VIABILITY AND MORPHOLOGY OF RAT-HEART CELLS AFTER FREEZING AND THAWING OF WHOLE HEART

Intact adult rat hearts were cooled in the presence of 10% DMSO [dimethyl sulfoxide] according to an external cooling program which approximated the optimal external 3-step cooling program for the isolated adult heart cells: 20 min at -20.degree. C, 0.2.degree. C/min from -20 to -25, -30 or -50.degree. C, and rapid cooling to -196.degree. C. Following rapid thawing, cells were isolated after perfusion with a 0.1% collagenase solution. Only cells which originated from the free wall of the right ventricle could be isolated, even after cooling to -20.degree. C. Most cells from hearts cooled to -196.degree. C did not survive. When the 3rd cooling step was omitted and the end temperature of the 2nd cooling step was -30.degree. C, 38% of the cells excluded trypan blue, 29% were morphologically intact, and 30% showed spontaneous contractions after thawing, expressed as percentages of the control. A much lower survival was found after cooling to -50.degree. C. Histological and EM study of the heart immediately after thawing revealed no differences between hearts cooled to -20, -30 or -196.degree. C. Also no marked differences were observed between the morphological integrity after freezing and thawing of the atrium, the left and right ventricle walls and the ventricular septum. The survival data suggested the presence of nonmorphologically detectable alterations in cells frozen to -196.degree. C, compared to cells frozen to -30.degree. C. The morphological investigations indicated no essential differences in resistance of atrial and ventricular cells to the freezing process. Experiments involving neonatal rat hearts cooled to -196.degree. C, according to the method which gave optimal preservation of the isolated cells, revealed that after thawing cells were present from which growing and contracting cultures can be derived. Cells in the neonatal rat heart are apparently more resistant to freezing to -196.degree. C than cells in the adult rat heart.