AJP - Heart and Circulatory Physiology, Vol 243, Issue 3 448-H455, Copyright © 1982 by American Physiological Society
Ca2+-tolerant adult canine myocytes: preparation and response to anoxia/acidosis
A. M. Spanier and W. B. Weglicki
A procedure is described for the production of large numbers of
Ca2+-tolerant adult canine ventricular myocytes. Gentle tissue dissociation
is achieved in Spinner flasks using 320 mosM enzyme buffer containing
collagenase hyaluronidase, and trypsin in the presence of millimolar levels
of Ca2+. The technique allows for the complete removal of erythrocytes, the
gentle removal of closely adhering nonmuscle cells (less than 3%
contamination), and the selective removal of damaged from nondamaged
myocytes. Total myocyte yields averaged 4-6 x 10(6)/g wet wt; 61.0 +/- 6.5%
of the cells have rod-shaped morphology and are "viable" based on trypan
blue exclusion. Only 3.9 +/- 1.1% of the rod-shaped cells beat
spontaneously when challenged with 2 mM Ca2+. Exposure to 2 mM Ca2+ at 37
degrees C results in minimal loss of viability (2.1 +/- 1.3%/h) based on
both trypan blue uptake and creatine kinase release. Simulation of cellular
(i.e., membrane) injury in vitro is performed by the separate application
of the perturbations of anoxia, acidosis, and low glucose to the canine
myocyte suspensions; the data suggest that these myocytes are affected
independently by these perturbations and are in good agreement with the
results obtained by others using the isolated rat myocyte system.
