Reanimation of cultured mammalian myocardial cells during multiple cycles of trypsinization-freezing-thawing

Summary

Isolated newborn rat heart cells were cultured for several days, then subjected to a standard procedure of trypsinization, slow freezing in 10% dimethylsulfoxide, storage at −180° to −190°C for 1 to 3 days, rapid thawing, and recultivation. The same cells were recycled two more times in identical procedures. Morphological observations were made by phase-contrast optics and cinematography between each cycle and at the end of every experiment. After comparing the cellular morphology and contractile patterns of treated cells with control cultures, it was shown from the results of more than 15 experiments that most myocardial cells survived the standard procedures of trypsinization, freezing, and thawing and regained the ability to contract normally and form synchronized networks. Evidence was obtained which indicates that a cycle of the standard trypsinization-freezing-thawing procedure permits a recovery rate of 83 to 91% viable cells, with myocardial cells surviving to the same extent as endothelial cells. Of the cells which were nonviable, approximately half the deaths were a result of prior damage by trypsin and half were due to the freezing-thawing procedures. The same proportion of spontaneously contracting myocardial cells was observed after a cycle of trypsinization-freezing-thawing as before. Occasionally, there was a delay of 24 hr after thawing before myocardial cells began contracting spontaneously in vitro. An experiment using Viokase (in place of trypsin) and glycerol (in place of dimethylsulfoxide) excellent results after one cycle of freezing-thawing. It was concluded that myocardial cells exhibited a remarkable recovery from the toxic effects of trypsin and the traumatic influences of multiple freezing-thawing procedures. Endothelial cells in the cultures survived the same procedures and proliferated normally in vitro.