Cryopreservation of Escherichia coli K12TG1: protection from the damaging effects of supercooling by freezing.

Injuries in living cells caused by water freezing during a freeze-thaw process have been extensively reported. In particular, intracellular water freezing has long been incriminated in cell death caused by a high cooling rate, but this supposition could not always be demonstrated. This work aims to discriminate the role of water freezing, dehydration and cold-induced injuries in cellular damage occuring during cryopreservation. For this purpose, Escherichia coli K12TG1 suspensions were maintained in a supercooled or frozen state at -20°C for times ranging from 10 min to 5 h. The supercooled state was maintained for a long period at -20°C by applying a non-injurious isostatic pressure (P<40 MPa). Next, viability and membrane damage were determined by agar plating and fluorescence staining with propidium iodide and bis-oxonol. It was clear that keeping the cell suspensions in the supercooled state had a detrimental effect on both viability and plasma membrane permeability. Conversely, when cells were subjected to cold stress by freezing, the survival rate remained high throughout the experiment, and the cell membranes suffered little damage. Moreover, cells subjected to 5h of osmotic treatments at -20°C, conditions that mimic cryoconcentration upon freezing, and subsequently diluted and thawed suffered little damage. Dehydration due to cryoconcentration upon freezing protects the cells against the deleterious effects of supercooling, especially in the plasma membranes. The decrease in membrane leakage upon dehydration at low temperatures could be linked to differences in the gel state of the membrane revealed by a higher Laurdan general polarization (GP) value.