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Micromachines (Basel)
2019 Feb 25;102:. doi: 10.3390/mi10020156.
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Multinucleation of Incubated Cells and Their Morphological Differences Compared to Mononuclear Cells.
Sugita S
,
Munechika R
,
Nakamura M
.
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Some cells cultured in vitro have multiple nuclei. Since cultured cells are used in various fields of science, including tissue engineering, the nature of the multinucleated cells must be determined. However, multinucleated cells are not frequently observed. In this study, a method to efficiently obtain multinucleated cells was established and their morphological properties were investigated. Initially, we established conditions to quickly and easily generate multinucleated cells by seeding a Xenopus tadpoleepitheliumtissue-derived cell line (XTC-YF) on less and more hydrophilic dishes, and incubating the cultures with medium supplemented with or without Y-27632-a ROCK inhibitor-to reduce cell contractility. Notably, 88% of the cells cultured on a less hydrophilic dish in medium supplemented with Y-27632 became multinucleate 48 h after seeding, whereas less than 5% of cells cultured under other conditions exhibited this morphology. Some cells showed an odd number (three and five) of cell nuclei 72 h after seeding. Multinucleated cells displayed a significantly smaller nuclear area, larger cell area, and smaller nuclear circularity. As changes in the morphology of the cells correlated with their functions, the proposed method would help researchers understand the functions of multinucleated cells.
Figure 1. Typical images of droplets plated on (a) glass bottom dishes manufactured by Fine Plus International (FPI) and (b) Matsunami and (c) a plastic dish. Image contrast was enhanced for visibility. Bars correspond to 2 mm calibrated at the surface of the dish.
Figure 2. Typical images of XTC-YF cells seeded on (a,b) less hydrophilic (FPI) and (c,d) more hydrophilic (Matsunami) glass bottom dishes. Cells were incubated with (a,c) normal medium containing 100 µM Y-27632 and (b,d) normal medium alone. Multinucleate cells are shown in red and their nuclei are shown in blue. Scale bars represent 100 µm.
Figure 3. The percentage of multinucleated cells (Rmulti, as defined in Equation (5)) of XTC-YF cells after seeding on less (FPI) and more hydrophilic (Matsunami) dishes and culture in the presence or absence of Y-27632. n, Number of cells.
Figure 4. Typical time-lapse images of the XTC-YF cells incubated in medium containing 100 µM Y-27632 and seeded on a less hydrophilic glass bottom dish. Bars in (h) = 30 µm and are applicable to all images. The numbers in the upper left of the panels indicate the time after seeding.
Figure 5. The number of nuclei in XTC-YF cells incubated with medium containing 100 µM Y-27632 and seeded on less hydrophilic glass bottom dishes.
Figure 6. Morphology of multinucleated and mononuclear XTC-YF cells 48 h after seeding on the less hydrophilic glass bottom dish. Morphological data, such as (a) the nuclear area, (b) cell area, (c) circularity of the nucleus, and (d) circularity of the cell, are shown. n, Number of nuclei; *, p < 0.05.
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