Extracellular Vesicles: EV Production, Characterization, And Quantification Methods | Corning

Extracellular vesicles act as a cell’s transfer signals – these nanoparticles, which contain protein, sugars, and other biological materials, communicate with adjacent cells and tissues to transmit messages, such as whether a cell is healthy or in distress. While this functionality represents significant opportunity for the biologics space, researchers are still building a foundation of knowledge surrounding their biology, application, and ultimate value.

Unlike whole cell therapies, extracellular vesicles are acellular products, the equivalent of text messages sent back and forth in order to elicit reactions. Because extracellular vesicles are much smaller than whole cells, they are able to cross biologic barriers with ease, in contrast to the relative mass of whole cells or the immunogenic responses triggered by certain synthetic products. Their small size confers other benefits, as well – too small to convey pathogens such as bacteria or viruses, extracellular vesicles are also incapable of the unwanted replication or tumor formation that whole cell therapies can occasionally provoke.

Extracellular vesicles also represent the potential for less intensive development and manufacturing. Unlike synthetic molecular therapeutics such as messenger RNA, which often require tweaks like sheathing or additional amino acids to prevent degradation, EVs are self-assembled and already possess the natural protections necessary to perform their designated purpose. Their promise as a potential new therapeutic strategy is clear; less clear is the fundamental understanding needed to produce, quantify, and characterize these materials. Yet the existing science surrounding extracellular vesicles implies a dynamic modality possessing a comparable or even superior therapeutic capacity than their parent cells. As biotherapeutic companies and contract development and manufacturing organizations (CDMOs) continue to investigate the potential for extracellular vesicles as therapeutics, they build on exciting insights around these acellular products, including their promise in delivering an abundance of regenerative medicine applications, oncological therapies, and other intractable disease treatments.

An Overview of Extracellular Vesicles (EV) Production

In synthetic biology, the addition of any inorganic element to a biologic has the potential to trigger an immune response. This reality creates a layer of challenge for drug developers as they work to identify and optimize synthetic lipids, polymers, or proteins lacking this immunogenicity. This represents part of the promise for EVs – as natural acellular products, everything inherent to their makeup is recognized by the body, rendering them essentially non-immunogenic. Additionally, for many other engineered or synthetic advanced therapies, the amount of therapeutic agent required to elicit an efficacious response is high, necessitating their conveyance in a larger nanoparticle or a microparticle much larger than an EV.

The Promise of Extracellular Vesicles

The potential EVs possess for the field of acellular therapies – treatments derived from acellular products that achieve the same therapeutic response without delivering entire cells – is an exciting one. As EVs continue to demonstrate this promise in the clinic, researchers are likely to pursue their utility along two distinct pathways. The first is as a therapeutic, addressing a wide range of disease states, from cancer to degenerative genetic conditions to infectious diseases such as COVID-19. The second, and potentially nearer avenue, involves EVs as a diagnostic tool: capturing a patient’s EVs through liquid biopsy or another technique in order to reveal their messaging, indicating problems such as cancer or other disease states. This application of EVs – potentially tracking the progression of a cancer or whether it has metastasized to other parts of the body – coupled with their treatment potential make them a crucial biomarker for exploration in clinical oncology in particular.

Naïve Extracellular Vesicles

Just as there are two major pathways for investigating their therapeutic potential, there are two ways that have been identified for therapeutic utilization of EVs. The one that has arguably received the most attention from industry are what have been referred to as “naïve” EVs, or unmodified EVs produced from stem cells. These exist in contrast to engineered EVs, which may undergo gene editing or require other engineered components to modify the resulting EVs for a specific purpose. Naïve EVs are produced by culturing the stem cells which secrete them into the surrounding liquid media environment; thus, the primary goal supporting their production is establishing an environment capable of yielding healthy stem cell growth.

Some scientists are utilizing space saving solutions such as Corning HYPER technology that supports a process capable of high-density cell culture in a small footprint leading to high yields of EVs. Others are investigating the potential of microcarriers to reap the benefits of an adherent cell culture with characteristics seen in suspension culture, while others still are working toward optimizing stem cells to grow in suspension. Corning HYPER technologies hold significant promise for helping EVs reach the clinic. For instance, Corning® HYPERStack® 12-layer cell culture vessels have demonstrated a capacity to produce up to seven trillion high-quality EVs from mesenchymal stem cells (MSCs), with the addition of mechanical movement of the Corning HYPERStack vessel resulting in a 4-fold increase in EV production compared to a static vessel.

Investigating Characterization and Quantification to Galvanize Innovation in Extracellular Vesicles

While many companies are working to optimize their EV production, many have commensurately lost line of sight to the biological or fundamental characterization necessary to achieving true optimization. For example, 2D cell culturing techniques hinder continuous batch processing opportunities as they are limited by cell confluency and time. EV production can be impacted once cells make physical contact with one another as they no longer require vesicle messaging for communication, thus, inconsistencies in production levels per cell can be fostered. EV production in 3D cell culture can help circumvent this issue and enable continuous batch processing. At present, there is limited technology available that can support bulk 3D cell culture; however, Corning has recently launched the Corning® Elplasia® 12K Flask that can support the culture of 12,000 spheroids in a footprint similar to a T-75 flask. This vessel is fit for EV production from 3D cultures as it designed with a single media reservoir to supply nutrient exchange for all 12,000 spheroids simultaneously resulting in equivalent environments. Though they share a common media environment, the spheroids are sequestered individually into each microcavity, preventing physical contact. This allows each spheroid to act as an independent population, fostering ample EV production with batch-mode capabilities given the appropriate cell line.

In addition to optimizing production, precise quantification and characterization of EVs remain critical for advancing both fundamental research and clinical applications. Traditional methods such as nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) have provided valuable insights into EV size distribution and concentration, yet these approaches often require extensive sample preparation and specialized expertise. The Corning Videodrop offers a streamlined, label-free approach for rapid nanoparticle size and concentration measurement. By utilizing interferometric light microscopy, Videodrop enables real-time, high-resolution analysis of EVs with minimal sample manipulation. This technology facilitates more efficient EV characterization, ensuring greater reproducibility and reliability in both research and therapeutic applications.

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Researchers are still pursuing the fundamentals of EVs, including why EVs are produced, how many are required to signal effectively in a given environment, and what signals are required to produce more EVs. While there are no vessels that have been innovated specifically for EV production, there are many entities pioneering reagents to promote EV production, as well as foundational research related to the impact of tangential considerations such as alterations in cell metabolism such as starvation and hypoxia. More and more clinical trials are documenting success of applied EVs for treatment in pertinent diseases such as acute respiratory distress syndrome from long-term COVID-19, cardiac tissue regeneration, and oncological applications. With the continued documented capacity of EVs outperforming conventional treatments, such synthetic therapeutic agents or whole stem cells, in clinical trials, EVs are positioning themselves as a modality to watch over the next several years.

In many of the therapeutic effects observed in EVs, the mechanism of action is not yet fully understood. Right now, the lack of standardization surrounding EV characterization and production is pervasive – consortiums are still working to define the protocols necessary to adequately study and develop these acellular products. The minimum requirements for production characteristics, isolation techniques, and final output need to be fully realized and deeply validated in order to progress the space and create an avenue for regulatory acceptance. There have been a number of advancements around adapting existing characterization techniques for EVs from other biomolecular workflows such as monoclonal antibody, viral particle, or protein production. Much of the current focus of this work has been on minimizing the number of steps or assays needed to determine fundamentals such as size, shape, concentration, surface marker expression, cargo content, and functionality. This pursuit, in conjunction with efforts to standardize characterization, quantification, and production for these acellular products, will serve to bolster additional innovation in the space in the coming years.

Extracellular Vesicles: Looking Ahead

As more clinical trials pursue EVs as a therapeutic modality, with the medical repercussions of the COVID-19 global pandemic driving many applications, their regenerative potential when compared to whole stem cell treatments is likely to continue to drive innovation. The rising number of publications and clinical trials involving EVs indicate a demand for standardized protocols and technologies to enable efficient scale-up, without compromised quality, for acellular therapies. By harnessing the power of EVs to inspire cellular response, the industry is poised to pioneer acellular therapies capable of achieving comparable or improved regenerative response when compared to other regenerative medicines.