EFFECT OF COOLING RATE AND OF DIMETHYL-SULFOXIDE CONCENTRATION ON ULTRASTRUCTURE OF NEONATAL RAT-HEART CELLS AFTER FREEZING AND THAWING

The ultrastructure of neonatal rat heart cells in suspension and in tissue culture after freezing at optimal, suboptimal and supraoptimal cooling rates with 2.5, 5, 7.5 and 10% DMSO [dimethylsulfoxide] was investigated. The effect of DMSO treatment only on the structure of the cells was also studied. A comparison was made with the survival in culture. Without freezing, increasing DMSO concentrations caused an increase of morphological damage, correlating with a decrease of the survival in culture. With 2.5% DMSO there was no difference with untreated cells. At higher DMSO concentrations, the ultrastructural damage increased from spaces between cell membrane and cytoplasm at 5% DMSO to interrupted cell membranes, swollen or destroyed mitochondria, and nuclei with clumped chromatin at 10% DMSO. After freezing at optimal or nonoptimal cooling rates with 5 or 7.5% DMSO, the ultrastructure correlated well with the survival. After freezing with 2.5 or 10% DMSO at optimal or nonoptimal cooling rates, differences in survival were found which were not reflected in the ultrastructure of the cell. After 8 days of culturing, cells which were frozen at all the different cooling rates and DMSO concentrations appeared to have a normal structure.