Time-course colony tracking analysis for evaluating induced pluripotent stem cell culture processes.

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.