The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor. Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions. Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 106 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process. A streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry.

中文翻译：

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在整合的悬浮微载体生物反应器中选择人诱导的多能干细胞系优化心肌细胞的分化。

大量心肌细胞的产生对于细胞疗法的需求至关重要。这项研究描述了在微载体搅拌釜反应器中干细胞扩增和分化的完全整合生物过程中，适合于生产心肌细胞的人诱导多能细胞（hiPSC）系的选择。首先评估了五个hiPSC品系在单层培养物中的心脏分化效率，然后评估了它们在连续搅拌条件下在微载体（MC）培养物中的扩增和分化相容性。三种细胞系具有很高的心源性，但是在连续搅拌的MC培养物中，只有一种（FR202）成功扩增。因此，在搅拌罐式生物反应器中连续搅拌下，在22天的集成生物过程中选择FR202用于心脏分化。总而言之，我们将基于MC的hiPSC扩增（第1阶段），CHIR99021诱导的心肌细胞分化步骤（第2阶段），使用基于乳酸的处理（第3阶段）和细胞恢复步骤（第4阶段）的纯化整合到一个流程中1个生物反应器，在受限的氧气控制下（<30％DO），并通过定期的间歇式介质交换进行连续搅拌。在扩增阶段可实现高密度的未分化hiPSC（2±0.4×106细胞/ mL）。通过控制生物反应器培养物中的搅拌速度和DO水平，在CHIR99021诱导的分化期中产生了具有> 80％肌钙蛋白T的7.36±1.2×106细胞/ mL心肌细胞。通过在无葡萄糖的纯化介质中添加乳酸，可以提高心肌细胞的纯度（> 90％的肌钙蛋白T），亚细胞G1期的增加和聚集体大小的减少表明细胞损失较小。最后，我们发现恢复期对于产生更纯净和功能性的心肌细胞（> 96％肌钙蛋白T）很重要。在300毫升工作体积中进行了三次独立运行，证实了该过程的稳定性。建立了一个简化且可控制的平台，用于在常规类型的搅拌罐生物反应器中的MC上大量生产纯功能性心房，心室和淋巴结心肌细胞，可以进一步扩大规模并将其转化为符合良好生产规范的生产工艺，以实现细胞治疗行业的数量要求。我们发现恢复期对于产生更纯净和功能性的心肌细胞（> 96％肌钙蛋白T）很重要。在300毫升工作体积中进行了三次独立运行，证实了该过程的稳定性。建立了一个简化且可控制的平台，用于在常规类型的搅拌罐生物反应器中的MC上大量生产纯功能性心房，心室和淋巴结心肌细胞，可以进一步扩大规模并将其转化为符合良好生产规范的生产工艺，以实现细胞治疗行业的数量要求。我们发现恢复期对于产生更纯净和功能性的心肌细胞（> 96％肌钙蛋白T）很重要。在300毫升工作体积中进行了三次独立运行，证实了该过程的稳定性。建立了一个简化且可控制的平台，用于在常规类型的搅拌罐生物反应器中的MC上大量生产纯功能性心房，心室和淋巴结心肌细胞，可以进一步扩大规模并将其转化为符合良好生产规范的生产工艺，以实现细胞治疗行业的数量要求。