No abstract

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 hybridthresholding 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. V C 2015 International Society for Advancement of CytometryKey terms Key terms: stem cells; spheroid formation; cell tracking; dynamic behavior STEM cells can differentiate into diverse specialized cells; this mechanism allows the human body to be repaired and replenished. They have become crucial in modern regenerative medicine, where cells are grown in vitro and transplanted into the human body to help self-recovery (1-3). To maintain the self-renewal ability of stem cells is a great challenge during cell culture, because stem cells tend to lose their stemness-the potential for self-renewal and multilineage differentiation propertiesowing to an insufficient microenvironment. The maintenance of self-renewal potential in adult human mesenchymal stem cells (MSCs) by a biomaterial substrate was first reported in Huang et al. (4). This study showed that when MSCs were cultured on chitosan (CS) membranes and those modified by hyaluronan (CS-HA) they could aggregate to form 3D cellular spheroids. Moreover, these 3D spheroids were found to be more strongly associated with the upregulated expression of stemness marker genes than MSCs in 2D culture. Because of their rich biological content and superior ability to mimic the in vivo environment than 2D cell culture, such multi-cellular

To grow stem cells in vitro is an important task in regenerative medicine. Cell motility that can be derived by cell tracking is a useful index to evaluate the viability and stemness of stem cells. The precision of cell tracking is highly dependent on correct detection of the cell centroids, which are usually determined by cell segmentation process. In this study, a cell segmentation method combining doublethresholding and disk-based reconstruction is proposed to solve the problems arising from shape deformation and uneven illumination during cell culture. The qualitative and quantitative comparisons show that the proposed segmentation method increases accuracy in detection the cell centroid.

We successfully engineered the CDM network labeled with fluorescent markers highlighting fibronectin -a protein of the extracellular matrix, and we also observed the dynamics of key components driving cell migration, such as the actin cytoskeleton and focal contacts. Our results reveal key differences between 2D and 3D cell migration. (i) We report new types of protrusions distinct from filopodia/lamellipodia reported on planar surfaces, which are driven by pressure. (ii) Our 3D network is deformed reversibly during migration and this allows the extraction of forces locally applied by cells. We correlate these local forces to the focal contacts dynamics, and our measures indicate a local pulling mechanism for forward cell motion and nucleus translocation. (iii) During migration, the nucleus local deformation by the cytoskeleton is needed to facilitate motion. These three phenomenapressure-driven protrusions, local forces correlated to local focal contacts, and nucleus deformation driven by the cytoskeleton -are reproduced in microchannels matching cell dimensions. Altogether, our results show that mechanical confinement of cell and nucleus is the main cause for differences between 3D and 2D motions.