Eatman_2000_J.Pharmacol.Toxicol.Methods_44_533

Chick cardiomyocytes cultured in fetal bovine serum (FBS)-supplemented media are phenotypically unstable, becoming noncontractile and unresponsive to stimuli after several days. We report a culturing protocol that preserves the differentiated cardiomyocyte phenotype for at least 9 days in culture. Cardiomyocytes isolated from 11-day chicken embryos, and cultured in either Dulbecco's Modified Earle's Medium (DMEM)/Ham's F12 medium with N-2 supplement or Medium 199 (M199) with 10% FBS continued to beat spontaneously for 4-5 days; only cells cultured in N-2-supplemented medium exhibited spontaneous beating beyond 5 days. Immunostaining for alpha-actinin after 9 days in culture revealed that myofibrils persisted in N-2-supplemented cells, while no myofibrils were observed in the FBS-supplemented cells. For cells in FBS-supplemented media, [3H]thymidine incorporation rates were 7.5 and 3 times greater than that of cells in N-2-supplemented media at Days 4 and 9 in culture, respectively. The effect of growth media on the binding parameters of the muscarinic antagonist, [3H]N-methyl-scopolamine (NMS), was also compared. While B(max) decreased 34% between Days 4 and 9 for cells maintained in N-2-supplemented media, a 77% decrease was observed for cells cultured in FBS-supplemented media. The phenotypic stability of this preparation makes it feasible for the first time to use these cells in experiments that require more than 4 days to complete.