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Fish cell lines are widely used for the studies of developmental biology, virology, biology of aging, and nutrition physiology. However, little is known about their physicochemical properties. Here, we report the phospholipid compositions and mechanical properties of cell membranes derived from freshwater, anadromous and marine fish species. Biophysical analyses revealed that fish cell lines have highly deformable cell membranes with significantly low membrane tensions and Young's moduli compared with those of mammalian cell lines. The induction of cellular senescence by DNA demethylation using 5-Aza-2'-deoxycytidine significantly reduced the deformability of fish cell membrane, but hydrogen peroxide-induced oxidative stress did not affect the deformability. Mass spectrometry analysis of phospholipids revealed that the level of phosphatidylethanolamine molecules containing polyunsaturated fatty acids significantly increased during the 5-Aza-2'-deoxycytidine-induced cellular senescence. Fish cell lines provide a useful model system for studying the changes in the physicochemical properties of cell membranes during cellular senescence. Abbreviations : 2D-TLC: two-dimensional thin layer chromatography; 5-Aza-dC: 5-Aza-2'-deoxycytidine; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; FBS: fetal bovine serum; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PS: phosphatidylserine; PUFA: polyunsaturated fatty acid; SA-β-gal: senescence-associated beta-galactosidase; SM: sphingomyelin.