Isolation and low temperature preservation of adult rat heart cells

Adult rat heart cells were isolated by perfusion of the coronary system of the heart with a 0.05% collagenase solution.In one method (A), cells were finally isolated by shaking the heart fragments in a collagenase solution, after which the cells were washed and suspended in a Ca- and Mg-free buffered salt solution. The effect of different DMSO concentrations, 5, 10, 15, 20, 25, and 30% and the effect of the addition and dilution rate of DMSO on the number of trypan blue-excluding, intact, and contracting cells were studied. The highest DMSO concentration which was tolerated by the isolated adult heart cells was 15%. Variation of addition rate and the dilution rate of DMSO had no effect. After freezing at external cooling rates of 1, 5, 10, 30, and 50 °C/min to ?100 °C, and then rapidly to ?196 °C, in the presence of 5, 10, or 15% DMSO, reanimation of these cells was not achieved.In another method (B), heart fragments, after collagenase perfusion of the heart, were first treated with 5, 10, or 15% DMSO, after which the cells were isolated. If these cells were frozen at 1 °C/min with 10% DMSO, 15% of the cells, expressed as a percentage of the control, remained morphologically intact and 38% of the cells were contracting after thawing. Significantly higher survival percentages of 30 and 61%, respectively, were obtained if the heart fragments were left intact during freezing.