In Part 1 of this series we covered some basic definitions and reviewed critical steps in the scale-up process for pluripotent stem cells (PSCs). In this post, we will take a deeper look at important considerations for process development and optimization of large-scale PSC culture.
Growing Adherent Cells in Suspension
PSCs typically grow in adherent, 2D culture. Since this format is not amenable to scale-up, it is desirable to transfer PSCs to suspension or 3D culture. This process is not trivial, requires considerable technical expertise, and will need to be customized depending on the type of PSC. There are multiple approaches to growing adherent cells in suspension, including encapsulation of PSCs in hydrogel, and growth of PSCs on microcarrier beads or hollow fibres. Each of these approaches has benefits and drawbacks; for instance, using microcarriers introduces an additional processing step to remove the microcarrier beads from the cell culture.
In response to this challenge, the team at CCRM has innovated around an alternative approach by which PSCs are coaxed into growing as multi-cell aggregates in suspension without the use of microcarriers.
How to Get Adherent Cells to Grow as Aggregates?
The ability of PSCs to grow in aggregates is dependent on the specific cell type. In other words, some types of PSCs are easily adaptable to this format, while others need some encouragement.
Different culture parameters can be optimized to promote aggregate growth. For example, agitation rate is a key parameter for the regulation of aggregate formation. Higher agitation rates increase shear force, making it harder for aggregates to form. Shear force can be modulated in a number of ways, including using different types of impellers if cells are being grown in stirred tank reactors. Agitation rates can also be tailored to the stage of the culture by making changes as early single-cell cultures progress into aggregate cultures.
Finally, different media compositions can be used to influence the number, size, and morphology of aggregates. It is important to note that all aggregate cultures benefit from the use of media containing Rho kinase inhibitor to reduce single-cell death and promote aggregate formation.
Analytics for Testing Key Cellular Attributes
Keeping close tabs on the purity, potency, and identity of PSCs is critical to ensuring a safe and effective final product. The focus is to determine if PSCs are differentiating and/or losing viability because of the culturing process.
Flow cytometry is an important method used to assay for markers of cell phenotype and pluripotency. Cells should be sampled at different stages of the culturing process to monitor the expression of markers of interest. In-process sampling for flow cytometry and functional assays introduces open and manual steps into the process.
The development of sensor technology is an area of rapid innovation that has the potential to allow for real-time evaluation of cell identity and viability. The most commonly-used sensors are immersed in the bioreactor to return information about pH and dissolved oxygen. While these readouts do not tell us directly about the identity or viability of the cells, they provide information about the overall health of the culture and could be used to create automated workflows that feed cultures based on real-time sensing.
Currently, work is underway across the industry to re-purpose sensors commonly used in well-established bio-processing applications for cell therapy workflows. A gap in the field is the lack of sensors that can effectively measure markers of cell identity currently measured by flow cytometry.
Requirements for Downstream Processing
To determine the appropriate starting volume for a culture, downstream processing steps must be considered.
For instance, when PSCs are grown in aggregates, a dissociation step is required during downstream processing. Hence, our team has developed a process solution that allows for isolation of aggregates from suspension culture and enzymatic digestion into single cell suspension. Generating a single cell suspension inevitably results in cell loss – something to consider at the beginning of the process.
Further, the end use of the cells, and the therapeutic dosing requirement, is a key consideration in determining an appropriate production volume. In the case of an allogeneic therapy, where many doses would be manufactured in a single batch, the final bioreactor volume could be up to 2,000 L. Accommodating a production bioreactor of this size would require careful planning at the seed train stage.
In summary, considering the technical and logistical challenges in scaling up manufacture of PSCs for differentiation to therapeutic cell types, working with a process development team with experience in this unique cell therapy application is highly recommended.
Contact us to find out how our services can help in the development of your PSC-derived cell therapy.