A comparative study of collagenase- and trypsin-dissociated embryonic heart cells: reaggregation, electrophysiology, and pharmacology

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Abstract
The electrophysiological and pharmacological properties of aggregates prepared from cells of 7-day-old chick embryo heart ventricles depend on the enzyme used for cell dissociation. The mean beat rate of aggregates formed from trypsin-dissociated cells was about 53 beats/min whereas aggregates formed from collagenase-dissociated cells had a mean beat rate of more than twice this value. Spontaneous activity of most aggregates formed from trypsin-dissociated cells was inhibited by elevating external potassium or by adding tetrodotoxin to the medium. A similar response to potassium was seen in all aggregates formed from collagenase-dissociated cells. However, approximately half of the aggregates formed from collagenase-dissociated cells were tetrodotoxin insensitive. Intracellular microelectrode recordings demonstrated that aggregates formed from collagenase-dissociated cells typically had reduced action potential maximal upstroke velocities and depolarized threshold potentials in comparison to those recorded from aggregates formed from trypsin-dissociated cells. In the presence of tetrodotoxin the maximal upstroke velocity of aggregates formed from either collagenase- or trypsin-dissociated cells decreased markedly. In the case of the collagenase-treated cells, the spontaneous activity which persisted in the presence of tetrodotoxin was abolished by the slow channel blocker D-600. Computer simulation of membrane depolarization supports the view that aggregates formed from collagenase-treated cells have a reduced fast inward sodium current and a significant leakage current. Aggregates prepared from trypsin-dissociated cells display properties which more closely resemble those of intact 7-day embryonic ventricular tissue. We therefore conclude that, contrary to previous reports, collagenase is not the enzyme necessarily best suited for cell dissociation in all tissue culture studies.