Using A Fixed Bed Reactor System For Expansion Of Human Embryonic Stem Cells

The following content was originally published by Bioprocess Online on October 18, 2022.

Emerging as a revolutionary treatment for a wide range of unmet medical needs, cell therapies are a rapidly growing area of the pharmaceutical industry that offers many opportunities to those companies pursuing them. However, many of the candidates in this space rely on adherent platforms for production, which present several limitations when it comes to scale-up capacity. Overcoming these challenges became a key focus for scientists at regenerative medicine company ViaCyte Inc., as they explored new technologies that would help enable expanded scales of culturing human embryonic stem cells (hESCs) for the development of cell replacement therapies as a functional cure for Type 1 diabetes. Using the Corning^® Ascent^™ Fixed Bed Reactor (FBR) system, ViaCyte was able to take advantage of the benefits of adherent cell culture platforms with the scale and automation capabilities traditionally seen in suspension manufacturing systems.

Diabetes mellitus is a debilitating disease characterized by elevated blood glucose levels because of insufficient production of insulin from pancreatic beta cells. While the discovery of insulin as a therapeutic treatment occurred 100 years ago, exogenous insulin administration remains a primary form of treatment for patients with Type 1 diabetes, an autoimmune condition affecting nearly two million Americans.¹ There is currently no known way to cure or prevent Type 1 diabetes.  The economic costs attributable to diabetes in the United States are significant with an estimated $237 billion for associated medical care and an additional $90 million related to time lost from work.² Disease management requires constant monitoring of blood glucose levels and frequent painful insulin injections or use of an insulin pump enable patients to survive.  However, neither can replicate the precise control of blood glucose levels via insulin secretion from beta cells and the disease can cause numerous health complications.

ViaCyte has been exploring alternative treatment options for those suffering from this debilitating disease, including the ability to reduce or even eliminate insulin injections altogether through the use of stem-cell-derived islet replacement therapy. As detailed on the company’s website, ViaCyte scientists were the first to describe directed differentiation of human pluripotent stem cells into insulin-expressing pancreatic cells and the first to demonstrate the ability of these cells to faithfully secrete insulin in response to increased blood glucose in pre-clinical models. ViaCyte has shown that these cells differentiate in vivo, in both preclinical and clinical studies, to all the constituent cell types of the human pancreatic islet.

Hannah Rasby, senior process development engineer at ViaCyte, explains that treatment using cell therapy has shown incredible promise, but a challenge of islet cell replacement therapy is producing enough for the patient population in need of this revolutionary product. “The field has seen success with beta cell infusion therapy using human islets from cadaver pancreases, but it is difficult to get enough viable cells and often more than one cadaver is needed. Those cadavers need to not only match but also appear at a similar time,” she explains. “So ViaCyte has developed a process to produce PEC [pancreatic endoderm cells] from stem cells .  The PEC are put into an immunoencapsulation device and implanted subcutaneously. These cells continue to differentiate once they’re implanted and are then able to generate insulin in response to glucose in the blood, as a pancreatic islet would.” While stem cells are significantly more readily available than donor organs, meeting the needs of millions of patients still has production challenges.

Pluripotent stem cells like hESCs are generally grown using an adherent substrate to preserve native biological function. Planar or 2D platforms, such as stacked vessels, have proven to be reliable for growing cells, but they are limited in scalability; producing a large volume of cells requires the use of multiple vessels in parallel. Not only does this labor-intensive task limit efficiency for any laboratory, but it also involves multiple rounds of manipulation for processing, increasing variability as well as the risk of contamination. This type of adherent cell culture system also limits the ability to monitor the condition of cells or control the environment within the layers of the vessel.

The alternative to adherent cell culture is a 3D suspension-based system, which may better facilitate scalability and offer more control during processing. However, depending on the cell line and process, moving from adherent to 3D can present significant challenges and may extend development and introduce risks as engineers must adapt the process to a new form of cell culture. Seeking a solution that could potentially address the limitations of these systems, ViaCyte scientists evaluated several technologies using a checklist of critical factors that would ultimately pave the way toward testing the Corning Ascent FBR system.

As Rasby explains, ViaCyte is focused on cutting-edge solutions that can help reach its goals of developing new cell therapy options for patients with diabetes. Rasby describes the three basic questions the team used to evaluate a scale-up technology, “’Will the cells grow [in this system]?’ ‘Are we able to monitor them as they grow?’ And, ‘Will we be able to get the cells back?’” she explains. “One of our team members identified the Corning Ascent at a very early stage and recognized its potential in scaling up adherent culture, including its ability to grow with our company’s needs, from the surface area of its smallest unit of one meter squared to the largest of one thousand meters squared, which is the equivalent of more than 1,500 10-layer stacked vessels. So, rather than having an army of these smaller plastic vessels, you can have a singular unit that is a completely closed system. The difference was night and day.”

Corning’s Ascent FBR system is an automated, closed-system bioproduction platform with inline monitoring and lot traceability for cGMP production needs. The bioreactor vessel is designed for optimal fluid dynamics, providing efficient nutrient delivery and waste removal. Its specially treated, woven, polymer mesh substrate is also designed to maximize cell growth throughout the bioreactor bed with consistent media exposure. The woven nature of the mesh enables uniform fluid flow and cell distribution, which helps to enhance cell health and productivity and achieve high yields of cells and cell-based products. Depending on the desired application observed, viable cell harvest can achieve greater than 90% yield.

The Ascent FBR platform is designed to provide linear scalability with multiple size options from process development to production scale. It is a closed system that is run and monitored outside of a laminar flow hood, while allowing easy aseptic sampling from the top of the bioreactor within a hood for cell counting during initial process development. Disposable inline sensors monitor key process parameters, such as pH, dissolved oxygen, and temperature, and a sampling port enables additional sampling of media for offline analysis of glucose and other analytics.  The ability to harvest viable cells from the Ascent FBR enables its use in other applications, such as cell therapy workflow, or other biologic production as well as seed train from small to large Ascent bioreactors.

While other scale-up technologies are available, the Ascent FBR system was the only adherent culture solution ViaCyte identified that provided both scalability and the cell harvest capability ViaCyte required. “There is very rarely a golden solution that is 100% perfect in every aspect, so knowing what is out there and understanding that in the context of our cells, and our process allowed us to be able to compare and contrast to see what fits best and what is most compatible with our cells,” says Rasby. “That is what made the Ascent FBR so attractive.”

Adding to this was the cooperation and collaboration between the teams at ViaCyte and Corning that began at the earliest stages, helping the process move forward very quickly. “That was exhibited throughout the entirety of our evaluation with them,” she adds. “Their representatives were on the ground helping to train us on the equipment and guide us through its use. This was especially critical because these were the first human embryonic stem cells to be grown in the system, which meant we had to figure out how to adapt the system to our process. Having that constant support from Corning personnel made a huge difference and allowed us to make a lot of progress in a short amount of time.”

Using the Ascent FBR system, ViaCyte conducted single passage runs where the cells were grown in the Ascent FBR system and then removed and evaluated. The team also executed a longer run where the same cells were seeded, expanded, harvested, and then put back into the reactor again multiple times, which provided insight into whether cell growth kinetics were affected by the Ascent culture and harvest conditions. The results of these runs showed the Corning Ascent FBR system demonstrated great promise as a potential avenue for scale-up of hESCs.

Specifically, the cells were evenly distributed throughout all mesh disks in the bioreactor chamber. Crystal violet staining at the end of cell expansion qualitatively showed evenly distributed cell presence across the bottom, middle, and top layers of the reactor (Figure 1).

The cells were able to grow and expand in the Ascent FBR system comparable to those in a traditional 2D culture vessel with less than 1% difference in average viability post-harvest between the Ascent FBR system and the Corning CellSTACK^®   vessel (2D control) and less than 1% difference in cell density post-harvest as compared to control cells cultured in a CellSTACK vessel (there was no significant difference between the two methods for viability or expansion kinetics). And finally, the cells were able to maintain pluripotency and genomic stability during culture and passage/harvest. After three passages, 99.6% of cells were triple positive for OCT4+, NANOG+, SOX2+, indicating pluripotency. Karyotyping showed normal chromosomal composition following each of three passages. This is a short time point but shows promise for more extensive testing.

Overall, the Corning Ascent FBR system is a high-density, fixed bed bioreactor platform that can offer a promising solution for stem cell scale-up while also achieving low-cost production and faster timelines. These advantages will serve as key benefits in an increasingly competitive market where current and future innovators explore the possibilities of these treatments and the dramatic impact they can potentially have on the future of patient care.

1. Banting, F. G., Best, C. H., Collip, J. B., Campbell, W. R. & Fletcher, A. A. Pancreatic extracts in the treatment of diabetes mellitus. Can Med Assoc J. 1922;12, 141‐146.
2. American Diabetes Association. Economic costs of diabetes in the U.S. in 2017. Diabetes Care. 2018, March. https://diabetesjournals.org/care/article/41/5/917/36518/Economic-Costs-of-Diabetes-in-the-U-S-in-2017