Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor
Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor
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DOI：
10.1002/bit.26359
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发表时间：
2017-10-01
影响因子：
3.8
通讯作者：
Hewitt, Christopher J.
中科院分区：
文献类型：
--
作者：
Rafiq, Qasim A.;Hanga, Mariana P.;Hewitt, Christopher J.
微生物反应器在工艺开发中起着关键作用，因为它们减少了试剂需求，并可以促进工艺参数和培养条件的高通量筛选。在这里，我们已经证明并详细解释了，第一次，自动ambr 15细胞培养微生物反应器系统的可扩展的贴壁人间充质多能基质/干细胞（hMSC）微载体培养过程的发展。这是通过首先改善微载体的悬浮和混合，然后改善细胞附着从而减少初始生长滞后期来实现的。后者通过在前24小时仅使用50%的最终工作体积的培养基并使用间歇搅拌策略来实现。与原始工艺（24小时无搅拌，100%工作体积）相比，这些变化导致24小时后活细胞密度增加>150%。使用与ambr 15中相同的方法，在更大规模的转瓶研究中获得了类似的改进。最后，将这种基于血清培养基的改进生物工艺方法应用于ambr 15中的无血清工艺，与基于血清的工艺相比，产量增加>250%。在两种规模下，培养期间使用的搅拌是微载体悬浮液N-JS所需的最低搅拌。与转瓶相比，ambr 15的使用及其改进的对照将含血清培养基中活细胞密度的变异系数从7.65%降低至4.08%，并且转换为无血清培养基进一步将其分别降低至1.06- 0.54%。与基于血清的手动转瓶工艺相比，无血清和自动化处理的组合将重现性提高了10倍以上。本研究的结果表明，ambr 15微生物反应器是一种有效的工具，用于hMSC微载体培养物的生物工艺开发，无血清培养基，控制和自动化的组合提高了工艺产率和一致性。Biotechnol. Bioeng. 2017;114：2253-2266. (c)2017年Wiley Periodicals，Inc.
Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24h compared to the original process (no agitation for 24h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in >250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, N-JS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06-0.54%, respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253-2266. (c) 2017 Wiley Periodicals, Inc.