Abstract
Mesenchymal stem cells from the synovium (synovial MSCs) are attractive for cartilage and meniscus regeneration therapy. We developed a software program that can distinguish individual colonies and automatically count the cell number per colony using time-lapse images. In this study, we investigated the usefulness of the software and analyzed colony formation in cultured synovial MSCs. Time-lapse image data were obtained for 14-day-expanded human synovial MSCs. The cell number per colony (for 145 colonies) was automatically counted from phase-contrast and nuclear-stained images. Colony growth curves from day 1 to day 14 (for 140 colonies) were classified using cluster analysis. Correlation analysis of the distribution of the cell number per colony at 14 days versus that number at 1–14 days revealed a correlation at 7 and 14 days. We obtained accurate cell number counts from phase-contrast images. Individual colony growth curves were classified into three main groups and subgroups. Our image analysis software has the potential to improve the evaluation of cell proliferation and to facilitate successful clinical applications using MSCs.
Highlights
Mesenchymal stem cells from the synovium are attractive for cartilage and meniscus regeneration therapy
Among the many Mesenchymal stem cells (MSCs) types, primary synovial MSCs are useful for cartilage regeneration in clinical situations[1] because synovial MSCs can be cultured from synovium and have a high chondrogenic potential[2]
Our software converted phase-contrast images to black and white images (Fig. 2A), followed by counts of the cell number per colony. The accuracy of this method was verified by comparison with cell counts obtained using synovial MSCs stained with 4′,6-diamidino-2-phenylindole (DAPI; Fig. 2A) on days 4 and 14
Summary
Mesenchymal stem cells from the synovium (synovial MSCs) are attractive for cartilage and meniscus regeneration therapy. We investigated the usefulness of the software and analyzed colony formation in cultured synovial MSCs. Time-lapse image data were obtained for 14-day-expanded human synovial MSCs. The cell number per colony (for 145 colonies) was automatically counted from phase-contrast and nuclear-stained images. Phase- contrast images have a critical problem of noise arising from the presence of red blood cells or tissue debris[4] We have addressed this limitation by the development of novel image analysis software that can distinguish living cells from other debris. Our second purpose was to analyze individual colony growth curves of synovial MSCs using our novel image analysis software. Our novel image analysis software enabled us to determine the distribution of cell numbers per colony based on time-lapse images taken during the culture period. Our third purpose was to investigate whether analysis of the cell number per colony distribution could determine which day of culture would best predict the cell yields at 14 days
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