THE INFLUENCE OF COOLING RATE ON SURVIVAL OF FROZEN CELLS DIFFERS IN MONOLAYERS AND IN SUSPENSIONS

The influence of cooling rate on the survival of corneal keratocytes f rozen as attached monolayers or as isolated cells in suspension was in vestigated. Cells were cooled to -80 degrees C at rates ranging from 0 .2 to 10 degrees C/min in the presence of 10% (v/v) cryoprotectant (Me (2)SO, propane-1,2-diol or glycerol). Cell survival was judged, after rapid warming and removal of the cryoprotectants, by trypan blue stain ing. None of the treatments caused actual loss of cells from the monol ayers. After freezing and thawing, there was a very strong influence o f cooling rate on cell survival in monolayers. Survival was highest (> 80%) after cooling at 0.2 degrees C/min, but declined sharply to <40% after cooling at 1 degrees C/min. Conversely, isolated cells showed hi gh survival at 1 degrees C/min but lower survival at 0.2 degrees C/min : indeed cell survival after cooling at 0.2 degrees C/min was lower in suspensions than in monolayers.