Elanzew Andreas, Nießing Bastian, Langendoerfer Daniel, Rippel Oliver, Piotrowski Tobias, Schenk Friedrich, Kulik Michael, Peitz Michael, Breitkreuz Yannik, Jung Sven, Wanek Paul, Stappert Laura, Schmitt Robert H, Haupt Simone, Zenke Martin, König Niels, Brüstle Oliver
Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, Germany.
LIFE&BRAIN GmbH, Cellomics Unit, Bonn, Germany.
Front Bioeng Biotechnol. 2020 Nov 9;8:580352. doi: 10.3389/fbioe.2020.580352. eCollection 2020.
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.
虽然人类诱导多能干细胞（hiPSC）为疾病建模提供了新的前景，但不同细胞系间存在的高表型变异性需要使用大量的hiPSC群体来解读个体基因变异的影响。因此，需要更高程度的并行化以及hiPSC生产的通量，而这只有通过实施细胞重编程和hiPSC扩增的自动化解决方案才能实现。在此，我们描述了干细胞工厂（StemCellFactory），这是一个自动化的模块化平台，涵盖了hiPSC生产的全过程，从成人成纤维细胞扩增、仙台病毒介导的重编程到hiPSC克隆的自动分离和平行扩增。我们开发了一种无饲养层、仙台病毒介导的重编程方案，适用于通过机器人液体处理单元进行细胞培养处理，该单元能以最先进的效率在3周内产生无足迹的hiPSC。不断生长的hiPSC集落会被自动检测、收获，并在24孔板中进行克隆繁殖。为确保高保真性能，我们配备了一台高速显微镜用于过程质量控制，并通过基于图像的汇合度测量来自动计算稀释比例。这种基于汇合度的传代方法能够在24孔板中对hiPSC进行平行和单独扩增，或在6孔板中进行放大培养，至少传代10次。自动扩增的hiPSC表现出正常的生长特性，并且在至少5周（10代）内持续表达多能性相关干细胞标志物TRA-1-60。我们的设置能够在完全确定的条件下对hiPSC进行自动化、无需用户干预的扩增，可用于在工业规模和质量水平上生成大量用于疾病建模和药物筛选的hiPSC系。
