“Form follows function” is a familiar phrase. Yet in extracellular vesicle (EV) translational science, the relationship is reciprocal, not unidirectional. What EVs and/or exosomes contain, and how they are structured, directly determines what they do biologically. [1, 2, 3]
If EV-based therapeutics are to succeed clinically, developers must move beyond counting particles and begin defining function in measurable ways.
That was the central message of our recent webinar, EV Content vs. Function where the RoosterBio team explored how orthogonal analytical methods can connect extracellular vesicle composition to biological activity.
Why Content Matters
Extracellular vesicles are inherently complex. [4] They carry proteins, nucleic acids, lipids, and metabolites. Their surfaces display specific markers. [5] And critically, their composition changes depending on:
- Cell source
- Culture conditions
- Downstream processing
As Elie Zakhem, PhD framed during the webinar, the complexity of their interdependent nature and nurture presents a CMC challenge. To be considered for trials a drug material, the final EV doses must be defined through identity, potency, purity, and safety.
Particle counts and size distributions are not enough. Developers need to understand which molecular components contribute to biological activity and how manufacturing decisions affect them. This need is becoming increasingly urgent.
Figure 1, above. Number of newly posted trials worldwide that employ extracellular vesicle or “exosome” preparations as the clinical intervention. [6, 7] Diagnostics and observational trials not included.
As trending in clinical trial tallies (Figure 1), EV-based trials have risen sharply since 2018, with MSC-derived EVs representing a large majority of programs. The field is maturing. Analytics must mature with it, not merely catch up.
CD73 as a Case Study in Linking Content to Function
To make this concrete, Stephen Lenzini, PhD presented a focused case study on CD73.
CD73 is an enzyme commonly found on MSC-derived extracellular vesicles (Figure 2). It converts AMP into adenosine, and has been linked to anti-inflammatory potency in multiple preclinical, in vivo disease models. [8]
Figure 2, above. CD73 is a canonical surface marker for MSCs. It is also expressed on the extracellular vesicles released by MSCs. This enzyme converts AMP into adenosine, an anti-inflammatory signaling mediator.
The question was simple:
Can we measure CD73 content in a way that meaningfully predicts CD73 function?
To answer this, the team used three orthogonal methods:
- Capillary Western blot to quantify CD73 protein levels
- Single-vesicle nano flow cytometry to measure CD73 molecules per particle
- A CD73 activity assay to measure enzymatic function
Each method measures the same biological feature using different physical principles. When independent methods agree, confidence increases.
What the Data Revealed
The most important finding was not just that CD73 could be measured. It was that downstream processing steps can reshape EV populations in ways that directly affect functional markers.
As shown in Figure 3, filtration reduced larger vesicles while tangential flow filtration (TFF) increased particle concentration.
Figure 3, above. Representative experiment measuring extracellular vesicle particle count by nanoparticle tracking analysis (NTA) across serial unit operation steps to harvest, concentrate (tangential flow filtration, TFF), and filter (0.45mm and 0.2mm MSC-EVs). Other experiments addressed how these steps affected EV size and CD73 activity.
But when CD73 was analyzed on a per-particle basis (Figure 4), an early filtration step led to a measurable drop in CD73 molecules per particle. Larger vesicles with more exposed CD73 molecules appeared to be selectively reduced. After that shift, however, CD73 content and activity remained stable through the remainder of downstream processing.
Figure 4, above panels. CD73 molecules per particle (y) as separated in view by size (x), measured by single-vesicle characterization via NanoFlow Cytometry across serial unit operation steps to harvest, concentrate, and filter samples of EV from MSC-conditioned medium. Note decrease in particle size counts after the 0.45mm filtration step. Below panels. Note decrease in calculated CD73 molecules in per particle after the 0.45mm filtration step. These data were corroborated by readings from quantitative Western blotting (see Webinar for details).
When the team compared protein-level measurements, single-vesicle quantification, and enzymatic activity (Figure 5), the results correlated strongly. Content tracked with function. That correlation from orthogonal assay systems is what gives analytics power.
Figure 5. Instrument readouts of CD73 protein level per billion extracellular vesicle particles (left, quantitative Western blot), calculated CD73 molecules per particle (middle, single-vesicle characterization by NanoFlow cytometry), and calculated CD73 enzymatic activity per particle (right, activity assay) all report similar trending data. CD73 activity from functional molecules correlates with numbers of CD73 per single particle and amount in whole samples in each process step.
Moving Beyond Particle Counts
Many extracellular vesicle programs still rely heavily on particle number as a dosing metric. But particle count alone is an incomplete measure of biological activity. If functional markers like CD73 drive therapeutic effect, then dose definition may need to reflect molecular mechanism, not just vesicle abundance.
By combining:
- Per-particle molecular measurements
- Whole-sample protein quantification
- Direct functional assays
Developers can begin shifting the conversation from “How many EVs do we have?” to “What biological activity do these EVs deliver?” That shift matters for both scientific understanding and regulatory readiness.
What This Means for Extracellular Vesicle Developers
Across the webinar, one theme stood out: analytics are no longer a final checkpoint. They are a development driver.
Many questions that followed the presentation reflected real-world concerns:
- How do filtration steps affect potency?
- How should scalable processes compare to ultracentrifugation?
- How much material is needed for meaningful characterization?
The case study demonstrated benefits of early integration of scalable unit operations with strong, orthogonal analytical frameworks. Doing this will help developers avoid surprises later.
As EV therapeutics move closer to clinical translation, the ability to connect molecular content with biological function will increasingly determine which programs scale successfully. [9]
Key Takeaways
- EV composition changes during processing and those changes can influence functional markers.
- Orthogonal analytical methods provide stronger confidence than any single assay alone.
- CD73 content correlated with CD73 enzymatic activity, demonstrating a clear link between content and function in this model system.
- Analytical strategies can help define potency in ways that go beyond particle count and support translational development.
What Comes Next
This blog focused on the data and the analytical framework.
In a follow-up post, we will examine the audience questions from the webinar — including practical concerns about scalability, filtration strategies, sample requirements, and regulatory alignment. As the field evolves, the ability to connect form with function will play a central role in turning complex biology into consistent, scalable therapies.
If you’re working to define potency, optimize downstream processing, or strengthen your extracellular vesicle characterization strategy, we encourage you to watch the full webinar when it becomes available on demand. When you’re ready to Contact Us For Your Analytical Needs, one great way is directly through the quick requirements capture page.
References
- Lenzini, Stephen, Zakhem, Elie. Watch That Car-go! Acceleration Towards MSC-EV Characterization. RoosterBio Blog 2025; Available from: https://www.roosterbio.com/blog/watch-that-car-go-acceleration-towards-msc-ev-characterization/.
- RoosterBio. Analytics: A Referee for the Tug-of-War Between MSC-EVs’ Nature & Nurture. RoosterBio Blog 2024; Available from: https://www.roosterbio.com/blog/analytics-a-referee-for-the-tug-of-war-between-msc-evs-nature-nurture/.
- Zakhem, Elie. Heterogeneous but Advancing: The Complex Journey of EVs Toward Clinical Translation. RoosterBio blog 2025; Available from: https://www.roosterbio.com/blog/heterogeneous-but-advancing-the-complex-journey-of-evs-toward-clinical-translation/.
- Lenzini, Stephen. Big Effects in Small Packages: What Are Extracellular Vesicles, Exosomes, & Microvesicles? And Why Are They En Route to the Clinic? RoosterBio Blog 2021; Available from: https://www.roosterbio.com/blog/big-effects-in-small-packages-what-are-extracellular-vesicles-exosomes-microvesicles-why-are-they-en-route-to-the-clinic/.
- RoosterBio. The Spirit of MISEV. RoosterBio Blog 2025; Available from: https://www.roosterbio.com/blog/the-spirit-of-misev/.
- Cuoto, Pedro. Exosome Clinical Trials 2011-2025. 2025; Available from: https://celltrials.org/public-cells-data/exosome-clinical-trials-2011-2024.
- Available from: https://clinicaltrials.gov/search.
- Cramer, Madeline. CD73: A Team Player Caught in the “AKT” of Wound Healing & Cell Survival via MSC Exosomes? RoosterBio Blog 2024; Available from: https://www.roosterbio.com/blog/cd73-a-team-player-caught-in-the-akt-of-wound-healing-cell-survival-via-msc-exosomes/.
- RoosterBio. RoosterBio’s Equinox Reflections from ISEV & ISCT 2025. RoosterBio Blog 2025; Available from: https://www.roosterbio.com/blog/roosterbios-equinox-reflections-from-isev-isct-2025/.