Many emerging cell and gene therapies are aimed at using cell replacement to regenerate damaged or diseased tissues. Pluripotent stem cells (PSCs) have the potential to generate any cell type in the body and can be considered the raw or input material for cell replacement therapies. In this two-part series of posts, we will look at the steps involved in large-scale manufacturing of PSCs.
What Kinds of PSCs Are Used in Cell and Gene Therapies?
Often the term PSC is used as a catch-all to refer to any cell type that is in an undifferentiated state. From a scientific standpoint, it is important to draw a distinction between PSCs and tissue-specific multipotent stem cells, like mesenchymal stem cells, which have more restricted lineage potential. For the purposes of this discussion, the term PSC will encompass embryonic stem cells and induced pluripotent stem cells.
Four Major Steps for PSC Scale-Up by Establishing a Seed Train:
Cell culture scale-up, regardless of the cell type, is based on the concept of a seed train. The goal of the seed train is to generate increasing cell numbers, in a step-wise process, to seed a larger production-scale bioreactor. Beginning with the end in mind simply means that to design an efficient seed train you must know the size of your final production bioreactor, in addition to any bottlenecks in your downstream process that might limit your maximum batch size.
- Adaptation from planar culture to suspension culture: According to standard laboratory technique, PSCs are most commonly grown in adherent culture (e.g. six-well plate format). The first step in the seed train is transferring a planar cell culture into a suspension-type culture (e.g. shake or spinner flasks) and determining if that cell line will grow naturally in suspension as aggregates or if manipulations/adaptations are required to accomplish growth in suspension.
- Early adaptation to small-scale suspension culture: PSCs can be scaled-up in suspension in different formats (e.g. stirred-tank or rocking bioreactors) depending on the cell type and what type of equipment is available. The key, at this step, is progressing cells through larger culture volumes ranging from 50 mL to 1 L. At this stage, optimization of culture conditions is performed to establish cell-specific parameters for oxygenation, mixing speed, pH and feeding rates.
- Scaled-down production model: Once the final bioreactor scale has been chosen, PSCs can be grown in a scaled-down format to optimize growth conditions in that specific type of bioreactor. This is a critical troubleshooting step that will help to avoid production failure at large scale. The scaled-down model is also useful for future process improvements, allowing new conditions to be tested at smaller scale before implementing them at the full production scale. Altogether, employing a scaled-down production model is beneficial for reducing process risk.
- Inoculation of production bioreactor: The size of the production bioreactor will depend on the final cell numbers required in the differentiation to final cell product. Production bioreactors can range in size from <1 to 2000 L, depending on whether a production process provides for an autologous or allogeneic therapeutic product.
Stay tuned to the CDMO Education Centre for the second post in this series. In it, we will look at each of these steps in more detail and help you to understand key considerations for PSC scale-up.