The StemCellFactory: A Modular System Integration for Automated Generation and Expansion of Human Induced Pluripotent Stem Cells.
The StemCellFactory: A Modular System Integration for Automated Generation and Expansion of Human Induced Pluripotent Stem Cells.
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DOI：
10.3389/fbioe.2020.580352
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发表时间：
2020
影响因子：
5.7
通讯作者：
Brüstle O
中科院分区：
文献类型：
--
作者：
Elanzew A;Nießing B;Langendoerfer D;Rippel O;Piotrowski T;Schenk F;Kulik M;Peitz M;Breitkreuz Y;Jung S;Wanek P;Stappert L;Schmitt RH;Haupt S;Zenke M;König N;Brüstle O
虽然人类诱导多能干细胞（hiPSC）为疾病建模提供了新的前景，但在不同细胞系中观察到的高表型变异性要求使用大型hiPSC队列来解读个体遗传变异的影响。因此，在hiPSC的生产中需要更高级别的并行化和通量，这只能通过实施用于细胞重编程和hiPSC扩增的自动化解决方案来实现。在这里，我们描述了StemCellFactory，这是一个自动化的模块化平台，涵盖了hiPSC生产的整个过程，从成人成纤维细胞扩增、基于仙台病毒的重编程到自动化分离，以及hiPSC克隆的平行扩增。我们开发了一种无饲养层的仙台病毒介导的重编程方案，适用于通过机器人液体处理装置进行细胞培养处理，该装置可在3周内以最先进的效率提供无足迹的hiPSC。在24孔板中自动检测、收获并克隆繁殖进化的hiPSC菌落。为了确保高保真性能，我们采用高速显微镜进行过程质量控制，并采用基于图像的汇合测量进行自动稀释率计算。这种基于汇合的分裂方法使得hiPSC能够在24孔板中平行和单独扩增，或者在6孔板中放大至少10代。自动扩增的hiPSC表现出正常的生长特征，并且在至少5周（10代）内显示出多能性相关干细胞标志物TRA-1-60的持续表达。我们的设置能够在完全定义的条件下自动化，用户独立的hiPSC扩增，并且可以用于产生大量的hiPSC系用于疾病建模，以及工业规模和质量的药物筛选。
While human induced pluripotent stem cells (hiPSCs) provide novel prospects for disease-modeling, the high phenotypic variability seen across different lines demands usage of large hiPSC cohorts to decipher the impact of individual genetic variants. Thus, a much higher grade of parallelization, and throughput in the production of hiPSCs is needed, which can only be achieved by implementing automated solutions for cell reprogramming, and hiPSC expansion. Here, we describe the StemCellFactory, an automated, modular platform covering the entire process of hiPSC production, ranging from adult human fibroblast expansion, Sendai virus-based reprogramming to automated isolation, and parallel expansion of hiPSC clones. We have developed a feeder-free, Sendai virus-mediated reprogramming protocol suitable for cell culture processing via a robotic liquid handling unit that delivers footprint-free hiPSCs within 3 weeks with state-of-the-art efficiencies. Evolving hiPSC colonies are automatically detected, harvested, and clonally propagated in 24-well plates. In order to ensure high fidelity performance, we have implemented a high-speed microscope for in-process quality control, and image-based confluence measurements for automated dilution ratio calculation. This confluence-based splitting approach enables parallel, and individual expansion of hiPSCs in 24-well plates or scale-up in 6-well plates across at least 10 passages. Automatically expanded hiPSCs exhibit normal growth characteristics, and show sustained expression of the pluripotency associated stem cell marker TRA-1-60 over at least 5 weeks (10 passages). Our set-up enables automated, user-independent expansion of hiPSCs under fully defined conditions, and could be exploited to generate a large number of hiPSC lines for disease modeling, and drug screening at industrial scale, and quality.
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影响因子：
64.8
作者：
Liu, Guang-Hui;Qu, Jing;Suzuki, Keiichiro;Nivet, Emmanuel;Li, Mo;Montserrat, Nuria;Yi, Fei;Xu, Xiuling;Ruiz, Sergio;Zhang, Weiqi;Wagner, Ulrich;Kim, Audrey;Ren, Bing;Li, Ying;Goebl, April;Kim, Jessica;Soligalla, Rupa Devi;Dubova, Ilir;Thompson, James;Yates, John, III;Esteban, Concepcion Rodriguez;Sancho-Martinez, Ignacio;Izpisua Belmonte, Juan Carlos
通讯作者：
Izpisua Belmonte, Juan Carlos
影响因子：
16.6
作者：
通讯作者：
--
影响因子：
14
作者：
Brafman, David A.;Chang, Chien W.;Fernandez, Antonio;Willert, Karl;Varghese, Shyni;Chien, Shu
通讯作者：
Chien, Shu
影响因子：
64.8
作者：
Kilpinen H;Goncalves A;Leha A;Afzal V;Alasoo K;Ashford S;Bala S;Bensaddek D;Casale FP;Culley OJ;Danecek P;Faulconbridge A;Harrison PW;Kathuria A;McCarthy D;McCarthy SA;Meleckyte R;Memari Y;Moens N;Soares F;Mann A;Streeter I;Agu CA;Alderton A;Nelson R;Harper S;Patel M;White A;Patel SR;Clarke L;Halai R;Kirton CM;Kolb-Kokocinski A;Beales P;Birney E;Danovi D;Lamond AI;Ouwehand WH;Vallier L;Watt FM;Durbin R;Stegle O;Gaffney DJ
通讯作者：
Gaffney DJ
影响因子：
48
作者：
Chen, Guokai;Gulbranson, Daniel R.;Hou, Zhonggang;Bolin, Jennifer M.;Ruotti, Victor;Probasco, Mitchell D.;Smuga-Otto, Kimberly;Howden, Sara E.;Diol, Nicole R.;Propson, Nicholas E.;Wagner, Ryan;Lee, Garrett O.;Antosiewicz-Bourget, Jessica;Teng, Joyce M. C.;Thomson, James A.
通讯作者：
Thomson, James A.