“Blivet” image from Wikimedia Commons depicting the comical paradox of illustrating a third dimension with only a concept of 2D

• 3D cell culture provides a more efficient and scalable production of adherent cells and exosomes/extracellular vesicles (EVs) than 2D planar cell culture. It requires less hands-on labor, reduces the risk of contamination, and lowers overall costs per patient dose.
• Scale-up with 3D entails minimal protocol adjustment between increased culture volumes with only subtle parameter changes between single vessels. Unlike “babysitting” dozens to 10s to 100s of 2D surfaces, 3D bioprocess timelines can be left alone, sparing operators their weekends.
• Clinical process development via 3D systems can be a sensible and even favorable approach for nascent cell and gene therapies, starting from the very outset of preclinical and clinical bioprocess program planning.
• RoosterBio provides specialized products and comprehensive guidance to help researchers and developers adopt streamlined and economical 3D culture systems.

From Squares to Spheres

RoosterBio can only be as successful as it is empathetic to customer needs. Our catalog of exosome/extracellular vesicle and mesenchymal stem cell (MSC) products is thus the natural ascension out of routine, empirical problem-solving during the brisk cadence of our development services programs. Today, repeat customers and serial visitors to our website may notice that RoosterBio increasingly gears its product engine for 3D bioprocess applications. Why is that?

Is it fair to digress into the story of “Flatland,” revisited with a droll yet enduring poignancy by the late Carl Sagan? In Flatland, all life and perception of it is in two dimensions. There, a two-dimensional (2D) square creature is plucked out of his environs into the third dimension (“Spaceland”) by a 3D Sphere. Life will never be the same for poor old “Square.” It may sound silly, but perhaps there’s a little bit of Square in all of us, we who transcend gravity for a vision above Earth’s curved horizon.

Ever since humans’ hominid ancestors fled the forests into the veld lands, vestigial instincts compelled us to thrill seek by climbing back up, beyond width (x) and length (y). We also stack things into the z-dimension to create order and efficiency. Advancements in steel frame technology in the 19^th Century led to skyscrapers in the 20^th, which allow urban environments to efficiently maximize the macro scale of human action. At nano scale, computer chips made with UV lithography contend with the laws of physics as they strain the limits of Moore’s Law. How are they achieving “More Moore?” Many strategies involve 3D integration, where circuits are addressed vertically, connecting more transistors within the same footprint to improve performance and reduce power consumption.

Returning the Flatland story, our Square has a tough time convincing his fellow 2D denizens of a multi-dimensional reality and is tormented for his disruptive heterodoxy. In contrast, RoosterBio has already inhabited both 2D (“Flatland”) and 3D (“Spaceland”) via more than a decade of expansion culture and conditioned media collection for advanced cellular therapeutics. We therefore fully empathize with those who solely work in planar, two-dimensional cell culture systems. But in this blog, we’ll aim to do a better job than Flatland’s Square to demystify for readers a universe of multiple dimensions for cell and exosome harvest. Unlike Square, you don’t have to be a spectator in bioprocessing’s embrace of 3D, scorned and alone. On the contrary, you can even optimize the best Sphere to assist you along the way. ^{1, 2} Moreover, we and other cell culture process innovators everywhere are opening our eyes anew, and enjoying the view!

An Expensive Hobby or a Calling Toward Improved Access for Patients?

In advanced therapies involving cells and/or genes, quality is the #1 contributor to a clinical developer’s insomnia. Cost-on-goods-sold (COGS), ³ an interdependent factor, comes in a close second place, for excess cash burn during drug development will infamously derail countless brilliant ideas. To plan for progress to run smoothly forward, we advise first strategizing from the program’s end and working backwards. During this exercise, identify key metrics such as number of harvested cells per mL of total media volume, number of necessary population doublings (PDs), cost per million cells, and finally, the number of doses required for development, clinical studies, and ultimate post market approval. Stating the obvious, it’s crucial to adapt for different scales and to adjust each scale for minimal disruption to the complex bioprocessing.

Believe it or not, a few cell therapy product innovators are fixated on what to make, while reflexively mentally blocking the question of how to make. They assume that although 3D cell culture systems exist in an abstract realm, making practical use of them would be too steep a climb beyond the 2D comfort zone.  True enough, it can be challenging to swap a scientist’s or clinician’s hat for an engineer’s hat.  “Stick to the knitting, stay of out trouble” is the mentality. Times have changed, however, due to a quiet revolution brought about by life sciences process innovators. The barrier to entry into 3D has now never been lower, and benefits of derisked bioprocess, lower cost per dose, and reduced timelines can be quickly realized, regardless of scale.

If staying small scale and/or approaching human health challenges is to remain just another research “hobby,” some Flatlander investigators could certainly begin an assortment of early-Phase clinical trials based on 2D. There’s no shame in that, considering the slim odds of any BLA approval. However, any first sign of clinical success will be heard by patient advocates who will cry out that this novel medicine no longer be relegated to hobby status. Are you and your team motivated by a “calling” to help them? Imagine a future where you overlook from the high ground, more in command of your product’s ultimate fate from end to end. What will success in abundance look like? It might mean scale-up and a time-consuming overhaul of the 2D bioprocess into 3D bioreactor culture systems. But as we’ll explain later, this doesn’t have to be the only way.

Figure 1 (above). Increasing cell culture surface area is needed to accommodate higher demand of cellular raw materials during scale-up. At least 1000-fold greater volumes of 2D flasks are necessary for manual passage of adherent cells grown on planar surfaces when compared with R&D and Phase III/GTM (go to market) bioproduction. By contrast, only one single culture vessel is needed for changes in volume unit scale when “working in 3D.”

First, consider a cautionary example of where 2D could lead us. At small scale, a 2D R&D process performed for ~100 million cells (Figure 1) needs very little time and effort to yield high-quality MSCs. 2D workflow is initially not unlike standard, R&D biolabs in small companies and universities worldwide, a map dotted with biological safety cabinets, fume hoods, incubators, CO₂ and l-N₂ banks, freezers, autoclaves, etc. However, with each ratchet in volume scale through clinical development stages, it’s often more laborious by an order of magnitude. By Phase III and market (GTM), the 2D effort is nearly prohibitive for adherent cells like MSCs.

2D scale-up involves increases in more plastic waste ⁴ and hands-on operator time, but also incubator footprint, and different kinds of—and more numbers of—vessel types. One standard CO₂ incubator cube can hold roughly 40 individual cell stack (or 4x CS10s) layers. Production scales needed for a Phase III trial (or beyond) would require a large site full of dedicated cell culture incubators and biological safety cabinets, as well as trained staff to feed and passage these cells across evenings, weekends, and holidays. These complications call to attention tens of thousands of dollars in “hidden” CapEx and square feet/meters of physical plant expansion.

Here’s one early “take home message” for readers: when stuck in a 2D Flatland mindset, the scientist-hobbyist will need to dismantle and rebuild the current process with each new increment of scale, while COGS and timelines will commensurately increase. Yet the advanced therapies leader—armed with the latest knowledge and foresight—understands 3D’s basic strength. While the single culture and/or collection vessel will grow, 3D’s built-in industrial features allow processes, manipulations, and timing to remain the same. COGS measured as Cost per Million Cells ($) would decrease, and with it, the burdensome costs passed on to the developer, the health system reimbursements, the patients, and their families (Figure 2). This is how the built-in efficiency of an industrialized economy of scale can reap benefits for all.

Figure 2 (above). RoosterBio (RBI) has periodically modeled Cost per Million Cells ($) as a key metric that drives access to advanced therapies and regenerative medicine, particularly with extracellular vesicles (EVs)/exosomes and mesenchymal stromal/stem cells (MSCs).  A standard, R&D-type, two-dimensional (2D) method to expand and prepare adherent MSCs costs more than $100 per million cells (T Flasks). When adapted to RoosterBio’s high-performance growth medium and cells (RBI 2D) this method can see costs per million cells reduced by ~40%. Adoption of 3D bioprocess to 3L, 15L, 50L, and 200L volumes results in further diminution of cost to ~$10 per million cells or less.

Less Hands-On = Less Risk

Figure 3 (above). Reduction in the contribution of labor and QC testing toward cost per million cells ($) is especially dramatic during scale-up to 3D processes from 2D T Flasks and adoption of RoosterBio (RBI) cells and media. Along with the decreased need for “hands-on time” and labor/testing across higher volume production scales, there can also be decreased risk of batch failure due to human error or variability in raw input materials.

We easily see how 3D cell culture could plausibly reduce direct, hands-on labor costs and testing during bioprocess scaleup (Figure 3). However, cell culture cost is also partly a function of risk, which can be less tangible to estimate up-front. In turn, risk is a function of hazard multiplied by exposure. Each new, hand-cultured 2D flask is an invitation for risk to enter a sequence of steps to prepare complex, cellular raw materials. For the small-scale clinical developer who aims to rapidly enter first-in-man trials, risks via 2D preparations of cellular materials could be deemed minor. The process to obtain them for early-Phase trials will seamlessly translate out of the previous R&D effort.

Other bioprocess risks, such as mycoplasma contamination, are major and could derail an entire batch. (It’s said that more hands make light work, but then there’s also the proverbial too many chefs in the kitchen!) RoosterBio’s media exchange-free system (whether in 2D or 3D) already greatly reduces such risks. However, moving to a 3D vessel enables the operator to quickly adjust to a right-fit vessel size and further decrease the number of major hands-on steps: Formulate – Seed – Feed – Harvest, That’s it!

With a plan to implement a more automated 3D bioprocess, the regenerative medicine innovator can now focus on training and cultivating the team’s higher-value talents, as they’ll henceforth spend fewer hours of drudgery behind a biological safety cabinet’s glass—or troubleshooting. Surely, this could translate to a “win-win” solution. Yet what can better flexibility and simplicity mean for the total workflow?

Enjoy the Weekend

When 3D cell culture reduces labor costs via lower hands-on time during bioprocess scaleup (Figure 3), this also tangentially involves minimized overhead “hands-off” time due to paperwork, reporting, red tape, dealing with complex supply chains, and “putting out fires” – the fodder for a 3AM panic. Yes, a biolab career might be a calling, but does it need to be a sentence? Notwithstanding delirious scientific curiosity to seize the winning result, weekends might also be important for phone calls to friends and family, banana pancakes, sports games and music recitals, physical and spiritual renewal, etc. So why can’t cellular scientists achieve a proper work/life balance, too?

To save these special times for its partners and customers, RoosterBio has already worked across the weekends, holidays, and evenings to optimize streamlined cell expansion and conditioned media collection in 3D (Figure 4). Today, once such process flow timelines are firmed, they do not need to change across late R&D through clinical manufacturing.

Figure 4 (above). What is needed to obtain 200 million MSCs? Implementing a high-performance cell culture expansion medium such as RoosterNourish™ allows the bioprocess technician to eliminate media exchanges and hands-on steps, use less total media volumes, and shorten duration of cell culture (upper panels). In turn, adjustment from a 2D planar batch culture system to a 3D fed batch culture reduces media volumes and manual handling even more. Note how the operator can walk away and enjoy both weekends: during the 3D fed batch bioreactor expansion, and then, during EV collection phases of an example process (lower panels).

MSCs are considered a plug-and-play cellular technology platform that’s instrumental toward more advanced regenerative medicines a la emerging “MSC 2.0” applications. ⁵ The last 5-10 years thus witnessed a large increase in EV or exosome related clinical trials and startup company incubations, many of which employ MSCs a bioproduction cell. MSCs are prolific producers of EVs, ⁶ and will secrete them in much higher numbers than in 2D amidst the churn of a 3D bioreactor’s fluid dynamics. ⁷ Using customizable, safe, and reliable MSCs, RoosterBio has adapted these cells for expansion and/or conditioned media collection via a variety of scalable, manufacturable, industry-leading 3D bioreactor formats, including PBS, Sartorius, Pall, Eppendorf, Univercells, Terumo, and others. Although our 3D MSC process technologies will allow the cell technician to fully enjoy evenings and weekends (Figure 4), this doesn’t mean we and our partners need sacrifice any momentum towards the clinic. We have assisted partners to proceed with clinical trials in months instead of years due to our ability to accelerate and streamline development timelines with GMP products and maintenance of Type II US FDA Biologics Master Files (MF).

Get Started… From Above & Beyond Flatland

For many, we’ve found that it can be perfectly adequate to expand MSCs in 2D culture for R&D/Discovery, preclinical development, and Phase I trials. A 2D bioprocess for a small number of human doses can easily recapitulate the initial material prepped for in vitro assays and in vivo IND-enabling animal studies. The “triathlon” from 2D to 3D bioreactor cell expansion for later-Phase trials may still require some non-trivial detouring through process development optimization to demonstrate comparability between final products. Nevertheless, we frequently observe that the basic product critical quality attributes (CQAs) ⁸ between 2D- and 3D-cultured cells can be the same, ⁹ so long as the critical process parameters (CPPs) ¹⁰ are monitored and controlled correctly.

This blog’s readers may wonder: Why not skip 2D process altogether and prepare MSCs via 3D modalities from the very outset?  Wouldn’t it seem practical to forego the time ³ and waste ⁴ in this way?

Historical barriers to entry for the first bioprocess pioneers to “leave Flatland” and adopt 3D have finally been cleared away. That is, RoosterBio now offers MSC expansion and collection media specially engineered for 3D expansion. These include cGMP bagged RoosterNourish™ product formats as well as complementary RoosterReplenish Bioreactor Feed. Recently, these were bundled together in one discounted starter kit offering, all ready to test drive.¹¹ Not only has RoosterBio developed special EV collection media in development grade as well as cGMP formats, but we’ve also made it simple for customers to get running today with protocols that need only modest capital investment at the small scale.^{12, 13, 14} We’re fully versed in what’s important to monitor, to leverage, and to troubleshoot, and so there is little that surprises us. ^{15, 16}

Another barrier to entry was hitherto limited guidance on how to tweak bioreactor CPPs to yield a 3D product with consistent CQAs. To address this, RoosterBio (founded 2013) ¹⁷ has spent its first decade of product launches and hands-on customer service to develop scalable bioprocess for MSCs via economical, miniaturized, practical, and off-the-shelf 3D formats. We’ve also worked with industry-leading consortia and organizations such as the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) and BioFabUSA, as well as plotted a strategic Roadmap with the National Cell Manufacturing Consortium. ¹⁸ RoosterBio has learned among the very best, and been fortunate to also teach the best…right up to the present day.

Our own track record of partnerships and collaborations, presentation of posters at major international conferences, and publication of journal articles speaks for itself. Although we recognize that each bioproduction scheme is a little different, we self-publish a library of protocols, quick reference guides, and white papers that detail how to rapidly begin your own journey into 3D. If that’s still challenging, we love speaking with new friends, assisting your definition of requirements for engineered cells or EVs, and helping you convert these sketches into full-fledged action plans. It’s simply never been easier to compose and execute on a cell or exosome therapy, with a rapid green light directing to an IND filing.

Like suffering through instant coffee after an upgrade to freshly ground beans, or using dial-up after a fiber optic connection, we concede that devolving back to 2D from 3D bioprocess might only be reversible under protest. Yet unlike our ill-fated Square character from Flatland, you won’t ever need to or wish to go back. Start from 3D! The “view from on high” will be as magnificent as can be from Day 1.

 

References

1. Yuan, X.; Tsai, A. C.;  Farrance, I.;  Rowley, J.; Ma, T., Aggregation of Culture Expanded Human Mesenchymal Stem Cells in Microcarrier-based Bioreactor. Biochem Eng J 2018, 131, 39-46. 10.1016/j.bej.2017.12.011
2. Rafiq, Q. A.; Coopman, K.;  Nienow, A. W.; Hewitt, C. J., Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors. Biotechnol J 2016, 11 (4), 473-86. 10.1002/biot.201400862
3. Lim, M., Candiello, J. Best Practices in RegenMed Product & Process Development – Know Your COGS. https://share.hsforms.com/1Ns3ZdJgFQKW05XATgdnfrQ3564o.
4. Agbojo, A., Lim, M., Lembong, J. Environmental Analysis of Therapeutic hMSC Manufacturing: A Comparison of Multiple Bioprocess Systems. https://www.roosterbio.com/blog/environmental-analysis-of-therapeutic-hmsc-manufacturing-a-comparison-of-multiple-bioprocess-systems/.
5. Olsen, T. R.; Ng, K. S.;  Lock, L. T.;  Ahsan, T.; Rowley, J. A., Peak MSC-Are We There Yet? Front Med (Lausanne) 2018, 5, 178. 10.3389/fmed.2018.00178
6. Yeo, R. W.; Lai, R. C.;  Zhang, B.;  Tan, S. S.;  Yin, Y.;  Teh, B. J.; Lim, S. K., Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery. Adv Drug Deliv Rev 2013, 65 (3), 336-41. 10.1016/j.addr.2012.07.001
7. Adlerz, K.; Trempel, M.;  Wang, D.;  Kirian, R. D.;  Rowley, J. A.; Ahsan, T., Comparison of msc-evs manufatured in 2D versus scalable 3D bioreactor systems. Cytotherapy 2019, 21 (5, Supplement), S58. https://doi.org/10.1016/j.jcyt.2019.03.434
8. Adlerz, K. Critical Quality Attributes (CQAs): Know Their Importance & Limitations in Product & Process Development. https://www.roosterbio.com/blog/critical-quality-attributes-cqas-know-their-importance-limitations-in-product-process-development/.
9. Kirian, R. Maintaining CQAs as Manufacturing Processes are Scaled from 2D to 3D Bioreactor Culture. https://www.roosterbio.com/blog/maintaining-cqas-as-manufacturing-processes-are-scaled-from-2d-to-3d-bioreactor-culture/.
10. Lembong, J. Identify & Define Your Cell Therapy’s Biomanufacturing Approach for Critical Process Parameters (CPPs). https://www.roosterbio.com/blog/identify-define-your-cell-therapys-biomanufacturing-approach-for-critical-process-parameters-cpps/.
11. RoosterBio XF Bioreactor MSC & EV Starter Kits. https://www.roosterbio.com/products/xf-bioreactor-hbm-msc-starter-kits/.
12. RoosterBio Recommended Expansion Protocol for Fed-Batch Culture Regimen (AMBR® 250 Expansion). https://www.roosterbio.com/wp-content/uploads/2024/02/K81005-AMBR250-Recommended-Protocol-2.0_v2.pdf.
13. RoosterBio Recommended Expansion Protocol for Fed-Batch Culture Regimen (PBS Mini Expansion). https://www.roosterbio.com/wp-content/uploads/2024/04/K81005-PBS-Mini-Recommended-Protocol-2.0.pdf.
14. RoosterBio Recommended Protocol for Fed-Batch hMSC Expansion and Extracellular Vesicle Production in Spinner Flasks https://www.roosterbio.com/wp-content/uploads/2024/04/K81005-125mL-Spinner-Flask-Recommended-Protocol-EV-Process-2.0.pdf.
15. Lembong, J.; Kirian, R.;  Takacs, J. D.;  Olsen, T. R.;  Lock, L. T.;  Rowley, J. A.; Ahsan, T., Bioreactor Parameters for Microcarrier-Based Human MSC Expansion under Xeno-Free Conditions in a Vertical-Wheel System. Bioengineering (Basel) 2020, 7 (3). 10.3390/bioengineering7030073
16. Willstaedt, T. M.; Walde, A.; Rowley, J. A., A FED-BATCH CHEMICALLY DEFINED HMSC-EV BIOPROCESS MEDIUM ENABLING 2-4X EV YIELD IMPROVEMENTS IN BIOREACTOR CULTURE. Cytotherapy 2024, 26 (6, Supplement), S59. https://doi.org/10.1016/j.jcyt.2024.03.105
17. Olsen, T. R.; Rowley, J. A., Corporate profile: RoosterBio, Inc. Regen Med 2018, 13 (7), 753-757. 10.2217/rme-2018-0092
18. NSF Engineering Research Center for Cell Manufacturing. Achieving Large-Scale, Cost-Effective, Reproducible Manufacturing of High-Quality Cells A Technology Roadmap to 2025. https://cellmanufacturingusa.org/sites/default/files/NCMC_Roadmap_021816_high_res-2.pdf.