Achieving High Yield, Versatility, and Scalability for Adherent Cell Culture with Circulation/Perfusion Systems | Corning CellCube | Corning

BILODEAU: Circulation/perfusion systems are a means to achieve adherent culture at scale with minimal variability and, ultimately, less labor versus scaling out static vessels. Circulation of conditioned medium allows for efficient gas and nutrient delivery and removal of metabolites, even when you have dense or compact growth surfaces. You can get higher cell yield and/or higher productivity versus just static culture with efficient media usage. In addition, there are the advantages of closed systems with process monitoring and control.

BILODEAU: That depends on the specific system and a couple of other things. One: whether the cell type that you’re using can proliferate well on the substrate with dynamic medium flow. Two: whether you want to harvest the cells or not. Addressing the first point, different dynamic platforms have different growth surface materials that may require some optimization or adaptation, as well as troubleshooting to determine a suitable circulation rate in that system. By contrast, let me reference the Corning CellCube system. The culture surfaces of the CellCube modules are tissue culture treated polystyrene, which means your standard T-flask culture seeding will translate well to the CellCube surface. This is especially true for cells that are typically grown in roller bottles because a single plate of the CellCube module is the same surface area of an 850 cm² roller bottle. Also, the CellCube modules are available in Corning CellBIND^® surface for enhanced detachment if your cells need that and are more loosely adherent.

Back to that second consideration for applications: the harvest of the cells can be an obstacle in some of these different dynamic culture platforms. Those specific systems would be better suited for harvesting the cell products—like extracellular vesicles or lentivirus—that are released into the circulating medium. There are other systems, like the Corning^® Ascent^® Fixed Bed Bioreactor system and the Corning CellCube system, where you can just as easily harvest the cells as you can the circulating medium. This means they work well for anything from stem-cell culture to viral-vector production for cell and gene therapy applications, and even vaccine production, as is the case with the CellCube system.

BILODEAU: Generically, you’ll have the cell-growth vessel and the medium-conditioning vessel, which are connected by tubing loops, or they could be one and the same, and a peristaltic pump to circulate medium throughout the system. Possibly, there are one or more peristaltic pumps for additional medium or solution feeds. For example, if you have a bicarbonate feed to control the pH. You also need a controller for process monitoring and control. There are also compressed gas feed lines for medium gassing. Finally—and this is an important one—there are sensors or probes for the different parameters that you want to monitor, like temperature, dissolved oxygen, and pH. Those could either be inline and/or they could be in the medium conditioning vessel.

BILODEAU: The most common controlled parameters are temperature, dissolved oxygen [DO], pH, and the circulation/perfusion rate to maintain sufficient oxygen and nutrient delivery to the cells during the expansion.

BILODEAU: At least during process development, satellite vessels that can be viewed under the microscope are often seeded in parallel as a surrogate for the cell growth. We do this routinely in the lab. For the Corning CellCube system, we use a handheld USB camera so we can view the outermost growth surface layers to directly monitor the cell growth because there isn’t always a one-to-one correlation with static vessels. The handheld microscope plugs into a smartphone; it’s inexpensive, easy to use, and has sufficient magnification to monitor the cell confluence and cell morphology during expansion. It works well for any large bioprocess vessel that’s difficult to get to the microscope. Some of the circulation systems on the market, the Corning Ascent FBR included, require sampling of a growth substrate to monitor the cell growth directly. However, visualization of the cells is only one means to monitor cell expansion. When you have well-characterized cultures, the parameters that are monitored during cell expansion—such as dissolved oxygen, glucose utilization, or lactate accumulation—become the surrogate to monitor how the cells are proliferating. They can be very predictive of the cell growth.

BILODEAU: As I mentioned earlier, there’s good translation from flat-stock cultureware to the CellCube because it’s the same growth surface material— treated polystyrene. There will be some necessary troubleshooting and optimization. The better you know your cells and the more you’ve characterized your cultures, the smoother that transition is going to be.

Specifically, it’s important to understand the timing of cell attachment and cell spreading in static culture before you move into the CellCube. It’s also helpful to understand the components of your medium and how they might change during cell growth. For instance, are the cells using a lot of glucose so that you can use glucose consumption as an indicator of expansion? Typically, we recommend starting with a CellCube 10- or 25-layer module, seeding with the standard cell density and medium that you use for maintenance cultures for your cell line. You plan for a typical expansion period and then start with middle-of-the-road medium conditioning. That would be a pH of 7.35 to 7.4 and 50% dissolved oxygen unless you have data to support different parameters. Monitor nutrient metabolite concentration offline regularly during the cell expansion. Harvest with your preferred association reagent and see what your yield is. Then, you would adjust the seeding, medium volume, medium-conditioning parameters to reach your desired yield per cm² on that small scale.

BILODEAU: Once you have determined your medium conditioning and all other process parameters, you’ve worked them out and dialed them in on a small scale. It’s linear to scale up from the CellCube 10 [10-Layer Module] to the CellCube 25 [25-Layer Module] to the CellCube 100 [100-Layer Module], as long as the volume that you use in that conditioning bioreactor has been scaled accordingly. For even larger yields, the CellCube 100 can be scaled out using our clean-room cart, which will hold four CellCube 100 modules. In that case, the four vessels can be fed from a single large bioreactor using a single four-head peristaltic pump to drive parallel circulation through all four of those CellCube modules.