Preservation of Viable Cells in the Undercooled State

Previous studies into the mechanisms governing the freezing of cells in the absence of extracellular ice have been extended to develop a method for the preservation of viable cells in the undercooled state. Deep undercooling of cells is achieved by suspending fine droplets of the cells in oil to make an emulsion, thus minimizing initiation of extracellular ice nucleation. Attempts to preserve yeast cells, cultured sainfoin cells, and dissected shoot-tips (pea and potato) in this way are described. The main findings are that yeast cells can be preserved undercooled at -20 degrees C for at least 16 weeks with no detectable loss of viability, showing that -20 degrees C is a low enough temperature for inhibition of significant biochemical deterioration and that the emulsions are stable over long periods. In preliminary experiments, sainfoin cells survived 24 hr at -10 degrees C, and shoot-tips survived 48 hr at -10 degrees C. Sainfoin cells, conditioned by growth in medium supplemented with sorbitol, showed enhanced survival after exposure to low temperatures and a lower intracellular freezing point than control cells. Possible reasons for this are discussed.