Phenotypic stability of chick cardiomyocytes in serum-free media - Preservation of muscarinic receptor expression

Chick cardiomyocytes cultured in fetal bovine serum (FBS)-supplemented medi a are phenotypically unstable, becoming noncontractile and unresponsive to stimuli after several days. We report a culturing protocol that preserves t he differentiated cardiomyocyte phenotype for at least 9 days in culture. C ardiomyocytes isolated from Ii-day chicken embryos, and cultured in either Dulbecco's Modified Earle's Medium (DMEM)/Ham's F12 medium with N-2 supplem ent or Medium 199 (M199) with 10% FBS continued to beat spontaneously for 4 -5 days; only cells cultured in N-2-supplemented medium exhibited spontaneo us beating beyond 5 days. Immunostaining for alpha -actinin after 9 days in culture revealed that myofibrils persisted in N-2-supplemented cells, whil e no myofibrils were observed in the FBS-supplemented cells. For cells in F BS-supplemented media, [H-3]thymidine incorporation rates were 7.5 and 3 ti mes 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 parameter s of the muscarinic antagonist, [H-3]N-methyl-scopolamine (NMS), was also c ompared. While B-max decreased 34% between Days 4 and 9 for cells maintaine d in N-2-supplemented media, a 77% decrease was observed for cells cultured in FBS-supplemented media. The phenotypic stability of this preparation ma kes it feasible for the first time to use these cells in experiments that r equire more than 4 days to complete. (C) 2001 Elsevier Science Inc. All rig hts reserved.