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March 04, 2021

A core regenerative medicine approach is cell replacement for the repair of damaged or diseased tissues. Transplantation of undifferentiated, pluripotent stem cells ( PSCs) would pose a significant safety risk due to the potential for unchecked cellular proliferation. Therefore, cell replacement therapies are based on the transplantation of cells that are no longer considered pluripotent and have progressed far enough along a differentiation pathway to be committed to a tissue-specific phenotype. For the purposes of this post, we will call the population of cells used for transplantation “differentiated cells. 

Determining the Optimal Cell Dose for Your Therapy  

For cell replacement therapy to be successful, you need a significant number of differentiated cells – sometimes upward of 1 billion cells per dose. Since differentiated cells have a limited proliferation capacity in comparison to PSCs, and some cells may not integrate into host tissue, high doses of cells are needed for cell replacement therapies 

It is worth noting that not all cell replacement therapies require large populations of differentiated cells. For instance, approaches for regeneration of the brain, eye and spinal cord require comparatively fewer differentiated cells for transplantation, -- somewhere in the tens of thousands of cells. The take home message here is that the target organ/tissue is an important factor in determining the number of cells needed 

How Our Experts Learned To Scale-Up  

The team at CCRM has tackled the challenges of manufacturing scale-up for PSC-derived cell therapies numerous times. We have built expertise working with cell therapies that require up to billion differentiated cells per dose and, in the process,  have identified key  considerations in the following areas 

  1. Determining how many PSCs are required: This is not straightforward and provides an opportunity for process development. First, we can estimate that the ratio of differentiated cells to PSCs is roughly 1:1. Therefore, to generate 1 billion differentiated cells, one would need approximately 1 billion PSCs. A huge step forward would be to optimize the process to maximize yield and generate many differentiated cells from a single PSC. This would require a deeper understanding of PSC biology and specialized molecular-genetic approaches that would need to be developed through basic R&D. Our team is tackling this problem from a process improvement standpoint by optimizing  upstream processing workflows to determine the best conditions for PSC scale-up.
  2. Switching from planarstatic PSC culture to aggregate culture supports scale-up: Our team has developed a unique process to accomplish this switch without the need for microcarriers or biological scaffolds. 
  3. Banking of PSCs: key advantage of first optimizing scale-up of PSCs is that large batches can be frozen to create a master cell bank. This highly-characterized population of PSCs will be used repeatedly to generate differentiated cellsthereby increasing the reliability and reproducibility of the process.  
  4. Determining critical quality attributes: The main goal for any process involving large-scale differentiation of PSCs is to create critical quality attributes, at multiple steps, to confirm that differentiation is proceeding properly. This approach helps us learn about the process and allows for the identification of manufacturing failures early.  
  5. The role of cell culture medium exchange and optimization in scale-up: A major cost driver for large-scale differentiation is the bioreactor feeding strategy. In static culture, a full medium exchange every day or every other day is standard, but not cost effective, because cells often do not require complete replenishment of all factors in the medium at such frequent intervals. The more we learn about our process, the more we can monitor cultures and feed based on need, eliminating waste and introducing cost savings. Further, custom media design, which can identify the minimal requirements to support cell growth, is a huge factor in reducing costs. Finally, developing a medium that better supports the transition from planar to aggregate culture would greatly improve this process. Our team has experience with media optimization for planar and aggregate culture and is well-equipped to provide insights into the best type of media for PSC scale-up.  

While our team has made incredible strides in manufacturing scale-up of differentiated cells, processes in this area remain largely manual and open due to the analytical techniques  needed to characterize differentiation at each stage of the process. To fully optimize, close and automate the differentiation process, sensors with the ability to measure the expression of differentiation markers in real-time would be an important advancement.  Overall, these process improvements would help to move PSC-derived cell therapies closer to the clinic.   

Contact us (cdmo@ccrm.ca) to find out how our services can help in the development of your PSC-derived cell therapy.

Cell image: Lilit Antonyan  

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