br Experimental Procedures br Acknowledgments

Experimental Procedures
Acknowledgments
Introduction Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold great promise in the fields of regenerative medicine and drug discovery. Although their practical usage requires large-scale cell culture, scaling up of conventional adherent cultures is extremely challenging, as uniform high quality, reproducibility, and low running and labor costs must all be achieved. Recently, hPSC suspension cultures (Amit et al., 2010; Chen et al., 2012; Olmer et al., 2010; Singh et al., 2010; Steiner et al., 2010) have attracted considerable attention. They can potentially be scaled up because attachment surfaces and adhesion molecules are unnecessary, resulting in reduced good-manufacturing-practice-grade components and production costs. However, the limitations of current suspension culture methods include suboptimal passaging procedures that require dissociation and reaggregation and uncontrollable spontaneous fusion between cell protease inhibitor (Amit et al., 2010; Olmer et al., 2010; Singh et al., 2010; Steiner et al., 2010). Enzyme treatments that dissociate hPSC colonies into single cells or small aggregates for subculturing induce considerable hPSC loss due to the sensitivity of these cells to physical stresses and single-cell dissociation (Singh et al., 2010; Steiner et al., 2010). Thus, enzymatic treatment may be the major reason for relatively low cell expansion ratios in suspension culture (Amit et al., 2010, 2011; Chen et al., 2012; O’Brien and Laslett, 2012; Olmer et al., 2010; Serra et al., 2012; Singh et al., 2010; Steiner et al., 2010; Zweigerdt et al., 2011). Another problem with suspension cultures is fusion between cell aggregates (Serra et al., 2012; Zweigerdt et al., 2011). Uncontrollable spontaneous fusion causes variation in sphere sizes; the formation of very large spheres may cause unwanted cell death and/or spontaneous differentiation (Bauwens et al., 2008). For the practical application of hPSCs in cell therapy or drug discovery, further refinements toward large-scale, 3D culturing systems are desired. Current versions of 3D culture systems for large-scale hPSC production include dynamic stirring of carrier beads or cell aggregates in spinner flasks or their equivalents (Abbasalizadeh et al., 2012; Amit et al., 2010, 2011; Chen et al., 2012; Krawetz et al., 2010; Olmer et al., 2010, 2012; Singh et al., 2010; Zweigerdt et al., 2011). Such stirring, however, needs to be fine-tuned to minimize detrimental shearing forces that cause significant physical damage to hPSCs (Abbasalizadeh et al., 2012; Amit et al., 2011; O’Brien and Laslett, 2012; Singh et al., 2010).
Results
Discussion Depending on the disease target or therapy type, a large number of cells may be required for clinical applications involving hPSCs. For example, whereas relatively small numbers of cells (around 105 or 106) will be required for treating macular dystrophy (Serra et al., 2012) or Parkinson’s disease (Lindvall et al., 2004; Serra et al., 2012), a much larger number of cells (between 109 and 1010) will be necessary for treating myocardial infarction, hepatic failure, or diabetes (Jing et al., 2008; Lock and Tzanakakis, 2007; Serra et al., 2012). This creates a need for a robust and reliable large-scale cell culture and production system for realization of cell-based therapy using hPSCs. Here, we provide proof-of-principle that a gas permeable culture bag can be used in the 3D culture system, obviating the need for stirring during gas and nutrient exchange. We succeeded to use a 200 ml culture bag with a thickness of 15 mm for cell production. Thus, it would be possible to use a 1 l medium capacity culture bag of the same thickness, with increased scale of production of 1 × 109 cells per 1 l bag. Moreover, our novel 3D sphere culture system may be utilized for other mass-production systems such as bioreactors after additional research and development of the necessary adaptation technology. In such potential new types of bioreactors, our 3D culture medium using the GG polymer would enable to use minimum dynamic agitation only for keeping sufficient exchange of gases and nutrients in a bioreactor but no strong stirring necessary for keeping the spheres in suspension. In addition, the low viscosity of the 3D culture medium would not disturb the monitoring of culture parameters such as pH and pO2. Thus, these advantages can greatly reduce cell damages caused by shear stress of dynamic agitation in application to the bioreactor systems.