Development of process analytical technology to monitor the cell secretome during differentiation of pluripotent stem cells

Background & Aim Induced pluripotent stem cells (iPSC) offers a renewable source for reliably and robustly generating off-the-shelf cell products for regenerative medicine. A number of differentiation protocols have been developed and PSC-derived cells have been used in a variety of animal models to treat disease and progressed through to clinical trials in Asia and more recently the United States. However, the manufacturing processes for expansion and differentiation of iPSC cells remain reliant on poorly scalable technologies with limited in process monitoring despite typically > 14 days of culture and manipulation required; increasing the risks and costs of batch manufacturing failure. The industrialisation of pluripotent stem cell-derived cell therapies requires the development of process analytical technologies (PAT) for the monitoring and control of large-scale cell differentiation bioprocesses. The extracellular microenvironment provides fertile hunting ground to determine the impact of processing conditions on process outcome with the myriad of molecules consumed and produced by cells during differentiation alluding to cell state and potential cell fate. PAT that targets the spent media enables control of critical processing parameters to maintain product critical quality attributes without requiring cell-based analysis to be conducted. Methods, Results & Conclusion Using two exemplar differentiation processes, going from induced pluripotent stem cells to, either, definitive endoderm or cardiomyocytes, we have characterised the cell secretome with respect to metabolites and secreted proteins, and performed multi-parametric analysis to determine potential biomarkers for monitoring future large-scale differentiation processes using cost- and time-effective PAT alternatives.