Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single-use bioreactors
Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single-use bioreactors
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DOI：
10.1002/term.2435
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发表时间：
2018-02-01
影响因子：
3.3
通讯作者：
Edenhofer, Frank
中科院分区：
文献类型：
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作者：
Kwok, Chee Keong;Ueda, Yuichiro;Edenhofer, Frank
通过标准贴壁培养以与基于细胞的疗法和装载细胞的植入物相关的量生产人诱导多能干细胞（hiPSC）是难以实现的，并且缺乏工艺可扩展性。克服这些障碍的一种有希望的方法是悬浮培养hiPSC。在本研究中，研究了搅拌悬浮培养容器在作为单细胞悬浮液接种的两个hiPSC系扩增中的适用性，其体积在50和2400 ml之间具有自由可扩展性。本文报道的简单而稳健的两步工艺首先在125 ml转瓶中（100 ml体积）在7天内产生直径为324 +/-71 m的hiPSC聚集体。随后将其解离成单细胞悬液，用于接种在3000 ml生物反应器（1000 ml体积）中，最终在另外7天后产生198 +/-58 m的hiPSC聚集体。在转瓶和生物反应器中，hiPSC可以作为聚集体在悬浮液中培养超过40天，保持未分化状态，如通过多能性标志物TRA-1-60、TRA-1-81、SSEA-4、OCT 4和SOX 2的表达所证实的，可以分化成所有三个胚层的细胞，并且可以被引导分化成特定谱系，例如心肌细胞。在100 ml体积下，hiPSC量增加高达16倍，相当于每天增加2.28倍;在1000 ml规模下，额外增加10倍。总之，在14天内将16 x10（6）hiPSC扩增成2x 10（9）hiPSC，每天增加8.93倍。这一数量的hiPSC很容易满足基于细胞的疗法的要求，并使其临床潜力更接近实现。
The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324 +/- 71m diameter in 7days in 125ml spinner flasks (100ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000ml bioreactors (1000ml volume), finally yielding hiPSC aggregates of 198 +/- 58m after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000ml scale, an additional 10-fold increase was achieved. Taken together, 16x10(6) hiPSCs were expanded into 2x10(9) hiPSCs in 14days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.