Segmentation and tracking of stem cells in time lapse microscopy to quantify dynamic behavioral changes during spheroid formation.

Dynamic behavior of stem cells during in vitro development is diverse. Previous cell tracking studies have focused more on cell proliferation than on cell aggregation. However, the enhancement of cell proliferation in association with cell aggregation has been reported. In a previous study, we also demonstrated that the aggregation of adult human mesenchymal stem cells to form three-dimensional (3D) cellular spheroids helped maintain the expression of stemness marker genes in the cells. However, the dynamic behavioral changes triggered by spheroid formation remain to be investigated. A scheme of image processing techniques is proposed to meet this need. A hybrid-thresholding technique was first developed for efficient segmentation of cell clusters, after which a cell tracking method based on pair-matching with topological constraints was designed. Two morphological indices were derived to track the timing of 3D spheroid formation during the cellular aggregation process. Five cell motility indices measured from single cells and 3D spheroids were then compared. After confirmation of more than 90% correspondence between the results obtained by manual tracking and the proposed methods, an analysis of cellular behavior reveals a significant increase in motility in association with spheroid formation, consistent with a previous report that used a gene expression approach. This study proposed a systematic image analysis method to quantify the dynamic behavior of stem cells for stemness evaluation during cell culturing in vitro. Results demonstrated the validity of the developed platform in investigation of the dynamic behavior of cell aggregation in stem cell cultures in vitro.