Abstract Title	Authors / Presenters	Conference (Abstract #)
Single Extracellular Vesicle Profiling: Harnessing NanoFlow Cytometry for Quantitative Surface Marker Analysis*	Madeline Cramer, Stephen Lenzini, Elie Zakhem, Jon A. Rowley	ISEV (#1189), ISCT (#37)
A Fed-Batch Chemically Defined hMSC-EV Bioprocess Medium Enabling 2-4x EV Yield Improvements in Bioreactor Culture	Terri Willstaedt, Amy Walde, Jon A. Rowley	ISEV (#472), ISCT (#329)
Scalable GMP-compatible process solution for MSC-EV purification with 10X yield improvements	Stephen Lenzini, Jae Jung, Madeline Cramer, Elie Zakhem, Jon A. Rowley	ISEV (#1227)
Analytical comparison of MSC-EV quality attributes across tissue sources and production platforms	Stephen Lenzini, Madeline Cramer, Elie Zakhem, Jon A. Rowley	ISEV (#305), ISCT (#312)
Elevating MSC-EV Analysis: Development and Qualification of a CD73 Bioactivity Assay	Madeline Cramer, Elie Zakhem, Jon A. Rowley	ISCT (#304)
*Top Scoring Exosomes Abstract Award at ISCT-2025. Podium presentation on Friday, May 9th, 8AM (CDT).

Waking Up the MSC-EV World: RoosterBio’s 3-Continent Spring Tour

Behold Spring! We’ve all made it to 2025, even with some positive news. With “Advanced Therapy Bio-Conference Season” kicking into high gear and greeted by earlier sunrises, RoosterBio is soon visiting new venues to present its latest abstracts. In Vienna, Austria, RoosterBio will show its recent work at ISEV-2025 (International Society for Extracellular Vesicles) on April 24-27. Here, instead of awakening to Maryland roosters “cock-a-doodle-doo!” perhaps we’d hear something more like “Kikeriki! Mei, scho wieda?” in the local Wienerisch. At ISCT-2025 (International Society for Cell and Gene Therapy) on May 7-10 in New Orleans, the RoosterBio team at Booth #531 might be roused to the sound of “Kikeriki! Laissez les bons temps crowler!”

Two of our team (Jon Rowley, PhD, Founder and Chief Product Officer; Nicholas Trotter-Mayo, PhD, Sales Director ) will also be found at ISSCR-2025 (International Society for Stem Cell Research) in Hong Kong on June 11-15, presenting at Booth #804, and might hear “喔喔喔 — 你喺啱時區未呀？”

Process Innovations Drive Translation Forward: A Preview of RoosterBio’s Conference Abstracts

But seriously, folks, we eagerly look forward to these events because we have notable progress to share of our own, and love to reconnect with old friends and to meet new faces. This is what you can expect when you interface with our five posters and presentations:

Firstly, RoosterBio is proud to announce “Single Extracellular Vesicle Profiling: Harnessing NanoFlow Cytometry for Quantitative Surface Marker Analysis” (@ISEV and ISCT) as an oral/podium presentation at ISCT, featuring speaker Madeline Cramer, PhD, Scientist in Analytical Development. This abstract was honored by ISCT to receive the Top Scoring Exosomes Abstract Award. Here, we learn how RoosterBio developed a calibrated NanoFlow cytometry method to quantify surface marker expression on single extracellular vesicles (EVs), enabling high-resolution profiling of MSC-EVs from bone marrow, umbilical cord, and adipose tissue. Optimized staining protocols and ERF-calibrated fluorescence quantification revealed that 50–70% of EVs expressed CD81, 40–60% expressed CD63, and 15–35% expressed CD73. Adipose-derived EVs showed the highest CD81 and CD63 expression per particle, while CD73 was enriched on larger EVs across all sources. This single-vesicle analysis advances EV characterization by revealing tissue- and marker-specific heterogeneity, supporting the development of targeted EV-based therapies and improved EV product consistency.

“A Fed-Batch Chemically Defined hMSC-EV Bioprocess Medium Enabling 2-4x EV Yield Improvements in Bioreactor Culture” (@ISEV and ISCT) demonstrates how RoosterBio developed a chemically defined, fed-batch bioreactor medium (RoosterHD™-EV) to streamline and scale the production of hMSC-derived extracellular vesicles (EVs). This single-medium approach eliminates the need for separate expansion and collection phases, simplifying the process and reducing raw material use. Compared to traditional methods, HD-EV increased EV yields 2–4x while preserving key quality attributes across multiple donors and tissue sources. Scale-up to a 3L bioreactor confirmed the system’s robustness. The approach offers a cost-effective, GMP-compatible solution for large-scale production of high-purity hMSC-EVs, supporting broader clinical and translational applications.

“Scalable GMP-compatible process solution for MSC-EV purification with 10X yield improvements” (@ISEV) presents data to show that—when applied to conditioned medium (CM) post-bioreactor harvest, AgentV™-DSP reduces filter fouling and enhances recovery during clarification, tangential flow filtration, and chromatography. In a 3L bioreactor system, AgentV treatment improved cumulative EV yield from ~5% to ~50% without affecting particle size, identity (CD63/CD9/CD81), or functional potency (CD73 activity). This streamlined downstream processing (DSP) approach significantly boosts MSC-EV production efficiency while preserving product quality, positioning AgentV-DSP as a breakthrough for clinical and commercial EV manufacturing.

In “Analytical comparison of MSC-EV quality attributes across tissue sources and production platforms” (@ISEV and ISCT), RoosterBio conducted a comparative analysis of MSC-derived extracellular vesicles (MSC-EVs) from bone marrow, umbilical cord, and adipose tissue across multiple scalable production platforms. Using a standardized media system, we found that 3D bioreactor platforms produced 4–5 times more EVs than 2D culture. Bone marrow and umbilical cord MSCs yielded higher particle counts than adipose-derived MSCs. EVs exhibited consistent identity markers (CD63, CD81, CD9, CD73), though particle size, RNA content, and CD73 activity varied by tissue source and donor. Despite donor-dependent variability, quality attributes were consistent across platforms, supporting robust process development for clinical-scale MSC-EV manufacturing.

With “Elevating MSC-EV Analysis: Development and Qualification of a CD73 Bioactivity Assay” (@ISCT), RoosterBio developed and qualified a CD73 bioactivity assay to assess the potency of MSC-derived extracellular vesicles (MSC-EVs) by measuring AMP-to-adenosine conversion. The team evaluated EVs from bone marrow, umbilical cord, and adipose sources. The assay met ICH Q2 guidelines, demonstrating linearity (3–18 µM AMP), specificity (over 80% inhibition with CD73 blocker), accuracy (75–125% spike recovery), and precision (CV <20% across replicates). This validated assay provides a robust, standardized method for assessing EV bioactivity and supports GMP-compliant testing, helping de-risk therapeutic EV development through more reliable potency measurements.

Please come visit Dr. Cramer’s talk as well as RoosterBio’s five posters at ISEV and ISCT. Collectively, they advance scalable, GMP-compatible manufacturing and analytical frameworks for MSC-derived extracellular vesicles (MSC-EVs). These include a 10-fold yield improvement using AgentV-DSP, cross-platform comparability of EV quality across tissue sources, single-vesicle surface marker quantification via NanoFlow cytometry, and a qualified CD73 bioactivity assay aligned with regulatory guidance. Together, these innovations support ISEV’s emphasis on rigorous EV characterization and ISCT’s focus on translational readiness, while directly addressing customer needs for standardized, high-yield, and potency-validated EV manufacturing solutions.

Not “Too Many Notes” at ISEV, Just the Sound of Progress

When visiting ISEV-2025, this scene in the movie Amadeus might fancifully intrude imagination while enjoying the “notes” of an evening glass of Gemischter Satz. The Austrian Emperor Joseph II allegedly remarked to an earnest, young Mozart that his Abduction from the Seraglio burdened his royal ears with “too many notes” and to “cut a few, and it will be perfect.” The young composer then cheekily replies, “Which few did you have in mind, Majesty?” As avid followers of extracellular vesicles (EVs; also, sometimes called exosomes) will testify, the EV field has exploded in the 15 years into an embarrassment of riches: new publications and new dedicated primary investigators, spanning from basic science to clinical translation. [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] With more pairs of eyes and hands devoted to this, the field has become crowded with complexities, [12] subtleties, distinctions, nuances, mysteries, and debates. I.e., “…too many notes?”

At this year’s ISEV, will there be a tug toward nostalgia for simpler times when EVs/exosomes were less known—yet perhaps seemed more understandable? Judging from a first glimpse of the Conference’s poster titles, it’s clear that the attendees are thinking lots about Translational & Therapeutic Applications; Manufacturing, Purification, & Characterization; Standardization, Quantification, & Biomarkers; Engineering & Targeting of EVs; EV Heterogeneity and Biogenesis Mechanisms; and explorations of Cross-disciplinary and Novel EV Sources. In other words, there’s no going back; complexity is here to stay! [13] We look forward to reporting soon on our experiences at this dynamic venue, and we, too, embrace the complexity of this bold new science.

From the Right Ideas to the Right Time: ISCT 2025 Hits Its Groove

At ISCT-2025, who could ignore the background groove of New Orleans’ own late Dr. John and his epic blues-funk jam, “Right Place, Wrong Time”? Cell and gene therapies (CGTs) are replete with examples of intrepid scientists who had all the right ideas, showed up at the right place to accomplish great things…and…discovered that they were either too early or too late for their time. This tune may be a reminder that even revolutionary clinical ideas need the right alignment of timing, infrastructure, regulatory support, and unmet clinical need to truly land. We predict that ISCT-2025 will convincingly show that NOW is the right place and the right time to execute on the CGTs that the world’s been waiting patiently for. The stage is set for Dr. Peter Mark’s and Dr. Scott Gottlieb’s prophecy to reach fruition: 10-20 new GCTs approved by the FDA by the mid-2020s. [14]

RoosterBio is thrilled to see mesenchymal stem/stromal cells (MSCs), EVs, and EVs from MSCs (MSC-EVs) find their rightful place among featured topics at ISCT, a thriving nexus of CAR-T, iPSCs, HSCs, AAVs, LNPs, CRISPR technology, cell engineering, and tissue engineering. Given the first MSC-based, FDA-approved therapy announced last December of Mesoblast’s Ryoncil, RoosterBio will surely be welcoming cross-pollination with many newcomers to help co-develop scalable ways to rapidly bring novel translational breakthroughs into clinical trials and beyond. At ISCT 2025, expect a clear shift from proving cell and gene therapies work to scaling them smartly — with cost, quality, and regulatory clarity dominating the conversation. Exosomes will emerge as the next serious modality, gaining traction as precision biologics with early clinical signals across niche indications. The vibe: industrialization is in, and platforms that can scale, adapt, or deliver off-the-shelf wins will define the next chapter.

Come See Us!

We invite you to follow along as RoosterBio helps shape this next phase — one grounded in rigor, scalability, and real-world impact. If you’re headed to ISEV, ISCT, or ISSCR, come say hello, catch a talk, and see how we’re building tools to meet this moment. The future of EV-based therapeutics and MSCs is taking form — and we’re proud to be part of the community that is perfecting its recipe.

 

References

1. Lenzini, Stephen. Big Effects in Small Packages: What Are Extracellular Vesicles, Exosomes, & Microvesicles & 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/.
2. Couch, Y., et al., A brief history of nearly EV-erything – The rise and rise of extracellular vesicles. J Extracell Vesicles, 2021. 10(14): p. e12144. 10.1002/jev2.12144
3. Kalluri, R. and V. S. LeBleu, The biology, function, and biomedical applications of exosomes. Science, 2020. 367(6478). 10.1126/science.aau6977
4. Tenchov, R., et al., Exosomes – Nature’s Lipid Nanoparticles, a Rising Star in Drug Delivery and Diagnostics. ACS Nano, 2022. 16(11): p. 17802-17846. 10.1021/acsnano.2c08774
5. 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/.
6. RoosterBio. The Spirit of MISEV. RoosterBo Blog 2025; Available from: https://www.roosterbio.com/blog/the-spirit-of-misev/.
7. RoosterBio. On the Exosome/Extracellular Vesicle Frontier, Choose Your Own Adventure. RoosterBio Blog 2023; Available from: https://www.roosterbio.com/blog/on-the-exosome-extracellular-vesicle-frontier-choose-your-own-adventure/.
8. RoosterBio. “Where Do We Come From? What Are We? Where Are We Going?” Know Your Process & Define Your Product with EV/Exosome Analytics. RoosterBio Blog 2022; Available from: https://www.roosterbio.com/blog/where-do-we-come-from-what-are-we-where-are-we-going-know-your-process-define-your-product-with-ev-exosome-analytics/.
9. RoosterBio. The New eXosome Files: ISCT 2024 Features 3 Abstracts from RoosterBio on Bioproduction of Extracellular Vesicles. 2024; Available from: https://www.roosterbio.com/blog/the-new-exosome-files-isct-2024-to-feature-3-abstracts-from-roosterbio-on-bioproduction-of-extracellular-vesicles/.
10. RoosterBio. Cytiva & RoosterBio’s Collaboration Yields Breakthroughs to Make Exosome Manufacture Easier from Benchtop to Bedside. RoosterBio Blog 2024; Available from: https://www.roosterbio.com/blog/cytiva-roosterbios-collaboration-is-yielding-breakthroughs-to-make-exosome-manufacture-easier-from-benchtop-to-bedside/.
11. Carson, Jonathan. EVs Answer to the CAR-Tease: Targeted Exosomes to Speed Toward Next-Gen Immunotherapies. RoosterBio Blog 2024; Available from: https://www.roosterbio.com/blog/evs-answer-to-the-car-tease-targeted-exosomes-to-speed-toward-next-gen-immunotherapies/.
12. Welsh, J. A., et al., Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles, 2024. 13(2): p. e12404. 10.1002/jev2.12404
13. Costa, L. A., et al., Functional heterogeneity of mesenchymal stem cells from natural niches to culture conditions: implications for further clinical uses. Cell Mol Life Sci, 2021. 78(2): p. 447-467. 10.1007/s00018-020-03600-0
14. Gottlieb, S., Marks, P. Statement from FDA Commissioner Scott Gottlieb, M.D. and Peter Marks, M.D., Ph.D., Director of the Center for Biologics Evaluation and Research on new policies to advance development of safe and effective cell and gene therapies. FDA Statement 2019 1/15/2019; Available from: https://www.fda.gov/news-events/press-announcements/statement-fda-commissioner-scott-gottlieb-md-and-peter-marks-md-phd-director-center-biologics.