The quality of stem cells obtained through serial subcultivation is the pivotal factor determining the therapeutic effectiveness of regenerative medicine. However, an effective quality monitoring system for cell culture is yet to be established. Detailed parameter studies of the migratory behavior of stem cells at different passages may provide insight into the deterioration of stemness. Thus, this study aimed to evaluate the feasibility of quantitative bioimage analysis for monitoring stem cell quality during in vitro culture and to explore the passaging effects on stem cell migration. An image-based analytical tool using cell tracking, cytometric analyses, and gating with time-lapse microscopy was developed to characterize the migratory behavior of human mesenchymal stem cells (hMSCs) isolated from human adipose tissue (hADAS) and placenta (hPDMC) cultured on chitosan membranes. Quantitative analysis was performed for the single cells and assembled spheroids selected from 15 videos of Passages 3, 5, and 11 for hADAS and those from 12 videos of Passages 7, 11, and 16 for hPDMC. These passages were selected to represent the young, matured, and degenerated stem cells, respectively. Migratory behavior varied with cell passages. The mobility of single hMSCs decreased at degenerated passages. In addition, enhancement of mobility, due to transformation from single cells to spheroids, occurred at each passage. The young hMSCs seemed more likely to move as single cells rather than as aggregates. Once matured, they tended to aggregate with strong 3D spheroid formability and increased mobility. However, the spheroid formability and mobility decreased at late passage. The increase in aggregation rate with passaging may be a compensatory mechanism to enhance the declining mobility of hMSCs through cell coordination. Our findings regarding the passaging effects on stem-cell migratory behavior agree with biochemical reports, suggesting that the developed imaging method is capable of monitoring the cell-culture quality effectively. © 2020 International Society for Advancement of Cytometry.

中文翻译：

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球体形成过程中对人类间充质干细胞迁移行为的传代效应的定量生物图像分析。

通过连续传代获得的干细胞质量是决定再生医学治疗效果的关键因素。然而，尚未建立有效的细胞培养质量监测系统。对不同代干细胞迁移行为的详细参数研究可能有助于深入了解干细胞的退化。因此，本研究旨在评估定量生物图像分析在体外培养过程中监测干细胞质量的可行性，并探讨传代对干细胞迁移的影响。一种基于图像的分析工具，使用细胞跟踪、细胞计数分析、并开发了带延时显微镜的门控，以表征从壳聚糖膜上培养的人类脂肪组织 (hADAS) 和胎盘 (hPDMC) 中分离的人类间充质干细胞 (hMSCs) 的迁移行为。对从 hADAS 的第 3、5 和 11 段的 15 个视频以及 hPDMC 的第 7、11 和 16 段的 12 个视频中选择的单个细胞和组装球体进行了定量分析。选择这些代分别代表年轻、成熟和退化的干细胞。迁移行为随细胞传代而变化。单个 hMSC 的迁移率在退化传代时降低。此外，由于从单个细胞向球体的转化，每次传代都会发生流动性增强。年轻的 hMSC 似乎更可能作为单个细胞而不是聚集体移动。一旦成熟，它们往往会聚集在一起，具有强大的 3D 球体可成形性和更高的流动性。然而，球体的可成形性和流动性在后期通过时下降。随着传代聚集率的增加可能是一种补偿机制，通过细胞协调来增强 hMSC 的移动性下降。我们关于干细胞迁移行为的传代效应的研究结果与生化报告一致，表明开发的成像方法能够有效监测细胞培养质量。© 2020 国际细胞计量学促进会。随着传代聚集率的增加可能是一种补偿机制，通过细胞协调来增强 hMSC 的移动性下降。我们关于干细胞迁移行为的传代效应的研究结果与生化报告一致，表明开发的成像方法能够有效监测细胞培养质量。© 2020 国际细胞计量学促进会。随着传代聚集率的增加可能是一种补偿机制，通过细胞协调来增强 hMSC 的移动性下降。我们关于干细胞迁移行为的传代效应的研究结果与生化报告一致，表明开发的成像方法能够有效监测细胞培养质量。© 2020 国际细胞计量学促进会。