Regulation of spontaneous activity and growth of embryonic chick heart cells in tissue culture

Abstract 1. 1. The percentage of embryonic heart cells that exhibit spontaneous activity (% BC) in tissue culture is dependent upon a wide variety of environmental parameters, including mode of tissue disaggregation, composition of the culture medium, density of cell inoculum, and other culture conditions, as well as the age of the embryo from which the cells are taken. 2. 2. Dissociating 7-day hearts with three 8-minute incubation cycles in 0.05% trypsin liberates a maximal number of viable cells; best results are obtained when enough tissue is used for dissociation to bring the resultant cell suspension to 2 to 3 × 106 cells/ml or more. 3. 3. To be able to determine % BC, clls must be plated at densities low enough to prevent extensive intercellular contacts. At densities above about 100 cells/mm2 many cells make contact with neighbors, making it impossible to distinguish whether a cell is beating because of its own electrogenic activity or as a response to an impulse conducted from a pacemaking neighbor. 4. 4. The serum used in a culture medium influences, to a marked degree, the percentage of cells which beat spontaneously. A total of 56 serum samples were tested, yielding a range of effects: from those sera which were clearly toxic to those which gave “optimal” results. A sample of serum was considered optimal if cells in standard low-potassium medium, made with that serum, were healthy in appearance, and consistently yielded 40–50% BC. 5. 5. At adult serum levels of K+ (4–5 meq/l) approximately 20% of the cells in a culture from 7-day heart beat spontaneously. Increasing extracellular K+ causes some cells to cease activity. Decreasing the K+ to 1 meq/l permits a maximal number of latent pacemakers to become active. Dose-response curves relating K+ concentration to % BC have been determined with cells from embryonic hearts aged 4, 7, 12, and 18 days. 6. 6. The greatest number of spontaneously active cells is obtained from 7-day hearts. Under optimal conditions, with 1 meq/l K+ medium, activity ranged between 40 and 50% BC, with a mean of 44% BC. The greatest number of active cells ever recorded in a single plate was 64% BC. Older hearts yielded progressively fewer pacemakers, to a minimum mean of about 10% BC at 18 days. Hearts earlier than 7 days also yielded fewer spontaneously active cells. 7. 7. Four-day heart cultures are relatively insensitive to elevated K+ in the culture medium. From this it is suggested that the high intracellular Na+-content and rapid Na+-flux characteristic of early embryonic hearts represent devices whereby early pacemaker cells can maintain a ratio of sodium: potassium conductance sufficiently large to permit the onset of spontaneous activity in the high-potassium environment provided by the egg before amnion formation. 8. 8. Two types of heart cells may be distinguished morphologically in cultures: F cells, which have the appearance of typical fibroblasts; and M cells, which resemble myoblasts of skeletal muscle. F cells comprise 40–50% of the cells; M cells 50–60%. Most overt pacemakers are M cells. However, a small proportion (less than 1%) of these which meet all the morphological criteria of F cells, are also spontaneously active. 9. 9. Mitotic activity in heart cell cultures can be induced or prevented merely by the presence or absence of chick-embryo extract. Cultures can be obtained which exhibit both rapid mitosis and high % BC, by dialyzing CEE to remove K+. It is concluded that no necessary antagonism exists between growth and the maintenance of a differentiated functional state in embryonic heart cells cultured under the conditions described.