High density continuous production of murine pluripotent cells in an acoustic perfused bioreactor at different oxygen concentrations
High density continuous production of murine pluripotent cells in an acoustic perfused bioreactor at different oxygen concentrations
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DOI：
10.1002/bit.24717
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发表时间：
2013-02-01
影响因子：
3.8
通讯作者：
Zandstra, Peter W.
中科院分区：
文献类型：
--
作者：
Baptista, Ricardo P.;Fluri, David A.;Zandstra, Peter W.
多能干细胞(PSC)的生产策略依赖于连续分离的贴壁或聚集培养，因此限制了细胞生产的强劲放大、在线控制和培养条件的优化。我们最近开发了一种方法，使连续(非连续分离的)悬浮培养介导的重编程达到多能性。在这里，我们使用这种方法来展示利用声学过滤技术实现连续氧气控制灌流培养的PSCs及其早期衍生物的可规模生产。在灌流速率为5.0d-1、比生长速率为0.61+-0.1d-1的条件下，培养7d后细胞密度可达1×107个/毫升以上。当降低介质溶解氧(5%DO)时，最大细胞密度增加了一倍(>2.5×107个/mL)。在延长的生产期内，细胞密度和存活率保持在80%，在此期间，多能性因子(SSEA-1和Nanog)的稳定表达以及分化为所有三个胚层的能力证实了多能性的维持。这项工作为在集成、可扩展和强化搅拌悬浮培养中生产多能细胞及其衍生物建立了一个通用的生物技术平台。生物技术。比昂斯。2013年；110%：648655。(C)2012年威利期刊公司。
Strategies for the production of pluripotent stem cells (PSCs) rely on serially dissociated adherent or aggregate-based culture, consequently limiting robust scale-up of cell production, on-line control and optimization of culture conditions. We recently developed a method that enables continuous (non-serially dissociated) suspension culture-mediated reprogramming to pluripotency. Herein, we use this method to demonstrate the scalable production of PSCs and early derivatives using acoustic filter technology to enable continuous oxygen-controlled perfusion culture. Cell densities of greater than 1?x?107?cells/mL were achieved after 7 days of expansion at a specific growth rate (mu) of 0.61?+/-?0.1?day-1 with a perfusion rate (D) of 5.0?day-1. A twofold increase in maximum cell density (to greater than 2.5?x?107?cells/mL) was achieved when the medium dissolved oxygen was reduced (5% DO). Cell densities and viabilities >80% were maintained for extended production periods during which maintenance of pluripotency was confirmed by stable expression of pluripotency factors (SSEA-1 and Nanog), as well as the capacity to differentiate into all three germ layers. This work establishes a versatile biotechnological platform for the production of pluripotent cells and derivatives in an integrated, scalable and intensified stirred suspension culture. Biotechnol. Bioeng. 2013; 110: 648655. (c) 2012 Wiley Periodicals, Inc.