Viability and morphology of rat heart cells after freezing and thawing of the whole heart

Abstract Intact adult rat hearts were cooled in the presence of 10% DMSO according to an external cooling program which approximated the optimal external three-step cooling program for the isolated adult heart cells: 20 min at −20 °C, 0.2 °C/min from −20 to −25, −30, or −50 °C, and rapid cooling to −196 °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 °C. Most cells from hearts cooled to −196 °C did not survive. When the third cooling step was omitted and the end temperature of the second cooling step was −30 °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 °C. Histological and electron microscopical study of the heart immediately after thawing revealed no differences between hearts cooled to −20, −30, or −196 °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 suggest the presence of nonmorphologically detectable alterations in cells frozen to −196 °C, compared to cells frozen to −30 °C. The morphological investigations indicate no essential differences in resistance of atrial and ventricular cells to the freezing process. Experiments involving neonatal rat hearts cooled to −196 °C, according to the method which gave optimal preservation of the isolated cells, revealed that after thawing cells are present from which growing and contracting cultures can be derived. It appears that cells in the neonatal rat heart are more resistant to freezing to −196 °C than cells in the adult rat heart.