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  • Due to the limited proliferative capability of CMs large

    2018-11-08

    Due to the limited proliferative capability of CMs, large scale production of CMs is based on the expansion of pluripotent hESC followed by a CM differentiation step (Chen et al., 2014). Scaling up of hESC expansion in monolayer (MNL) culture platform is problematic mainly due to surface area limitation. One of the approaches to overcome this hurdle is the use of suspended 3D microcarriers (MC) which can provide large surface area per culture volume (Oh et al., 2009; Chen et al., 2011). Oh et al. demonstrated that hESC can be expanded to densities of 3.5×106cells/mL (18 fold expansion) in MC spinner flask culture while retaining hESC pluripotency, ability to differentiate into the 3 germ layers, and normal karyotypes (Oh et al., 2009). In comparison, parallel hESC expansion on MNL platforms achieved densities of 0.8×106cells/mL (4 fold expansion) (Oh et al., 2009). Induction of CM differentiation can be achieved by using small molecules (Lian et al., 2013; Graichen et al., 2008; Minami et al., 2012) or growth factors (Sa and McCloskey, 2012; Kattman et al., 2011). RSVA 405 High differentiation efficiencies can be achieved by both methods, however, the small molecule approach provides a distinct advantage in terms of cost and reproducibility and thus has the ability to be amenable to GMP standards (Kirouac and Zandstra, 2008). One example of a highly efficient small molecule CM differentiation protocol is reported by Lian et al. (2012, 2013). In this protocol, CM differentiation is initiated by WNT pathway activation at day using CHIR99021 (CHIR) or 6-bromoindirubin-3′-oxime (BIO) and is followed by its repression at day 3 using inhibitors, IWP-2 or IWP-4. Differentiation efficiencies of up to 98% cardiac troponin-T positive RSVA 405 were reported for MNL cultures. CM differentiation in 2D MNL cultures can achieve high differentiation efficiency but is limited in scalability (Rowley et al., 2012). In comparison, protocols using 3D suspended embryoid body (EB) cultures achieve lower differentiation efficiencies (Chen et al., 2014) but have the ability to be scaled up volumetrically (Niebruegge et al., 2008). Generating EB structures is problematic though, since it requires extensive cell handling as pluripotent cells need to be dissociated and re-aggregated to the appropriate size, in order to create efficient EBs for further differentiation. This causes difficulties in maintaining reproducibility and cell viability, thus limiting its ability to be developed into an efficient and robust bioprocess (Placzek et al., 2009). In the present study, we attempt to integrate MC suspension based pluripotent hESC expansion followed by CM differentiation using the WNT modulation protocol (Lian et al., 2012) as a continuous process in order to increase the efficiency of CM differentiation. Hydrodynamic shear stress applied during culture agitation was found to drastically affect CM yields. Expansion of HES-3 in the vigorously agitated MC spinner culture resulted in 38.1% reduction of CM yield in the following differentiation step, as compared to static control MC cultures. While hESC expanded in the gentler rocker platform achieved an increase of 32.1% CM yield. Moreover, agitation applied during the first 3days of the differentiation process was also observed to have an inhibitory effect on CM generation. Thus, an efficient CM production platform was developed in which pluripotent cells were expanded in continuously agitated rocker culture followed by differentiation during which intermittent rocking was applied only on days 1–3 followed by continuous agitation until day 12. This MC based platform provides 18.89±2.82 folds of cell expansion and CM differentiation efficiencies of 31.75±9.74 CM/hESC having 65.73±10.73% and 59.73±9.17% expression levels for cTnT and MHC respectively. In total, this platform was capable of producing 190.5±58.8×106 CMs per run. The robustness of the integrated MC rocker platform was further demonstrated with an additional human induced pluripotent stem cell (hiPSC) line, achieving 19.56±0.44 CM/hiPSC. The CMs obtained from this process were examined for structural and functional properties through immunohistology and exposure to inotropic substance, Isoproterenol, respectively. The new method offers a simple means for the scalable production of CMs in large quantities through the integrated bioprocess of cell propagation and differentiation.