Suspension Culture and Cardiomyogenic Differentiation of Human Pluripotent Stem Cells in Stirred Bioreactor Systems

Human pluripotent stem cells (hPSC) present an attractive source to generate large amounts of lineage specific progenies for innovative cell therapies, tissue engineering, in vitro disease modelling and drug screening assays. The therapeutic and industrial applications of hPSCs will require large cell quantities generated in defined conditions. We have recently established single cell inoculated suspension cultures of hPSCs (Zweigerdt et al., Nature Prot. 2011) which form aggregates in stirred tank reactors (Olmer et al., Tissue Eng. 2012) in the defined medium mTeSR^TM (STEMCELL Technologies). This work enabled the translation of conventional, adherence-dependent “2D” culture of hPSC to “3D” suspension culture. Since stirred tank bioreactors allow straightforward up scaling and comprehensive monitoring of process parameters these systems are widely used for the mass culture of conventional mammalian cell lines. Application of stirred reactors to hPSC culture, however, is in its infancy. Aiming at low medium consumption but integration of all probes relevant for process monitoring (pO₂, pH, biomass) a parallel “mini bioreactor system” consisting of individually controlled vessels (DASGIP / Eppendorf) was utilized. After establishing stirring-controlled aggregate formation up to 2x10⁸ hiPSCs were generated per run in 100 ml culture scale applying batch-feeding. Yet, only linear cell growth was achieved suggesting suboptimal process conditions. Here we will present how perfusion-feeding results in substantially improved process characteristics and hPS cell yields. Expanded cells were directly used for the efficient differentiation into cardiomyocytes in stirred tank reactors in a chemically defined medium resulting >80% cardiomyocyte purity without additional lineage enrichment. Technical modifications of the bioreactor system will be highlighted including: impeller design, online biomass sensor integration, establishing a cell retention system and utilization of disposable bioreactor vessel (BioBLU® 0.3) combined with the DASbox® culture control system.

Funding sources: REBIRTH Cluster of Excellence (DFG EXC62/3), BMBF (VDI grant no. 13N12606), BIOSCENT (FP7/2007-2013, grant no. 214539) and StemBANCC (Support from the Innovative Medicines Initiative joint undertaking under grant agreement n° 115439-2, resources of which are composed of financial contribution from the European Union (FP7/2007-2013) and EFPIA companies’ in kind contribution). STEMCELL Technologies (Vancouver, Canada), DASGIP / Eppendorf (Germany)