This work studied the viabilities of five types of cells (two yeast cells, Saccharomyces cerevisiae CBS 1171 and Candida utilis; two bacterial strains, Escherichia coli and Lactobacillus plantarum; and one human leukemia K562 cell) as a function of cooling rate during freezing. The range of investigated cooling rates extended from 5 to 30,000°C/min. Cell viability was classified into three ranges: (i) high viability for low cooling rates (5 to 180°C/min), which allow cell water outflow to occur completely and do not allow any intracellular crystallization; (ii) low viability for rapid cooling rates (180 to 5,000°C/min), which allow the heat flow to prevail over water outflow (in this case, cell water crystallization would occur as water was flowing out of the cell); (iii) high viability for very high cooling rates (>5,000°C/min), which allow the heat flow to be very rapid and induce intracellular crystallization and/or vitrification before any water outflow from the cell. Finally, an assumption relating cell death to the cell water crystallization as water is flowing out of the cell is made. In addition, this general cell behavior is different for each type of cell and seems to be moderated by the cell size, the water permeability properties, and the presence of a cell wall.The freeze-thawing process remains the principal method of cell preservation to date, and the high survival rates achieved by this method are of interest from both the biophysical and practical points of view. This is to ensure that the recovery of entire cell populations is free from the risk of possible subsequent alteration of its genetic composition.Cell cryopreservation, which is commonly used in the food and pharmaceutical industries, requires optimization for each type of microorganism. Moreover, each type of cell has its own protocol for freezing. Numerous researchers have attempted to develop methods that permit 100% preservation of freezethawing of diverse cellular specimens (3, 6), but some microorganisms cannot yet be preserved by freezing.For a better cell preservation some cryoprotectants such as glycerol or dimethyl sulfoxide can be used (8). These molecules improve the cell preservation by minimizing the cell water content (6) and/or supporting the vitrification occurrence (1) and finally by protecting the cell's constitutive macromolecules (2, 5).The freeze-thawing process constitutes a double stress for the cell, i.e., thermal and hyperosmotic stresses, which act simultaneously during cooling (15, 16). The scenario of cell evolution during slow freezing is well known (13). The water surrounding the cell freezes before the cell contents, because the cytoplasm is more concentrated than the growth medium, and because thermodynamically, the component with the largest volume will nucleate first (6, 16). This freezing increases the osmotic pressure of the medium, and the extracellular solutes become concentrated in the remaining liquid extracellular water. Consecutive osmosis will then dehydrate cells as water diffuses from the cy...

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The survival of cells subjected to cooling at sub-zero temperature is of paramount concern in cryobiology. The susceptibility of cells to cryopreservation processes, especially freeze-thawing, stimulated considerable interest in better understanding the mechanisms leading to cell injury and inactivation. In this study, we assessed the viability of cells subjected to cold stress, through long-term supercooling experiments, versus freeze-thawing stress. The viability of Escherichia coli, Saccharomyces cerevisiae, and leukemia cells were assessed over time. Supercooled conditions were maintained for 71 days at -10 degrees C, and for 4 h at -15 degrees C, and -20 degrees C, without additives or emulsification. Results showed that cells could be inactivated by the only action of sub-zero temperature, that is, without any water crystallization. The loss of cell viability upon exposure to sub-zero temperatures is suggested to be caused by exposure to cold shock which induced membrane damage. During holding time in the supercooled state, elevated membrane permeability results in uncontrolled mass transfer to and from the cell maintained at cold conditions and thus leads to a loss of viability. With water crystallization, cells shrink suddenly and thus are exposed to cold osmotic shock, which is suggested to induce abrupt loss of cell viability. During holding time in the frozen state, cells remain suspended in the residual unfrozen fraction of the liquid and are exposed to cold stress that would cause membrane damage and loss of viability over time. However, the severity of such a stress seems to be moderated by the cell type and the increased solute concentration in the unfrozen fraction of the cell suspension.

The effects of slow chilling (2 degrees C min(-1)) and rapid chilling (2,000 degrees C min(-1)) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.