Phenotypic stability of chick cardiomyocytes in serum-free media. Preservation of muscarinic receptor expression.
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.
Eatman
D
D
Department of Pharmacology and Toxicology, School of Medicine, Wright State University, Dayton, OH 45435, USA.
Arthur
T M
TM
Ahmed
S
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Grubbs
R D
RD
D000185:Actinin / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002642:Chick Embryo; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006352:Heart Ventricles / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D007150:Immunohistochemistry; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism*; D019832:N-Methylscopolamine / Q000378:metabolism / Q000494:pharmacology; D010641:Phenotype; D011976:Receptors, Muscarinic / Q000032:analysis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D013997:Time Factors; D016276:Ventricular Function
D016895:Culture Media, Serum-Free; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D000185:Actinin; D004247:DNA; D019832:N-Methylscopolamine