If There’s a Fit, Can hMSCs Commit to EV/Exosome Bioproduction?

Some things are extremely compatible: Milk and cookies… apples and cinnamon… yet sometimes the compatibility is more elusive, such as between cats and dogs; a true-to-life good fit must thus be determined empirically. In the life sciences, progress similarly advances by trial and error and can be challenging to predict. From a recent 30-minute webinar ¹ hosted by CELL AND GENE THERAPY INSIGHTS and presented by Effie Huang, M.S. (Univercells Technologies By Donaldson) and Stephen Lenzini, Ph.D. (RoosterBio), we’re happy to report on experiments showing that Univercells Technologies’ scale-X™ bioreactor platform is highly compatible with RoosterBio cells and media for the robust upstream production of extracellular vesicles (EVs) and exosomes.

Univercells Technologies scale-X Bioreactors Maintain High Process Efficiency Across Different Volumes of Conditioned Media

Belgium’s Univercells Technologies, recently acquired by Donaldson Company (DCI), is a global provider of cell manufacturing solutions and products to empower cost-effective, lower-footprint viral production from R&D to commercial scales. This company is in contact with over 110 collaborators via its team of bioprocess experts who work with its hydro, carbo, and nitro fixed-bed bioreactor systems that accommodate projects for R&D/Proof of Concept (POC), intermediate/process development, and large commercial scales, respectively. These systems contain in-line sensors for continuous monitoring and real-time optimization of the bioreactor environment. As part of the NevoLine Upstream™ platform, the scale-X nitro bioreactor benefits from the automation and integration offered by the platform.

Huang described aspects of the scale-X systems that make it attractive for clinical developers of viral vaccines, gene therapy vectors, and oncolytic viruses. The system is automated for control of dissolved oxygen (DO), pH, and temperature, and it enables either suspension or adherent cell cultures to expand into a high density and under low shear conditions. With its two-layered surface “rolled” like a cylinder within the chamber, cells such as Vero and HEK293 attach and spread along one surface while a second layer is solely for media flow. Fresh media is added through inlet ports on the vessel top and there are outlet lines adjacent to a low shear impeller at the bottom.

This unique, cGMP-friendly, and scalable modular design allows for sensor-regulated diffusion of media nutrients, gas, and controlled temperature. Consequently, the cells are homogeneously dispersed across both length and depth of the bioreactor. Uniform growth kinetics are also extended across each grade of production scale: via the (i) “hydro” (2.4m² of culture growth surface); (ii) the “carbo” (10 & 30 m² growth surface) and (iii) the “nitro” (200 & 600 m² growth surface). scale-X nitro bioreactors used in NevoLine Upstream platform enables intensified processing by integrating all steps from inoculation to midstream processing to deliver a concentrated, clarified bulk ready for downstream.

Given Univercells Technologies successes with viral production, ^{2, 3, 4} it followed naturally that our collaboration would hypothesize a good fit between exosome/EV production and cell processing technology. That is, lipid-bound exosomes are similar in size to enveloped viruses like lentivirus and may even share aspects of biogenesis with them. ^{5, 6} Like conventional HEK293s and Vero cells for virus and vaccine production, mesenchymal stem/stromal cells (MSCs) are adherent and require the right substrate to survive and expand.

Unlike transformed cell lines, however, MSCs as non-suspension primary cells could conceivably throw a few curve balls. Among other challenges, there may be a large footprint of the culture vessels, need for multiple batches, and protracted hands-on time through manual operations. Univercells Technologies addresses each of these through a structured fixed-bed design of its bioreactors, which provides a homogeneous, low shear environment. This design allows for low working volume, higher productivity, and linear scalability. Also, it encloses the impeller for recirculation and automated control of key process parameters. Because RoosterBio and Univercells were looking to demonstrate a preliminary POC, the scale-X hydro bioreactor was pragmatically chosen for the first round of studies shown in this webinar.

RoosterBio Test Drives the Univercells Technologies’ scale-X hydro system – It’s Not Just for Viruses & Vaccines!

How did this POC for the scale-X hydro system turn out? Dr. Stephen Lenzini ⁷ of RoosterBio thus began the second half of the webinar to describe our work to evaluate this platform for EV/exosome bioproduction via MSCs. Stephen introduced RoosterBio’s thought leadership with scalable extracellular vesicle bioproduction, first validated in a variety of low volume systems and then, in many larger ones. ^{8, 9, 10, 11, 12, 13, 14} During its multi-year development of extracellular vesicle bioprocesses, RoosterBio has navigated roadblocks along the way by innovating novel, fit-for-purpose products and by standardizing its hard-won applied knowledge to provide first-in-class EV related analytical services. One such product is RoosterCollect™-EV. Unlike standard basal media, it is “clean,” having virtually no basal particles to confound EV analytical metrics. It arrives to customers off the shelf in both RUO and GMP formats. Other products include paired media and cell vials that are accordingly tech-transferred into a GMP bioprocess for extracellular vesicles with ease, supported by Type II Master Files with the FDA.

For purposes of this study, RoosterBio employed a protocol where it first expanded a seed train of cells in 2D flasks with RoosterNourish™ medium. On Day 0, the requisite number of cells was transplanted into the scale-X hydro bioreactor. No media change was required, although an additive of RoosterReplenish™ media was supplemented on Day 3 via then hydro’s input line. On Day 5, the media was exchanged from a cell culture spreading/expansion formulation (RoosterNourish™) to RoosterCollect-EV and maintained in the bioreactor until Day 12. As shown in Figure 1 (below), the Calcein-AM-stained MSCs attached firmly to the nonwoven polyethylene terephthalate (PET) fiber scaffold material and were maintained with strong viability through Day 10.

 

Figure 1. RoosterBio MSCs remain viable on the structured fixed-bed all through cell seeding, cell spreading, and secretion phases of an example conditioned media collection bioprocess for extracellular vesicles.

In comparison with MSCs grown solely on 2D cell flasks, the MSCs could be maintained for a longer duration using the scale-X hydro bioreactor—for 7 days versus 3 days. Further, the extracellular vesicles in conditioned medium from the hydro 3D bioreactor were 2-2.5-fold more abundant than EVs from MSCs grown in parallel seeded 2D planar flasks. Finally, the productivity of EVs per cell was also considerably higher via the 7 days of Univercells Technologies process than the 3 days of 2D process, yielding over ~2.4 x 10⁴ EVs/cell instead of ~4 x 10⁹ EVs/cell as per the 2D (see Figure 2, below).

Figure 2. In side-by-side experiments, MSC-EV productivity per volume (top) and per cell (bottom) was much higher when cells were maintained in the scale-X hydro bioreactor instead of on 2D planar cell culture flasks.

Conditioned media from collection phases via the 2D planar and 3D hydro bioreactor process yielded extracellular vesicle products with similar quality attributes. As shown in Figure 3 (below), nanoparticle tracking analysis (NTA), Western blotting for a canonical EV/exosome surface marker (CD63), and analysis of RNA content yielded qualitatively similar results between the preps. That is, their exosome-like size distributions, expression of known extracellular vesicle constituents, and encapsulation of intact nucleic acid all were comparable to one another.

 

Figure 3. In two side-by-side experiments, MSC-EV characterization parameters from conditioned media collected between 3D scale-X hydro bioreactors and 2D planar culture were similar according to size distribution (left), presence (-/+) of immunoreactive CD63 by Western (middle), and quantitation of internal particle RNA content (right).

At this stage of POC studies, we’re fortunate to report on a successful pairing between RoosterBio’s cell and media products and bioprocess and Univercells Technologies’ scale-X systems. Critical quality attributes of a benchmark 2D process are maintained, and this translation from this 2D platform to the 3D brings the added benefit of substantially improved extracellular vesicle productivity by way of a smaller, automated instrument footprint that may demand less hands-on time.

Questions & Answers

The webinar ended by addressing a few questions which are paraphrased below. To learn how they were answered, you can watch the recording.

1. For Univercells: What cell type was used to perform the homogeneous cell distribution data in the 600M² system?
2. For Univercells: How do you get a cell count during the cell growth phase of the process or when ready to infect or transfect?
3. For RoosterBio: Why is it important to have a particle free collection medium?
4. For Univercells: How can scale-X provide higher extracellular vesicle production yield?
5. For RoosterBio: Have you done functional tests on extracellular vesicles produced by bioreactor and 2D platforms?

Thank You

RoosterBio is most pleased to have participated in this collaboration with Univercells Technologies and the scale-X bioreactors. We stand ready to assist with your further questions and requests related to scalable bioproduction of extracellular vesicles and exosomes. If you’ve not yet had a chance to view this webinar, we invite you to take a brief moment from your day to check it out! We think it could be highly-compatible with your time.

 

References

1. Huang., E., Lenzini, S. Redefining extracellular vesicle production: proof of concept study evaluating structured fixed-bed bioreactor for high-capacity production CELL & GENE THERAPY INSIGHTS [Online], 2023. (LINK)
2. Berrie, D. M.; Waters, R. C.;  Montoya, C.;  Chatel, A.; Vela, E. M., Development of a high-yield live-virus vaccine production platform using a novel fixed-bed bioreactor. Vaccine 2020, 38 (20), 3639-3645. 10.1016/j.vaccine.2020.03.041
3. Nikolay, A.; Bissinger, T.;  Granicher, G.;  Wu, Y.;  Genzel, Y.; Reichl, U., Perfusion Control for High Cell Density Cultivation and Viral Vaccine Production. Methods Mol Biol 2020, 2095, 141-168. 10.1007/978-1-0716-0191-4_9
4. Leinonen, H. M.; Lepola, S.;  Lipponen, E. M.;  Heikura, T.;  Koponen, T.;  Parker, N.;  Yla-Herttuala, S.; Lesch, H. P., Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production. Hum Gene Ther 2020, 31 (5-6), 376-384. 10.1089/hum.2019.247
5. Gould, S. J.; Booth, A. M.; Hildreth, J. E., The Trojan exosome hypothesis. Proc Natl Acad Sci U S A 2003, 100 (19), 10592-7. 10.1073/pnas.1831413100
6. Pastuzyn, E. D.; Day, C. E.;  Kearns, R. B.;  Kyrke-Smith, M.;  Taibi, A. V.;  McCormick, J.;  Yoder, N.;  Belnap, D. M.;  Erlendsson, S.;  Morado, D. R.;  Briggs, J. A. G.;  Feschotte, C.; Shepherd, J. D., The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer. Cell 2018, 172 (1-2), 275-288 e18. 10.1016/j.cell.2017.12.024
7. Lenzini, S.; Bargi, R.;  Chung, G.; Shin, J. W., Matrix mechanics and water permeation regulate extracellular vesicle transport. Nat Nanotechnol 2020, 15 (3), 217-223. 10.1038/s41565-020-0636-2
8. From Manufacturing Through Characterization: Simplifying EV/Exosome Research & Development RoosterBio.com [Online], 2023. (LINK)
9. Manufacturing Advances Unlock the Therapeutic Potential of MSC-Derived Exosomes RoosterBio.com [Online], 2023. (LINK)
10. ISCT22: RoosterBio, ShiftBio partner to harness the power of MSCs, exosomes The Science Advisory Board [Online], 2022. (LINK)
11. Rowley, J., Boychyn, M., Kelly, T. Why Cell Manufacturing Matters: How Bioprocess Innovations Have Laid the Foundation for a Cell-Based Products Revolution BioProcess International [Online], 2022. (LINK)
12. Cruz, C. From Benchmark to Airborne: MISEV Guidelines Inspire Fledgling EV/Exosome Analytics Services RoosterBio blog [Online], 2023. (LINK)
13. Lenzini, S. Extracellular Vesicle/Exosome Upstream Process Development: Maximizing Productivity to Accelerate Clinical Adoption RoosterBio blog [Online], 2022. (LINK)
14. Jung, J., Lenzini, S. Extracellular Vesicle/Exosome Downstream Process Development Part I: Leveraging Filtration Technologies for Scalable EV Preparation RoosterBio blog [Online], 2022. (LINK)

 

About Univercells Technologies by Donaldson

Univercells Technologies by Donaldson is a global provider of innovative biomanufacturing technologies to achieve cost-effective viral production from R&D to commercial scales. The company offers a comprehensive technology portfolio leveraging the strengths of process intensification and chaining as a direct answer to the growing demand of viral vectors and viral vaccines. Univercells Technologies by Donaldson is committed to helping customers increase performance with minimized footprint and costs today, while anticipating the needs of tomorrow.

Building upon years of expertise and capitalizing on technology vetted by world leaders, Univercells Technologies by Donaldson was incorporated in Belgium in 2020 and is now part of Donaldson Company.