Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8602, Japan.
Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8602, Japan.
J Biosci Bioeng. 2019 Aug;128(2):209-217. doi: 10.1016/j.jbiosc.2019.01.011. Epub 2019 Feb 7.
Increasing the yield and maintaining a high quality of induced pluripotent stem cells (iPSCs) is necessary for manufacturing iPSCs at the industrial scale. However, because iPSCs are delicate, it is important to evaluate their quality during processing. To examine the status of cultured iPSCs non-invasively, morphology-based iPSC colony evaluation may be an efficient technology for cellular status monitoring and analysis. In this study, we examined the effectiveness of time-course colony tracking analysis for evaluating the iPSC culture process. Particularly, we obtained detailed time-course data to evaluate the effect of the pipetting technique on cell dissociation before seeding. Although the pipetting process causes severe shear stress to cells, which affects their quality, these effects have not been quantitatively analyzed because of their complex and uncontrollable parameters. By analyzing the heterogeneity and time-course responses of individual colonies, our colony tracking analysis revealed a critically damaged population caused by pipetting stress which could not be detected in conventional bulk analysis. Moreover, by comprehensively analyzing colony tracking data, which links the time-course morphology and marker staining results with each colony, we found that colony morphology is only highly correlated with the undifferentiated marker in the final stage, with a lower correlation in the early stages. Thus, colony tracking analysis provides a way to quantify cellular morphological information when evaluating complex iPSC manufacturing processes.
提高诱导多能干细胞（iPSCs）的产量和保持其高质量对于在工业规模上制造 iPSCs 是必要的。然而，由于 iPSCs 很脆弱，在处理过程中评估其质量非常重要。为了非侵入性地检查培养的 iPSCs 的状态，基于形态的 iPSC 集落评估可能是一种用于细胞状态监测和分析的有效技术。在这项研究中，我们研究了时间进程集落跟踪分析评估 iPSC 培养过程的有效性。特别是，我们获得了详细的时间进程数据，以评估在播种前细胞分离过程中的移液技术的效果。虽然移液过程会对细胞造成严重的剪切应力，从而影响细胞的质量，但由于其复杂和不可控的参数，这些影响尚未得到定量分析。通过分析单个集落的异质性和时间进程响应，我们的集落跟踪分析揭示了由移液应激引起的严重受损的细胞群体，这在常规的批量分析中无法检测到。此外，通过综合分析集落跟踪数据，将时间进程形态和标记染色结果与每个集落联系起来，我们发现集落形态仅与最终阶段的未分化标记高度相关，而在早期阶段相关性较低。因此，集落跟踪分析提供了一种在评估复杂的 iPSC 制造过程时量化细胞形态信息的方法。
