Multinucleation of Incubated Cells and Their Morphological Differences Compared to Mononuclear Cells

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 tadpole epithelium tissue-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.