Eureka! The glowing data speak for themselves; it’s time to graduate from HTS, petri dishes and mice, and time to help actual human patients in need. Your company or translational center wishes to join more than 1253 therapeutic developers across East Asia, Europe, or the USA, into which $23B has been invested to launch over 2000 clinical trials involved with advanced therapies (aka regenerative medicine). [1, 2] Given over 60 such therapies now on the market in nations monitored by the Alliance for Regenerative Medicine (ARM), your team would be entering a crowded race—not for the sprinter, but for the flexible and hardy endurance triathlete.
To cross the finish line, you’ll not only need sound strategy for years of trial design and management, but also for the cellular materials to manufacture (or directly comprise) the administered drug. Just as triathlon racers are seen in all shapes, sizes, and ages, your team will need a manufacturing process that best dovetails your capabilities and core strengths—and also, an idea of how you’ll need to “stretch.” [3, 4, 5] Bioprocess for advanced therapies involves many steps: tissue acquisition, cell isolation, cell expansion, harvest, volume reduction/washes, formulation & filling, QC & release, storage & inventory, and shipping/logistics.  Throughout these, testing of quality, potency, viability, and sterility is critical.  Although it’s important to control and document how each step is handled end-to-end, [8, 9] this blog will focus on cell expansion.
As any triathlete-in-training can tell you, “You get out of it what you put into it.” Like athletes, biomanufacturers also cross-train, breaking in their process “shoes” on liquids, machines, and solid surfaces. Hence, choice of versatile “upstream” cellular starting materials underlies an effective manufacturing plan. We’ve written extensively on the innate portability of hMSCs (human mesenchymal stromal/stem cells) into an assortment of expansion platforms from two-dimensional, planar growth (2D) to three-dimensional (3D) suspension growth on microcarriers. [10, 11, 12, 13]
MSCs are easy to grow and manipulate—and boast a proven track record of safety in more than 1500 posted clinical trials worldwide.  Regulatory-friendly, GMP-certified hMSC cell banks with defined donor characteristics can routinely and robustly expand from frozen vials into clinically relevant dose sizes when paired with a system of high-performance media, in less than 10 days. With some of these linked to Type II Drug Master Files (DMFs) from the FDA, [15, 16] simple, pre-validated bioprocess templates from standardized materials can allow a fast track to human doses, shortening development times from years to months. [17, 18]
Like a triathlete between two legs of the race, it’s often sensible—if not expected—for a bioprocess to “change shoes” after successfully completing one phase of competition. That is, some specialized cell expansion formats are more suited for manual and shorter duration needs (e.g., the “foot race”), whereas others are mechanically oriented and longer in length (e.g., the “bike race”).
To start the “foot race” with basic science or discovery phase R&D, hMSCs cells grow well in simple 2D tissue culture flasks for R&D purposes. We and our collaborators have found that they can be scaled up into 2D multi-stack flasks from Corning,  or scaled out into 96 well plates for bioassay under high content imaging systems.  Under our optimized growth conditions, no media change is required, and cells can be plated at low density to yield more than 100-fold expansion after a single passage. In terms of overall final cost, the media matters; high-performance media can achieve lower waste and substantially higher cells per liter. [21, 22, 23]
To zoom through the “cycling race” with clinically relevant doses of hMSCs for 1000s of patients with non-rare diseases and chronic health conditions, a trail to greatly increased CDMO capacity, cell volume, and operator bandwidth must be blazed. The transition from small scale, 2D flask-grown MSCs—e.g., for Phase I trials—to bioreactors for bigger patient cohorts is going to be all but necessary for full commercialization of some MSC therapeutics. [24, 25, 26] That is, when juggling batches of more than 10-20 billion cells, it becomes increasingly more practical and less costly to switch to a “3D” or bioreactor set-up.
A proverbial change to “bike shoes” may be necessary to deal with gears, wheels, and spin for the longer-distance portion of the cell manufacturing “triathlon”—i.e., later-Phase clinical trials and post-BLA distribution to patients. Fortunately, our team repeatedly demonstrates that hMSCs not only grow prolifically in standard 2D flasks and plasticware but also in different bioreactor 3D culture contexts. These platforms often involve the “cycling” of media—combining impellers, agitators, or wheels with cell suspension tanks and microcarrier growth substrates. We observe close comparability between MSC product made by either 2D or in “3D” bioreactors using the RoosterBio cell and media system. 
With our company’s cell and media system, we consistently achieve densities of more than 0.5 million cells per mL between scales of 0.1L (PBSmini) to 50L (PBS80) via PBS Biotech’s Vertical Wheel® technology.  This can translate to 31 billion cells per run in a 50L format, or enough for dozens of average-sized MSC doses for humans in an early-Phase clinical trial.  Sartorius—long known as a gold standard in the bioprocess industry for full-scale manufacture of adherent and non-adherent cells—offers a graded approach of options from small (<1L) or pilot scale (>1L) to giant (1000L+). Applied to Sartorius’ 3D suspension stirred-tank bioreactors, our team’s product systems have shown successful scaling between the small, modular Ambr®250  and the larger, Biostat STR® 50L systems.  With >25B cells per lot attainable, linear scalability to the Biostat STR® 2000L system could potentially yield one trillion cells per run, sufficient for thousands of MSC doses. To accommodate liter-scale, closed-system production volumes, we and others offer GMP media in bagged formats.
MSCs are, by definition, plastic-adherent cells.  Thus, when grown “3D” in bioreactors, they still require a surface on which to attach and spread out, whether “floating” or fixed within a complex, compact matrix. Perhaps extending the triathlete analogy a bit too far, other cell expansion systems are much like running and cycling on a single pair of “shoes”—no change of format required. That is, cell numbers required for autologous cell therapy batches tend to be lower than for allogeneics. These starting materials from autologous sources can readily “run” through hollow-fiber perfusion systems that allow the operator to carefully fine-tune critical process parameters in small-scale development mode or at full-scale.
Work with our collaborators demonstrate successful hMSCs growth in hollow-fiber (HFB) perfusion systems from FiberCell  and the Quantum® cell culture cartridges from Terumo.  These systems enable high cells per volume culture of up to 2.1m2 of surface area, closed system automation, and constant input/output flux of nutrients for stable growth conditions. Although the hollow fiber technology does not yet reach the large scale of other bioreactors, these devices are already outstanding for extracorporeal cell therapies for human patients, with cell numbers that may approach 1B per cartridge.  Further, intriguing options can be explored to harness cell-secreted products in a continuous mode, instead of via batch productions.
Other single-use bioreactor formats with demonstration in hMSCs include fixed bed, hollow fiber, rotary cell culture systems (RCCS), rotating bed, or rocking motion (e.g., WAVE™) configurations, as recently expertly reviewed by Nogeuira, et al. (2021; see Table 1, below).  These platforms vary in scalability, degree of adoption by large instrument makers, clinical readiness, reusable componentry, and range of stresses leveraged on the expanding cell populations. To best match hMSCs to a new bioreactor system, some empirical trial and error may be involved to best optimize mixing forces, composition/coating, and porosity of microcarriers, media constituents, and origin of cell by donor or tissue type. 
Table 1, below, adapted from by Nogeuira, et al. (2021) 
|Major Manufacturers||Culture volume or area for MSCs, L or m2||
|Stirred tank||Eppendorf, EMD Millipore, Satorius Stedim||
0.01 – 2000*L+
|• Long-established industry know-how across diverse cell systems, including hMSCs
• Wide range of scale from R&D to pilot to commercial
• Predictable scalability of cell yield per volume scale
|Vertical-Wheel||PBS Biotech||0.1 – 70L||• Low shear stress & efficient mixing
• Wide range of mfg. scales
• Unique design avoids cell entrapment under impeller
|Hollow fiber||Fibercell, Terumo||to 2000m2||• Low shear stress
• Very small footprint with high surface growth area
• Very compatible with perfusion, extracorporeal, and allogeneic clinical applications
• Disposable, single-use cartridges for closed systems
|Fixed bed||Pall/iCELLIS®,or misc. DIY (“do-it-yourself”)||0.003 – 1000L+||• Low shear stress
• Small footprint with high surface growth area
|Rotary cell culture system||DIY||to 0.01L||• Low shear stress
• No air bubbles
• Simulates low-gravity for NASA/space!
|Rotating bed||Zellwerk GmbH||to 0.6m2||• Low shear stress
• Small footprint with high surface growth area
• Intermittent contact with medium and headspace
|Rocking motion (e.g., “WAVE™”)||Cytiva (WAVE™ Cellbag™)||0.05 – 0.6L||• Low shear stress & efficient mixing
• Wide range of mfg. scales, and popular among cell biomanufacturers
• No air bubbles
• Very compatible with autologous cell therapies
hMSCs don’t just comprise the main starting material as ready-to-inject cellular therapeutics. They also facilitate product systems that are portable into processes to harvest their prolific secretomes, i.e., EVs (extracellular vesicles)/exosomes. [19, 34] At 15L pilot scale using the PBS system, we recently reported EV yields of 3.31 trillion EVs from the culture, or about 3000+ EVs per cell. Similarly, using Sartorius’ Ambr®250 scalable model for stirred tank systems, we’re presently observing ~5000 EVs produced per cell.  Assuming a stringent purification scheme that discards ~90% of the EVs during DSP, this productivity level alone could supply tens of doses to human patients for diverse clinical indications. 
In downstream processing (DSP) of biologicals, it’s rare that you can “have your cake and eat it too;” although purity and potency are closely related, more stringent purification can negatively correlate with yield.  Considering the challenges of downstream production with EVs, it’s prudent to ask where standard 2D flask culture of MSCs can fit into the scheme of human EV trial material. Recent modeling studies have argued that “when lot size is sufficiently large, EV harvest can become cheaper than cell expansion.”  Our empirical data that evince higher cell densities via 3D tend to bear this out.  Comparing a 2×10-layer CellStack process (> 3L) to 3D bioreactors, we’ve reported about 500M cells and 9.8×1011 EVs of media via this 2D approach. This is less efficient than the 3L Vertical Wheel system from PBS, which cranks out 1.5B cells and 6.0×1012 EVs. Efficiency is also less than what we recently observed with a stirred tank model system.  Nevertheless, if we consider time to be a scarcity compounded by equally scarce funds for new bioreactor capex, it’s certainly feasible to proceed quickly via 2D flasks for EV/exosome preclinical work, IND-enabling studies, and small-scale Phase I trials. We and our collaborators continue to improve our EV processes by facilitating improved downstream processing—and also improved productivity of EVs on a per-cell basis. 
It’s not just how you grow hMSCs that matters, it’s what you do with them! Why not use them in a form that requires as little upstream effort as possible, e.g., for bioprinting?  Large, cost-efficient, post-expansion volumes of 50M cell vials may be prepared that are “ready to print” to use for various bioinks for tissue engineering applications, [24, 41] such as the CellInk BioX instrument series. [42, 43]
Much like bio-“3DP,” genetic engineering of hMSCs unlocks a whole new universe of preclinical and clinical possibilities. With advancing gene transfer and genome editing techs, each cell can be engineered into a micro-delivery system for a genetic medicine. To enhance transduction [44, 45] or transfection [46, 47] of these, complete media that easily “drops-in” one’s scalable bioprocess may be necessary, given that hMSCs and other primary cells are less amenable to conventional gene transfer modalities than transformed cell lines.
Asked publicly whether it’s masochism or motivation that inspires the rare triathlete or advanced therapeutics developer—and they’ll probably answer with something like “It’s just what we do…the easy road of mediocrity is simply more tedious to bear than the hard road toward excellence.” Privately, however, both know that it’s much easier to stay motivated (and less injured) by bringing the right tools into the right leg of the journey. Whether it’s the perfect shoe, or the lightest bike frame—or the best-fitting cell expansion bioprocess—the race can be open to nearly all, and there are hundreds of ways to win.
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