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

Jiang, Ching‐Fen; Hsu, Shan‐hui; Tsai, Ka-Pei; Tsai, Ming‐Hong

doi:10.1002/cyto.a.22657

Cited by 6 publications

(11 citation statements)

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“…The present study demonstrated that the spheroids maintained viability and stemness during the experimental period using polydimethylsiloxane-based concave micromolds. It has also been demonstrated in previous that the aggregation of adult human mesenchymal stem cells to produce three-dimensional cellular spheroids helped to maintain the expression of stemness marker genes in cells, which was reiterated by the present findings (4,6). A recent study has also indicated that rabbit corneal stromal-cell-derived spheroids positively expressed mesenchymal and stem-cell phenotypes, which were immunopositive for vimentin and cluster of differentiation 34 (a mesenchymal cell marker and a stem cell marker, respectively), as well as the mRNA expression of nestin and Nanog (a neural stem cell marker and a stem cell marker, respectively) (7).…”

Section: Discussionsupporting

confidence: 87%

“…Due to their rich biological content and superior ability to mimic the in vivo environment compared with two-dimensional cell cultures, such multi-cellular spheroids are currently receiving increased attention with regard to their applications and production (6). Typically, cells in a spheroid culture exhibit various properties that are distinct from monolayer cells (7).…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids

Lee¹,

Ko²,

Park³

2017

Experimental and Therapeutic Medicine

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Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×105 (group A) or 8×105 (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular deposits were observed following Alizarin Red S staining at days 14 and 21; oil globules were increased at day 18 when compared with day 6; and Alcian blue staining was more evident at day 18 when compared with day 6. Within the limits of this study, stem-cell spheroids from gingival cells maintained the stemness, viability, and differentiation potential during the experimental periods. This method may be applied for a promising strategy for stem-cell therapy.

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Section: Discussionsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids

Lee¹,

Ko²,

Park³

2017

Experimental and Therapeutic Medicine

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“…Tracking of the cell movement path was based on cell matching with minimum distance between two sequential frames. A high correspondence of segmentation using the proposed method with that achieved by manual drawing had been reported earlier with high tracking accuracy of the proposed method.…”

Section: Methodssupporting

confidence: 71%

“…The output of the analysis is an excel file with the path of the tracked cell with its label and the corresponding measured parameters. The package, written in MATLAB, is based on our previously developed segmentation and tracking framework for nonrigid cells in phase‐contrast microscopy . Here, we present an advancement of this framework to detect and track different cell patterns in the video, as well as to quantify the dynamic behavior during cell clustering.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, the highly mobile spheroids were usually associated with well‐maintained or up‐regulated expression of stemness marker genes ( Oct4 , Sox2 , and Nanog ) . In our latter study, a scheme of image processing techniques was developed to compare the dynamic behavioral difference between the individual cell and the 3D spheroids . Morphological and movement parameters were automatically measured to quantify the dynamic behavior of stem cells during in vitro cell culture.…”

mentioning

confidence: 99%

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Quantitative Bioimage Analysis of Passaging Effect on the Migratory Behavior of Human Mesenchymal Stem Cells During Spheroid Formation

et al. 2020

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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 Key terms migratory behavior; passaging effect; quantitative bioimage analysis; stem cell Stem cell therapy is an emerging treatment for neurodegenerative diseases, diabetes, and heart diseases. Stem cell isolation and in vitro culture are the primary steps before cell transplantation into the patient. The limited population of stem cells usually needs to be expanded, for a particular application, through extensive subcultivation. However, the successful candidate cells for transplantation into a patient must be capable of undergoing extensive subcultivation while maintaining their differentiation capabilities. Irrespective of the disease being treated, the quality of candidate cells obtained from serial subcultivation is the pivotal factor determining therapeutic effectiveness. To maintain the differentiation potential of cultured stem cells in vitro, various culture substrates have been developed to provide an optimal microenvironment for p...

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Need for winning combinations of modalities in cytometry of stem cells

Bacsó

2017

Cytometry Pt A

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

Cited by 6 publications

References 0 publications

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids

Quantitative Bioimage Analysis of Passaging Effect on the Migratory Behavior of Human Mesenchymal Stem Cells During Spheroid Formation

Need for winning combinations of modalities in cytometry of stem cells

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