cytometry can measure changes in cell proliferation, apoptosis, differentiation, or other functional responses.
• Intracellular signaling: By using flow cytometry in combination with intracellular staining techniques, researchers can see which downstream intracellular signaling pathways are activated by extracellular vesicles.
• Cargo delivery: Labeling of exosome cargo components, such as fluorescently labeled miRNA mimics, allows for monitoring their uptake by recipient cells. This is also useful for evaluating extracellular vesicles engineered as drug delivery vehicles for targeting and efficacy.

Advanced flow cytometry streamlines workflows

The iQue^® Advanced Flow Cytometry Platform, re-invents the standard flow cytometry workflow to make it easier, faster, and less expensive across applications, including extracellular vesicle analysis. 

Thanks to its ability to detect both cells and beads in the same well, the iQue^® Platform can deliver a large amount of data, with exceptional speed. Users also have the benefit of analyzing complex screening datasets with one, integrated Forecyt^® software, instead of multiple disjointed programs.

In addition to the iQue^® Platform, Sartorius has entered key partnerships with Rooster Bio, Evotect, and Curexsys to support robust, scalable, manufacturing of clinical-grade human mesenchymal stem/stromal cell (hMSC)-based exosomes for therapeutic applications.

Follow the cargo with advanced flow

Several research groups have used the iQue^® Platform in recent published studies featuring extracellular vesicles. In a 2019 publication, Das et al. used the platform to evaluate a versatile way of engineering exosomes for drug delivery without the need for complex genetic modifications of exosome-secreting cells. 

A different study used advanced flow cytometry to analyze the cellular uptake of extracellular vesicles. Here, Dekker et al. (2021) tested different tracers to track the biodistribution of extracellular vesicles, which is crucial for developing extracellular vesicle-based therapeutics.

Learn more about the iQue^® Platform, and its streamlined protocols and validated mix-and-read assay kits.