Pre clinical studies suggest that doses

Pre-clinical studies suggest that doses of up to one billion CM will be required to achieve therapeutic benefit after transplantation (Chong et al., 2014; Laflamme and Murry, 2005). In order to meet the current CM demand for pre-clinical studies and the anticipated demand for foreseeable clinical studies, development of a robust, scalable and cGMP-compliant differentiation process for the production of both hPSCs and hPSC-derived CM is essential. Suspension cell culture is an attractive platform for large scale manufacture of cell products for its scale-up capacity. Application of a suspension culture platform to support hPSC growth in matrix-free cell SB202190 has been well established (Amit et al., 2010; Krawetz et al., 2010; Olmer et al., 2010; Singh et al., 2010; Steiner et al., 2010; Chen et al., 2012). We previously also reported the development of a defined, scalable and cGMP-compliant suspension system to culture hPSCs in the form of cell aggregates (Chen et al., 2012). With this suspension culture system, hPSC cultures can be serially passaged and consistently expanded. In the present study we adapted our suspension culture system to establish a robust, scalable and cGMP-compliant process for manufacturing CM. We were able to use hPSC aggregates generated in the suspension culture system directly to produce CM with high efficiency and yield in suspension with various scales of spinner flasks. We optimized various critical process parameters including: small molecule concentration, induction timing and agitation rates for differentiation cultures in spinner flasks with scales up to 1L. In this study, we integrated undifferentiated hPSC expansion and small molecule-induced cardiac differentiation into a scalable suspension culture system using spinner flasks, providing a streamlined and cGMP-compliant process for scale-up CM differentiation and production.
Materials and methods
Results
Discussion In this study we present a scalable suspension culture system to generate high purity and yields of CM from hPSCs in defined, matrix-free, serum-free and cGMP-compliant conditions. The cardiac differentiation process is a seamless extension of our hPSC suspension culture system, streamlining the manufacturing process from undifferentiated cell expansion to cardiac differentiation. The process can consistently produce CM with a purity of 96±3% and overall CM yields averaging 1.4±0.4×106 cells/mL with scale up to 1L spinner cultures. A fully suspension-based process avoids the scale limitations and extensive labor associated with adherent cultures. Suspension culture also eliminates the need for vessel coating for cell attachment. This reduces not only reagent costs, but also space and operators, further reducing production costs. These advantages render this manufacturing process more manageable, cost-effective, labor-effective, and practical for large-scale production of hPSC-derived CM. EB formation method in suspension has been commonly used for hPSC differentiation. However, low efficiency of EB formation from hPSC adherent cultures has hindered the practical application of this method for large scale production of differentiated cells. In addition, EB formation in static suspension is not a reproducible process, particularly in the control of EB size, which has been shown to affect differentiation potentials (Niebruegge et al., 2009; Bauwens et al., 2008). The suspension culture system we developed previously allows grows hPSCs in sphere-shaped aggregates with relatively homogeneous size (Chen et al., 2012). Use of these cell aggregates as a starting cell substrate for cardiac induction avoids the EBs formation step. Aggregate size in our suspension culture system is readily controlled by seeding density, agitation rate and time in culture. With these advantages, the hPSC aggregate suspension culture is an ideal system for the production of CM.