Sheila F. Mathias

Ice nucleation and freezing in undercooled cells

DSC has been employed to study the effect of cooling on a range of cells under exclusion of extracellular ice and in the absence of chemical cryoprotectants. In contrast to earlier reports, all the cells studied were found to freeze at temperatures above that indicated for homogeneous nucleation of ice in undercooled liquid water. In the case of human erythrocytes this temperature difference was only 0.5 degrees, but for yeast cells and cells of plant origin the difference amounted to congruent to 9 degrees. Nucleation of ice within the cell (or at the cell wall/membrane) must therefore be initiated by a heterogeneous mechanism. A kinetic analysis of the temperature dependence of nucleation shows the rates to be consistent with the dimensions of the plant cells (or organelles), if these were to be the active nucleators. However, the nucleation kinetics of human erythrocytes are extremely temperature sensitive, and the kinetic parameters only differ by small, though significant, extents from those of the suspension medium. Possible nucleation mechanisms are discussed in terms of the experimental data and the cell dimensions. Finally, one of the underlying assumptions of the kinetic analysis, i.e., that ice growth must be rapid compared to nucleation, has been tested and validated by freeze-fracture electron microscopy.

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.

Nucleation and growth of ice in deeply undercooled erythrocytes.

Previous studies of the mechanism of freezing of erythrocytes in the absence of intracellular ice have been extended to define the catalytic sites responsible for promoting nucleation. The following aspects have been investigated: (1) the freeze propagation between undercooled erythrocytes, (2) the nucleation of ice in undercooled erythrocyte ghosts, and (3) the freezing behavior of undercooled hemoglobin solutions. The main findings are: (1) no cross-nucleation occurs between individual cells packed within the same emulsified water droplet; (2) the differential scanning calorimetric power-time curves of intact cells and ghosts are identical, indicating that hemoglobin does not affect ice nucleation; (3) the nucleation temperature of ice in an aqueous solution of hemoglobin (isolated from the cells) is substantially lower than that for the same solution when contained in the intact cell; (4) the threefold freeze concentration which accompanies the freezing of a 25% hemoglobin solution does not cause denaturation of the protein.

The nucleation of ice in undercooled water and aqueous polymer solutions

Abstract The theory of ice nucleation in undercooled water is reexamined in the light of recent experimental measurements and new specific heat and viscosity data on undercooled water. By the incorporation of these data in the calculation of the ice-nucleation rate, it is found that the nucleation model based on stepwise growth of a cluster fails at temperatures below 230 K. Results are reported of ice-nucleation rates in concentrated solutions of oxyhaemoglobin; they are compared with data for other aqueous polymer solutions.

Differential scanning calorimetric study on ice nucleation in water and in aqueous solutions of hydroxyethyl starch

Abstract An experimental investigation has been performed on ice nucleation rates over a range of degrees of undercooling, in water and in 15%, 25% and 35% w/w aqueous solutions of hydroxyethyl starch. An emulsion of droplets of the aqueous phase to be studied was cooled in a differential scanning calorimeter. The heat of crystallization was measured from which the nucleation rate was deduced after the droplet size (assumed uniform) had been determined. All the results obtained are in the main consistent with classical nucleation theory. The presence of hydroxyethyl starch is found to lead to pronounced increases in the exponential factor; this effect may be explained by changes in the interfacial free energies between ice and the aqueous phases. Discrepancies seen at both the high and the low temperature ends of the scans can be attributed to contributions to the exotherms by droplets of volumes larger and smaller, respectively, than the average.

Nucleation Kinetics of Ice in Undercooled Yeast Cells: Long-term Stability against Freezing

SUMMARY: Undercooling and ice nucleation in yeast cells exposed to increasing hypertonic polyvinyl-pyrrolidone (PVP) concentrations were measured. Ice nucleation rates were analysed in terms of classical nucleation theory. Contrary to earlier reports, nucleation was found to be of the catalysed type, by active catalytic sites within the cell. With increasing PVP concentration, the nucleation temperature tends to a limiting value (approximately 236 K), whereas the homogeneous nucleation temperature of the extracellular PVP solution continues to decrease with increasing PVP concentration. The calculated parameters in the nucleation equation indicate that the nucleation mechanism within the cell is unaffected by hypertonic stress. The experimentally determined freezing kinetics of undercooled water (in oil emulsions) were found to parallel closely previously reported death rates of yeast cells as a function of temperature. The observed kinetics are compatible with a slow crystallization or precipitation of emulsifying agent at the oil/water interface, to yield catalytic sites capable of promoting ice nucleation. Experiments with water- (or yeast)-in-oil dispersions containing no emulsifying agents led to long-term freezing resistance and high recoveries of viable cells.