Challenge Theme R&D spotlight: Development of an end-to-end closed iPSC expansion process

In this interview, Juline Guenat, Associate Lead Scientist at the Cell and Gene Therapy Catapult, highlights one of our Challenge Theme research projects, demonstrating a scalable, intensified and automated expansion process to generate high quality induced pluripotent stem cells (iPSCs).

1. Please provide an overview of the work presented in this poster.

This poster presents the development of a closed, scalable, semi-automated and cost-effective upstream process for the expansion of pluripotent stem cells (PSCs) in high-density, aggregate based culture in stirred tank reactors (STR).

2. What are the difficulties involved with developing scalable, closed and controlled pluripotent stem cell (PSCs) expansion processes?

PSC-derived therapies have the potential to serve as “off-the-shelf” allogeneic treatments for a wide range of chronic indications, such as cardiovascular and neurodegenerative diseases. These therapies target large patient populations and require high doses of ≥109 cells making them unsuitable for manufacturing with traditional 2D culture-ware.

These 2D systems rely heavily on manual, open, and poorly scalable processing with limited scope for process monitoring. All of this contributes to high manufacturing risk and cost of batch failure. The commercial success of allogeneic PSC-derived therapies relies on the design and development of controlled, robust, and cost-efficient processing platforms for large-scale manufacture.

3. How did you overcome these challenges in this research?

Production of PSCs at scale requires a seed train to generate the cell numbers needed to seed a bioreactor. This step is typically open, manual, labour intensive and at high risk for microbial contamination and concomitant batch failure. We show this process step can be closed and automated effectively using a hollow fibre bioreactor system, able to produce up to 2 × 109 cells. Cells harvested from this system were seeded into a STR as single cells, then expanded using periodic settling to allow for automated medium exchange.

An effective 3D-aggregate expansion was achieved with a 12-fold cell expansion over six days, with retention of pluripotency markers and trilineage differentiation potential. However, we found periodic settling reduces the ability to control aggregate size - a critical parameter that impacts expansion and differentiation.

To overcome this, we investigated an acoustic cell retention system to enable STR process intensification with controlled and automated medium exchange. We show cell retention ability, and increased control on aggregate size, which consistently yields a 20-fold cell expansion over four days while maintaining a pluripotent phenotype. Finally, integrated technologies were used for PSC concentration and wash to allow automated passages into STR, which allowed continued PSC expansion with similar growth characteristics at earlier growth cycles. We also demonstrate the ability of the resulting aggregates to spontaneously differentiate into the three germ layers: endoderm, mesoderm and ectoderm.

4. Improving the scalability and control of iPSC development processes is one of our key Challenge Themes - how does this project help us address this challenge?

This project looked to adapt traditional 2D iPSC cell culture to more innovative 3D suspension. Therefore, it offers the advantages conferred by using a STR. Cell therapy manufacturing is a complex field that requires skilled operators with open and manual processes; introducing closed automated systems would reduce labour, increase standardisation and efficiency. STR are also more compatible with process analytical technologies (PAT) which facilitate greater control through increased process understanding and process feedback. STR provides the possibility to scale-up, as opposed to scale out, which can help further reduce the footprint and closed processing allows a lower grade of clean room manufacturing.

5. What have been the positive outcomes of this project for the advanced therapies industry?

We were able to demonstrate for the first time a scalable, intensified, and automated expansion process to generate high quality iPSCs for two key industry barriers:

  • Cell bank generation
  • Producing large quantities of high-quality starting material for downstream differentiation

6. What are your plans to build on the achievements of this project to continue increasing the effectiveness of iPSC expansion processes

Our experienced team of biologists and bioengineers intend to build upon the learnings and knowledge gained during this project to establish a fully controlled system, integrating advanced analytical technologies such as PAT to enable real time monitoring and feedback control.

Click here to collaborate with us to improve scalability and control of allogeneic cell therapies