• This blog recaps Elie Zakhem, PhD’s key insights from ISEV2025, the premier global conference on extracellular vesicles (EVs) recently attended and presented at by RoosterBio.
• EVs are powerful but complex — their natural diversity makes them tricky to study and standardize.
• New tools like nanoflow cytometry help reveal hidden differences between EV types.
• MSC-derived EVs are leading candidates for future therapies but have faced manufacturing challenges.
• Engage early with regulators, as those conversations can smooth the path forward.

ISEV 2025 kicked off in April hosted in the charming city of Vienna, Austria. [1] Over five packed days, the conference brought together researchers, clinicians, industry leaders, and vendors from around the world to dive deep into all things extracellular vesicles (EVs).

From education day to plenary sessions, satellite meetings, poster presentations, and a busy exhibitor floor, the event was well-organized, easy to navigate, and most importantly, offered plenty of opportunities to learn, share, and network.

The Many Faces of EVs: Insights into EV Biogenesis & Tackling Heterogeneity Head-On

Day One started with Education Day, and despite the name, it wasn’t just for newcomers to the extracellular vesicle field. It was a solid warm-up for the entire week, filled with engaging talks and lively discussions. One of the early themes was EV heterogeneity; a hot topic and a critical challenge that continues to complicate research, standardization, and ultimately, regulation.

We were reminded that while extracellular vesicles are often described as lipid bilayer-bound nanoparticles (typically 50–1000 nm in size) expressing markers like CD63, CD81, and CD9, the reality is far more complex. Heterogeneity can stem from a variety of sources: the type of the secreting cell, the EV biogenesis pathway, the EV’s functional role, or even the composition of the EV “corona.” Add in donor variability and state of the secreting cell, and the challenge deepens.

The distinctions between different types of extracellular vesicles, like exosomes (endosomal origin) and ectosomes (plasma membrane origin), might sound clear on paper, but in practice, it’s messy. There’s no single, definitive marker that separates one from the other. For instance, exosomes are often thought to have more CD63, while small ectosomes might be enriched in CD9. Apoptotic bodies are typically associated with phosphatidylserine (PS) exposure, but even that isn’t a strictly exclusive feature. All of this can vary widely depending on the cell type that is secreting the EVs, so it is important to note that conclusions drawn from studies conducted on EVs secreted by one cell type may not be applicable to EVs secreted from other cell types. Clearly, there’s still a lot we don’t fully understand. Heterogeneity of EV populations complicates their study, as traditional bulk analysis methods fail to capture individual differences in size, content, and surface markers.

On the other hand, single-vesicle analysis promises a more precise approach, enabling detailed characterization of extracellular vesicle subpopulations. At ISEV, RoosterBio showcased recent groundbreaking work [2] utilizing nanoflow cytometry for single vesicle analysis, enabling precise quantification of CD63, CD81, and CD73 on distinct extracellular vesicle populations. This innovative approach revealed tissue- and marker-specific differences in EV characteristics, offering high-resolution insights into EV heterogeneity. Such detailed profiling has the potential to deepen our understanding of EV diversity, enhance interpretation of EV bioactivity, and inform strategies for enriching specific EV subtypes.

The themes of extracellular vesicle biogenesis and heterogeneity were also prominently featured in plenary and introduction sessions throughout the conference.

In the plenary session, “The ESCRT Machinery in Biogenesis of Intraluminal and Extracellular Vesicles,” Harald Stenmark, PhD, detailed the central role of the Endosomal Sorting Complexes Required for Transport (ESCRT) in membrane remodeling and vesicle formation. [3] The ESCRT machinery is essential for facilitating reverse-topology membrane scission, a key process in the formation of intraluminal vesicles (ILVs) within multivesicular endosomes (MVEs), and in the budding of ectosomes directly from the plasma membrane. While alternative, ESCRT-independent mechanisms—such as those mediated by tetraspanins—can support ILV formation, ESCRT remains the primary and most efficient pathway.

A critical component of this machinery is the adaptor protein ALIX, which contributes significantly to exosome biogenesis. ALIX interacts with syntenin-1 to regulate the fate of exosomes, determining whether they are secreted into the extracellular space or directed toward lysosomal degradation. Interestingly, while ESCRT machinery enables vesicle formation, its components do not enter the vesicles themselves. This is reflected in TSG101 being more frequently detected in extracellular vesicles compared to ALIX, possibly because ALIX remains associated with ESCRT components outside the vesicle.

Professor Stenmark’s insights underscore the sophistication of ESCRT-mediated vesicle biogenesis and its crucial interplay with accessory proteins like ALIX and syntenin-1, helping to delineate the molecular decisions that govern whether ILVs become exosomes or are degraded. This understanding advances both basic science and the development of EV-based therapeutics.

In his presentation “The Winding Road of CD63,” Dr. Guillaume van Niel explored the critical role of the tetraspanin protein CD63 in intracellular cholesterol trafficking, particularly within the endosomal system. CD63 is evolutionarily highly conserved, underscoring its fundamental biological importance. His findings demonstrated that CD63 is essential for sorting cholesterol into ILVs. [4] In the absence of CD63, cholesterol is misrouted out of MVBs and redirected to the Golgi apparatus, leading to cholesterol accumulation in the Golgi, a characteristic of certain lipid storage disorders. Quantitative assays and transmission electron microscopy (TEM) confirmed that CD63 knockout (KO) reduces cholesterol content in ILVs. CD63 deletion did not alter EV number, size, or canonical EV markers, and no consistent changes were observed in the proteome of cells or derived EVs. However, lipidomic analysis revealed subtle differences in lipid composition between wild-type and CD63-KO conditions. These findings position CD63 as a key regulator of cholesterol partitioning within endosomal compartments, with implications for understanding lipid homeostasis and associated diseases.

MSC-EVs: Power & Pitfalls of a Leading Therapeutic Candidate

Beyond extracellular vesicle biogenesis and heterogeneity, it was both exciting and encouraging to see a strong focus on MSC-derived EVs (MSC-EVs) across numerous talks and sessions. A key theme that emerged was the inherent heterogeneity of MSCs, which is inevitably reflected in their EVs. MSC-EV heterogeneity was also featured in a session chaired by Dr. Bernd Giebel, where this topic was addressed in depth. [5] Discussions highlighted how EVs derived from 2D versus 3D MSC cultures differ significantly, with 3D-derived EVs showing greater functional potency.

Professor Giebel also addressed several challenges associated with extracellular vesicles from primary MSCs, such as reduced efficacy in later cell passages, donor-to-donor variability, and inconsistencies in cell density during EV production. [6] In response to these limitations, he introduced the potential of using immortalized MSCs (iMSCs) as an alternative solution for MSC-EV manufacturing. Whether primary MSCs or iMSCs, all cell types present themselves with challenges of optimal culture conditions (optimal media, scalable manufacturing process and reproducibility). While opinions on the optimal cell source varied in the room, there was a broad consensus on the need for standardized and consistent manufacturing processes. The concept of iMSCs is certainly appealing; however, it does not fully resolve the heterogeneity issues related to selecting the optimal donor for generating the immortalized cell line, nor the variability that may persist across different passages. An alternative approach is the use of induced pluripotent stem cells (iPSC)-derived MSCs for EV production, which could potentially address these challenges. While opinions on the ideal cell source differed, with the recognition that each option presents its own set of challenges, there was broad agreement on the importance of standardized and consistent manufacturing processes.

RoosterBio’s extracellular vesicle production platform was highlighted as an example of such standardization. RoosterBio’s scalable 3D bioprocess involves an initial phase of MSC expansion using optimized media, followed by a complete media change to a low-particulate, defined formulation for EV collection over five days in bioreactors. This approach aims to maximize both yield and consistency.

At RoosterBio, we recognize the critical challenge posed by the heterogeneity of MSC-derived extracellular vesicles (MSC-EVs). To better understand this variability, we conducted a comprehensive study examining how EV characteristics can differ based on MSC tissue sources, donor variability, and commonly used production platforms, including 2D cultures, stirred-tank bioreactors, vertical-wheel bioreactors, and spinner flasks. [7, 8] The findings from this work were presented at ISEV2025. While EVs from different tissue sources and donors shared some characteristics, functional assays were better suited to reveal biological distinctions. A compelling example is the CD73 activity assay, which demonstrated that CD73 enzymatic activity varied significantly depending on the MSC tissue origin, donor, and even the day of EV harvest. [9]

EVs Meet Regulation: Clearing the Path to Clinical Translation

One topic that stood out this year was the regulatory landscape for EV-based therapeutics. Regulatory considerations came up in multiple sessions and were widely discussed among attendees, clear signs that the field is maturing and edging closer to clinical translation. MSC-EVs are currently seen as the front runners for market approval, particularly in their unmodified form, and they remain among the most promising therapeutic candidates, despite some challenges around heterogeneity. Engineering or loading EVs adds complexity in terms of characterization and regulation.

In an especially impactful session, Dr. Mario Gimona discussed GMP-grade MSC-EVs, where he provided critical insights into the regulatory and scientific considerations surrounding their clinical development. [10] Good Manufacturing Practices (GMP) are essential to ensuring that therapeutic products are consistently produced and controlled according to established quality standards. This framework applies equally to EVs intended for therapeutic use. For developers of EV-based products, it is critical to clearly define the product, establish a reproducible manufacturing process, identify and monitor critical quality attributes (CQAs), develop and validate appropriate analytical assays, and ensure consistent product testing. At the core of all these steps is a single unifying principle: consistency.

Maintaining open, ongoing communication with regulatory authorities is strongly recommended. Developers should proactively share details of their manufacturing processes and the scientific rationale behind their analytical methods. These components form a key part of the Chemistry, Manufacturing, and Controls (CMC) section of regulatory submissions. Although the regulatory framework for EVs is still evolving, developers can draw on lessons from cell and gene therapy to help guide the regulatory path for EV-based therapeutics.

Analytical characterization remains a cornerstone of regulatory evaluation and includes assessments of identity, purity, potency, and safety. Traditionally, EV identity has been defined by the presence of specific markers such as CD63 and TSG101. In the case of MSC-derived EVs, it’s also important to demonstrate MSC identity, typically by showing the presence of MSC-specific surface markers, thereby confirming the EV source.

Purity, however, continues to pose a significant challenge in the field. The field agreed that there is currently no consensus on what constitutes a “pure” extracellular vesicle preparation, whether it involves removing all associated surface proteins or accepting the EVs along with their naturally occurring protein corona. The most practical and regulator-acceptable approach is to establish a robust, well-controlled manufacturing process, paired with reproducible product characterization.

A critical and recurring theme in regulatory discussions is the demonstration of a mechanism of action (MoA). The CD73 activity assay, for example, was frequently highlighted during the conference as an important tool for characterizing MSC-EVs. CD73 on MSC-EV surfaces catalyzes the conversion of AMP to adenosine, a key molecule in promoting anti-inflammatory responses. No extracellular vesicle product characterization is considered complete without defining how the EVs exert their therapeutic effect.

Dr. Gimona emphasized the utility of the CD73 activity assay as a solid first-line quality control. Measuring adenosine levels and correlating them with anti-inflammation response may be beneficial during discussions with regulatory agencies. However, such assays must be validated. Developers were encouraged to engage regulators even in early development stages, especially when discussing potential MoAs and potency metrics.

Current analytical tools and techniques add another layer of complexity due to their limitations in sensitivity, specificity, and reproducibility. For instance, there are multiple methods available to quantify extracellular vesicles, yet each often yields different results. Some techniques fail to detect smaller EVs, while others struggle to distinguish EVs from protein aggregates or other particles. As a result, it’s generally recommended to report a range of values rather than relying on a single measurement. Protein quantification presents similar challenges, especially since it’s often a prerequisite for downstream analyses such as Western blotting or proteomics. The use of various protein assays only adds to the inconsistency, and it remains difficult to determine whether the measured protein is truly EV-associated or simply a contaminant.

Despite these obstacles, employing orthogonal techniques to assess the same parameter is essential for achieving a comprehensive and accurate characterization of EV samples.

At RoosterBio, we are committed to accelerating our partners’ progress by anticipating challenges and delivering solutions ahead of the curve. Our assay services include a comprehensive portfolio of EV characterization tools designed to approach the stringent requirements of CMC filings. All our assays have been developed and qualified, and ready to become compliant with GMP, supporting the successful translation of EV therapies from bench to clinic.

Future Thoughts: The Road Ahead for EV Science & Therapeutics

ISEV 2025 underscored just how rapidly the EV field is evolving, from technical refinements in characterization to serious discussions about regulatory frameworks and clinical translation. Despite heterogeneity challenges, the focus on standardization, reproducibility, and regulatory alignment reflects a field that’s not only expanding in scope but also maturing in its ambitions.

Single-vesicle analysis and tools like the CD73 activity assay are helping us peel back the layers of extracellular vesicle complexity, revealing the need for more precise characterization methods and standardized manufacturing processes, particularly for MSC-EVs. As our understanding deepens, so does the need for functional, mechanism-driven assays that can meet regulatory expectations.

Looking ahead, the path to EV-based therapeutics will hinge on consistency, collaboration, and early regulatory engagement. With MSC-EVs emerging as front-runners for clinical translation, the field is shifting from exploration to application. But success will depend on robust GMP-compliant processes, validated assays, and open dialogue with regulators. The road may be complex, but the direction is clear: we’re moving steadily toward making EV therapies a clinical reality. At RoosterBio, we’re proud to support this journey by equipping developers with the tools, processes, and foresight needed to navigate the next stage of EV innovation.

References

1. RoosterBio. Right Place, Right Time: RoosterBio’s Spring 2025 Conference Gigs Across 3 Continents. RoosterBio Blog 2025; Available from: https://www.roosterbio.com/blog/right-place-right-time-roosterbios-spring-2025-conference-gigs-across-three-continents/.
2. Cramer, M., et al., Single Extracellular Vesicle Profiling: Harnessing NanoFlow Cytometry for Quantitative Surface Marker Analysis. Cytotherapy, 2025. 27(5): p. S28. 10.1016/j.jcyt.2025.03.042
3. Vietri, M., M. Radulovic, and H. Stenmark, The many functions of ESCRTs. Nat Rev Mol Cell Biol, 2020. 21(1): p. 25-42. 10.1038/s41580-019-0177-4
4. Palmulli, R., et al., CD63 sorts cholesterol into endosomes for storage and distribution via exosomes. Nat Cell Biol, 2024. 26(7): p. 1093-1109. 10.1038/s41556-024-01432-9
5. Tertel, T., et al., EV products obtained from iPSC-derived MSCs show batch-to-batch variations in their ability to modulate allogeneic immune responses in vitro. Front Cell Dev Biol, 2023. 11: p. 1282860. 10.3389/fcell.2023.1282860
6. Giebel, B. and S. K. Lim, Overcoming challenges in MSC-sEV therapeutics: insights and advances after a decade of research. Cytotherapy, 2025. 10.1016/j.jcyt.2025.03.505
7. RoosterBio. When Data Blooms: Insights into MSC-EV Bioprocessing. RoosterBio Blog 2025; Available from: https://www.roosterbio.com/blog/when-data-blooms-insights-into-msc-ev-bioprocessing/.
8. Zakhem, E., Lenzini, S. Exploring MSC-EV Attributes: Analytical Comparison of Tissue Sources & Production Platforms. 2025; Available from: https://share.hsforms.com/1tRJSy9V_Tw-LiJHvli1PLw3564o.
9. Cramer, M., E. Zakhem, and J. A. Rowley, Elevating MSC-EV Analysis: Development and Qualification of a CD73 Bioactivity Assay. Cytotherapy, 2025. 27(5, Supplement): p. S82. https://doi.org/10.1016/j.jcyt.2025.03.153
10. Shekari, F., et al., Cell culture-derived extracellular vesicles: Considerations for reporting cell culturing parameters. J Extracell Biol, 2023. 2(10): p. e115. 10.1002/jex2.115