Minimizing variability and maximizing quality in iPSC cultures: an interview with Nathan Frank

Nathan Frank, Senior Scientist at Terumo Blood and Cell Technologies (CO, USA), discusses the limitations of T-flask-based iPSC expansion and explores how automated systems like the Quantum Flex hollow-fiber perfusion bioreactor address these challenges. Nathan also highlights strategies for a successful transition to automated systems and shares insights on the future of iPSC manufacturing.

This interview is part of the RegMedNet In Focus on scaling iPSC cultures. Discover expert opinions on this topic by visiting our feature homepage.

What are the limitations of expanding iPSCs in T-flasks?

Expanding large numbers of iPSCs using flasks can involve a lot of time and effort on behalf of the operator as multiple flasks must be set up identically and tended individually. How this is accomplished can vary from procedure to procedure and even operator to operator, but manual handling is necessarily involved and this will increase process variability. The probability of contamination in the final product can also rise as each flask must undergo multiple open events during the expansion process. When the final product is pooled, a single problem with one flask can contaminate the entire final batch. In static culture, the cells’ microenvironment changes as the culture proceeds with many cytokines and growth factors being used up and waste products and other metabolites building up as the cells expand. This means that the cells will experience a highly variable microenvironment that can swing between extremes of nutrient availability, pH and exposure to waste products like lactate, all of which can negatively affect cell proliferation and cell quality and may lead to regions of spontaneous differentiation. Even though tissue culture-treated flasks can be used, a substrate of some kind is almost always used for iPSC culture and this is one more place where flasks can vary from one another as the culture is being initiated.

How does working in a hollow-fiber perfusion-based bioreactor overcome these limitations?

Automation can help eliminate a lot of process variability and automated hollow fiber-based culture doubly so. The high surface area of a hollow fiber bioreactor means that one bioreactor is equivalent to many flasks, depending on what scale is desired by the user. Perfusion-based cell feeding and gas exchange helps to keep the microenvironment consistent over time with the ability to increase the perfusion rate of the medium in tandem with the needs of the expanding cell population and fresh oxygen being provided through the semi-permeable hollow fiber membrane. Waste products like lactate and CO2 are removed simultaneously with the addition of fresh medium further stabilizing the culture milieu. Maintenance of a homogonous microenvironment reduces the probability of spontaneous differentiation and can increase the overall quality of the final cell product. The chance for contamination is also significantly reduced using hollow fiber bioreactors as one can manufacture an entire batch of iPSCs with only three open events during the entire culture; compare this to 5–10 open events per flask during manual culture and the risk reduction is clear.

What is the biggest challenge when transitioning from manual to automated iPSC expansion?

Frequently, transitioning from manual to automated expansion can require the operator to change how they monitor and interact with the cells. Small-scale, manual cell culture is an intimate experience. When working at a small scale, the culturist has time to scan each flask for appropriate colony morphology and other indicators of cell health. This will be fine if only a few plates or flasks are in play. However, as one requires larger batches of cells, these artisanal methods are no longer sufficient. Automated cell culture requires a cultivation of new methods for in-process monitoring and understanding the behavior of large populations of cells without going colony by colony under a microscope. In hollow fiber, we monitor glucose consumption and lactate generation rates over time. This gives a more holistic picture of the entire culture over time as it expands and, combined with quality metrics on the harvested cell population, a clear picture of the process can be derived.

What steps can be taken to minimize challenges?

Understanding some of the cell populations’ dynamics prior to moving into any bioreactor can be helpful. Knowing what doubling times are typical in your cell populations is helpful as it gives you context for how your cells should behave in a novel system. Understanding a bit about glucose consumption and lactate production in manual culture will help set the user up for understanding how to use those values when the switch to bioreactor culture is made. Finally, knowing that there is likely going to be some optimization once the transition from manual to automated cell culture is made; the systems are different and it is not surprising when things may not be perfectly aligned on the first try.

How does working in the Quantum Flex affect the seed train?

Culturing iPSCs in Quantum Flex can bring a cell population from 15M to 500M in under four days. This means that a single 500M-cell bank can be produced from that initial inoculum that will provide cells for up to 30 more similar runs that all make use of the same starting material. Those numbers are using Quantum Flex’s small bioreactor set, and the standard set has approximately 10x the surface area of the small, implying that a harvest could be in the 5–6 billion cell range if the population dynamics from the small bioreactor hold constant.

How do you ensure an even distribution of cells across the surfaces of the bioreactor?

Fluid handling in the Quantum Flex system is highly manipulatable relative to manual culture where shaking the vessel on the X and Y axes and swirling are the primary options for distributing cells. In this case, a novel cell loading and distribution protocol was developed for the small bioreactor to make sure that the entire seeding inoculum is introduced into the fibers and distributed both along the length of the fibers and on the ‘top’ and ‘bottom’ of each fiber. This is accomplished by using the appropriate chasing volume to move the cell population into the bioreactor fibers while minimizing the loss to the non-fiber portion of the fluid circuit, slowly moving that cell bolus back and forth along the length of the fibers, and repeating a similar process with the bioreactor oriented in both the right-side-up and upside-down positions so that the use of the interior lumen of the fiber is maximized. The fibers are precoated with the ECM protein or attachment substrate of choice to facilitate adherence and integrin stimulation. Finally, hollow fibers have the unique advantage of being able to use ultrafiltration force to drive the cells to the coated membrane to encourage attachment.

Maintaining pluripotency is crucial for iPSCs. How do you ensure that iPSCs retain their pluripotency throughout the expansion process?

By working to achieve as uniform of a distribution as possible, maintaining a consistent microenvironment without dipping glucose levels or spiking lactate levels too high, perfusion gas exchange, using short culture times at higher seeding densities as in manual culture, and through the use of high-quality reagents.

How do you see the role of Quantum Flex and similar technologies evolving in the large-scale production of iPSCs? What are the potential applications, and how might this impact the cost and risk of iPSC-based therapies?

I think the future is bright for hollow-fiber expansion of iPSCs as there is quite a bit of unexplored territory at this moment. These data were generated in the small bioreactor and moving up to the standard-sized bioreactor will clearly bring the same process consistency at a larger scale. Beyond this, there is the potential for iPSC differentiation inside of the fibers meaning that an iPSC population could potentially be expanded and differentiated within the same vessel, thus keeping the process highly consistent and with an extreme minimum of opportunities for contamination. The Quantum Flex hollow fiber bioreactor can contain and expand both adherent and suspension cell cultures and this can potentially be done consecutively or simultaneously allowing for the adherent-to-suspension transition that would be required for iPSC differentiation into suspension cell types like T cells or natural killer cells. Additionally, the possibility for 3D culture of iPSC in suspension aggregates also exists, which might help maintain cell quality even more effectively while also allowing for increased cell harvests as surface area limitations are transcended.

The opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of RegMedNet or Taylor & Francis Group.

This content was produced in association with Terumo Blood and Cell Technologies.