This podcast is the third in a series of articles that was published in the eBook
“Cell Therapy Production”.
You can download all the articles in the series, by downloading the eBook.

Cellular therapies are therapeutics that utilize cells as the therapy. As the industry has grown, an increasing number of cell therapies have entered clinical trials, and some have achieved remarkable results. The potential of these therapeutics to treat diseases and conditions that were previously thought untreatable is inspiring. Several of the most promising candidates in the pipeline use mesenchymal stromal cells and pluripotent stem cells, both of which require an adherent substrate for native biological function. Thus, utilizing an adherent platform for production of these cell types provides several advantages, including shorter process development and optimization time, no need to adapt cells to suspension, and the ability to implement various surface modifications that promote the relevant biology.

When it comes to scale-up and commercial production, the industry typically thinks of suspension culture. This is based on the success of traditional protein-based therapeutic manufacturing. However, cell therapies are significantly more complex to manufacture and thus require more sophisticated manufacturing platforms. While suspension has traditionally been seen as the way to reach economies of scale, new adherent platform technologies such as stacked vessels, microcarriers, and fixed bed bioreactors, are making it possible for adherent cells to approach similar economies of scale while meeting the unique biological requirements of cell therapies.

We hear the term cellular therapy being used more and more frequently. Can you describe what a cellular therapy is and why it is an important therapeutic modality?

Austin Mogen
The broad term “cellular therapy” is used to describe a therapeutic modality that utilizes a cell as the therapy instead of something like a small molecule, protein or viral vector. In some ways, cells are like complex miniature machines that can be engineered by scientists to provide a therapeutic function such as killing tumor cells, reducing inflammation, or producing insulin, to name a few.

Ben Josey
Cellular therapies are important because of the impressive efficacy that some cell therapies have demonstrated in clinical trials. Diseases and conditions that were previously thought untreatable or incurable now have potential treatments and in some cases cures. Cell therapies harness the body’s own natural mechanisms by utilizing cells that are naturally found in your body. When these cells are partnered with scientific advancement, they can be programed to use your body’s natural processes to help treat and cure itself.

What kinds of cellular therapies would utilize an adherent platform?

Ben Josey
Some of the most promising front runners in the adherent cell therapy manufacturing space currently include mesenchymal stromal cells and pluripotent stem cells, both of which require an adherent substrate for native biological function. This would be in comparison to something like an immune cell that naturally exists in the blood and floats around in suspension. These stem cells can then be differentiated into a variety of other cell types (neurons for treating a brain condition, cardiomyocytes for heart diseases, islet cells for diabetes, etc.), many of which are also attachment dependent. So, utilizing an adherent platform in this context connects more closely to cells’ natural biological origin.

Can you layout for listeners the benefits of adherent vs. suspension cell cultures and when you might want to use each in cell therapy manufacturing?

Austin Mogen
To a large extent, cell culture platforms seek to mimic the native environment for whatever type of cell is being grown. For example, cells from solid tissues (like muscle) are more amenable to adherent culture, and cells from liquid tissues (like blood) are better suited for suspension culture. While it is possible to coax some cells to shift modalities, deviating from this basic premise creates challenges. It often requires extensive adaptation and sometimes genetic modification of adherent cell lines to make them amenable to suspension culture conditions. Suspension cultures have long had the advantage of being able to be grown in traditional, large-scale stirred tank bioreactors, which reduces the processing steps and lowers the facility footprint, but most stem cell-derived therapies require an adherent substrate. Newer technologies such as stacked vessels, microcarriers, and fixed bed bioreactors, are making it possible for adherent cells to approach similar economies of scale as suspension processes.

We hear a lot about the trend of transitioning from adherent platforms during research and trials to suspension when scaling up biological therapies to commercial production. Why do you think this is happening? To me, it seems that some cellular therapies are better kept in adherent production.

Ben Josey
This is mostly driven by economics and outdated facility designs. Another component is the lack of awareness about the advances in adherent platforms currently available. For traditional adherent platforms, such as T-flasks and roller bottles, the labor and space

required for scaling up isn’t feasible. While they work well for research and development, the number of flasks and bottles required for scale up to production levels makes them impractical. In addition, existing bioproduction technologies were developed with the transition from microbial fermentation to single component protein production. This process utilized large tanks to achieve economies of scale. Cell therapies are more complex and require a more complex and more adaptable or modular solution and the industry is moving towards this realization. Novel adherent technologies have been developed to try to address the need to balance economies of scale and the unique biological requirements of cell therapies.

Austin Mogen
Some of the developments in terms of technology that are making it possible to scale up the adherent cell culture workflows include microcarriers, as well as novel technologies in fixed bed bioreactor design. There has been an increase in the development of technologies that are pushing the boundaries of what we can do in terms of culturing adherent cells at large scale.

What is unique about adherent platforms for stem cell applications?

Austin Mogen
In the context of stem cell culture, one significant advantage that adherent platforms have is the ability to utilize various surface modifications. Biologically, most stem cells exist in contact- dependent niches, wherein their direct contact with other cells and extracellular matrices play major roles in the maintenance of their stemness. This is important to their ability to differentiate into different cell types, which is particularly relevant when we talk about the therapeutic value of these cells.

Anoikis is a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding extra-cellular matrix (ECM). It is a well-recognized issue in stem cell cultures. When using adherent platforms, it is possible to apply coatings that mimic the relevant ECM components. This is not possible in suspension and in some ways microcarriers bridge this gap. Microcarriers are small spherical beads that can be used in a suspension system like a bioreactor and they can be coated with relevant ECM proteins or synthetic mimetics that are helpful for culturing stem cells.

Ben Josey
Microcarriers do bridge this gap to some extent and they are a great solution for many situations. However, they also introduce their own set of challenges around process optimization. Cells that are coming from a traditional 2D vessel need to be adapted when moving into a larger format. Process development time is an important consideration, so a company must decide if microcarriers are a good solution or if one of the other advanced adherent platforms would be a better fit.

It seems to me there are many adherent cell culture platforms to choose from, can you describe some of them?

Ben Josey
In the context of stem cells, most people are familiar with 2D planar vessels like T-flasks. These are widely used in research, but quickly become limited at production scales. The next level up from T-flasks would be a stacked vessel like the Corning® CellSTACK®. These are essentially just big T-flasks that are connected and stacked in layers. The top tier in the stacked vessel evolution is the Corning HYPER technology, which uses propriety gas-permeable film technology to maximize the amount

of stacked growth surface area achievable in a given footprint. What all of these systems have in common is that the growth environment is static, meaning that the cells sit in a stationary media that is then exchanged at intervals.

Moving up in scale from 2D planar vessels are microcarriers, which are considered a 3D system utilizing small spherical beads paired with a suspension bioreactor. This is a hybrid adherent/ suspension system.

Lastly are the structured support bioreactors such as packed bed, hollow fiber, and fixed bed bioreactors, such as the new Corning Ascent FBR. These systems combine a fixed substrate with bioreactor technology, wherein you are able to grow adherent cells attached to a surface while in some fashion flowing fresh media past the cells. These systems add in some additional process optimization challenges, but they also provide huge benefits in the ability to monitor culture conditions, such as dissolved oxygen, pH, and metabolites. This can be very useful in optimizing a process and bringing it to production capacities.

What are the considerations for manufacturing cell therapies using some of the adherent systems described above?

Austin Mogen
There are advantages and disadvantages to each platform technology. For 2D planar stacked vessels, they have the advantage of a very low barrier to entry with low up-front investment costs and a high degree of process flexibility. Typically, these are single use vessels, so the primary costs are purchasing the vessel, incubator space, and clean room space. Thus, requiring minimal capital investment. The process development is minimized because the cells behave very similarly as they did in T-flask culture. This provides a simple transition from R&D to scale up. Take the Corning® HYPERStack®  vessel for example, cells typically perform the same in a T-flask as they do in a HYPERStack vessel with minimal process development required. It is a good choice when time to market and minimizing the process development time as much as possible are key considerations. Whether you use one stack or 60 stacks, your cells are consistently exposed to the same 0.22 mL of media per cm2 growth area and you can easily add or subtract modular HYPERStack vessels based on the needs of a given production run.

On the other hand, 2D stacked vessels are limited in their compatibility with inline process monitoring and the labor required to handle these at commercial scale can become significant. One alternative is the use of microcarriers, which offer the combination of maximized surface area combined with the process control of a bioreactor. However, these systems can be difficult to optimize, and process development can be much more extensive than for a 2D process. For cell therapy research applications, a dissolvable microcarrier such as Corning’s DMCs can provide substantial benefit for cell harvest. Another technology option that is becoming more popular is a structured support bioreactor, which offers a nice balance between 2D systems and 3D microcarriers. These bioreactors offer the high surface area to volumetric footprint of a microcarrier, thereby reducing space requirements and handling, as well as the process control of a bioreactor. For some applications, similar process development time to a 2D vessel system can be achieved. Historically, it has been difficult to effectively harvest cells from these systems, however, recent advances in technology are now making this possible. The new Corning Ascent™ FBR System is one example of a fixed bed reactor system designed to enable cell harvest.

What are the challenges of scaling up stem cells for clinical and commercial cell therapy manufacturing?

Ben Josey
There are many challenges facing companies in this space. The challenges are different depending on the various parts of the system. Across the board, there is a need to produce large numbers of cells (tens to hundreds of billions of cells) without altering the underlying biology of the cells that would impact the therapeutic effect. There is the need to accomplish this within a commercially viable cost of production. Many of these cells are not infinitely expandable. They differ from more traditional protein production cell line expansions, so achieving efficiency with doubling times and passage numbers becomes much more critical. Being able to achieve scale while maintaining biological function and relevance all within reasonable production costs is a big challenge that the industry faces.

Austin Mogen
Other challenges that are specific to scaling up stem cells include decreasing process variability and increasing the consistency of the bioproduction process. Cells are complex, composed of nucleic acids, proteins, and lipids; they provide quite a challenge in selecting the right production system. It is critical to have a robust production process so you can maintain consistency throughout that process both within the individual process and also between production lots. When you are producing a therapeutic you must ensure that what you produce in one lot is comparable to future lots. Automating some of the parts of the process to reduce the number of manual steps and opportunities for operator variability would greatly improve consistency. In addition, having the right analytics in place to properly measure the cellular characteristics or critical quality attributes permits insight into the process and provides parameters by which to measure whether the process is consistent throughout.

What recommendations do you have for scientists looking to scale up their stem cell culture?

Austin Mogen
In some situations, the best advice is to start at the beginning and take things one step at a time. This is not one of those situations. Start with the end goal in mind and be prepared to adapt. For example, it is important to define upfront the scale required to meet the necessary therapeutic dosage for that therapy. This can be applied to clinical trial and commercial scale-up operations and will help in choosing the scale-up platform and its attributes. Additionally, it is essential to define your critical quality attributes for the cellular therapy being developed and the methods of measuring these attributes. Maintaining these attributes throughout the scale-up process will ensure success. Ensuring that you have robust assays in place and can take accurate measurements throughout your process to maintain consistency is a good starting place.

How do you see the future of stem cell therapy manufacturing evolving and specifically adherent based manufacturing?

Ben Josey
It will probably end up going a couple different routes. Very interesting and specialized technologies are being developed to address situations that are just as unique as the cells being grown. In spite of all the technological advancements, current bioreactors are still primitive. In the body, these cells exist in a biologically relevant, anchorage-dependent context, within a constantly monitored and updated recirculation system. I see the future moving in this direction, with scalable modular bioreactor units that possess internal biocompatible scaffolding and are connected to mostly automated recirculation systems with adaptable online monitoring.

Austin Mogen
One of the future focuses of the cell therapy manufacturing space is automation and online monitoring. We are getting to a point where we have developed robust, future-looking technologies for scaling up and culturing these cells at large scale. However, many of these processes are still quite manual, and that can introduce variability into the process. So, automating many of the process steps, as well as online monitoring, will permit quantification of the cell characteristics online and in real time instead of taking days to generate that data. Lastly, the downstream process must be able to process a large amount of cells after they have been harvested from the cell culture scale up system. Work still needs to be done to increase the scale of downstream processing technology.

Do you have anything else that you would like to add for listeners?

Austin Mogen
It is a very exciting time in human medicine with advanced therapies being developed to overcome many previously untreatable diseases. The field is moving very quickly.

Research scientists and suppliers, like Corning, are moving as fast as they can to develop the best possible cellular engineering and bioprocessing tools to make the process of generating these therapies much smoother. I urge scientists to work with your supplier partners, such as Corning, and utilize their expertise to help develop and implement novel technologies into scale-up workflows.

Ben Josey
My favorite part of my job is working with scientists to try and help them meet their goals. I’d urge them to keep pushing the boundaries of science and please reach out to the Corning team and other suppliers to let them know about any trouble they are facing. By reaching out they will be able to see if suppliers have solutions or will work with them to overcome their challenges. There are so many exciting therapies in the pipeline and on the horizon that are just barely out of reach. If we all work on it together, we can push the industry forward and make them a reality.