DISSOCIATION OF ADULT MAMMALIAN HEART INTO SINGLE CELL-SUSPENSION - ULTRASTRUCTURAL-STUDY

  • 1 January 1979
    • journal article
    • research article
    • Vol. 129  (OCT) , 541-559
Abstract
Adult rat heart was dissociated into single cell suspension by a perfusion technique, which included 0.05% collagenase and 0.1% hyaluronidase in Krebs-Ringer phosphate buffer (KRP). The non-muscle cells of the cell suspension were separated from the myocytes by centrifugation of the cell suspension through 3% Ficoll solution in KRP with 0.01 mM Ca2+; .apprx. 90% isolated single muscle cells were obtained with this method. The successive stages of dissociation and the effect of dissociation procedure on the ultrastructure of the heart were studied by scanning and transmission electron microscopy. After 30 min of enzyme digestion, the dissociation of the inner endothelial lining of the ventricle into single cells or small groups of cells became apparent. The disruptions of the underlying cardiac skeleton as well as linear arrays of cardiac muscle cells were observed. After 45 min of enzyme digestion, the number of released single cells was higher with the separation of intercalated discs. The majority of non-muscle cells were dissociated from the walls of muscle cells. The widening out of lateral intercellular spaces between the myocardial cells was followed by or concomitant to separation of desmosomes. The majority of myocardial cells became separated from one another after 60 min of enzyme digestion. The separation of gap junctional sites was observed. The separation of gap junctions could take place in 2 ways: by unzipping gap junctional complexes with enzyme treatments or by tearing junctional complexes with the shearing force of perfusion and mechanical agitation. The dissociation of the heart was ineffective when perfused with media containing 1.0 or 2 mM Ca2+. The internal structural organization of isolated single myocardial cells retained the characteristic features of its counterpart in situ.