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

Cryobiology 70 (2015) 115–121

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Cryopreservation of Escherichia coli K12TG1: Protection from the damaging effects of supercooling by freezing H. Simonin ⇑, I.M. Bergaoui, J.M. Perrier-Cornet, P. Gervais UMR Procédés Alimentaires et Microbiologiques (PAM), équipe Procédés Microbiologiques et Biotechnologiques (PMB), Université de Bourgogne/AGROSUP Dijon, 1 Esplanade Erasme, Dijon, France

a r t i c l e

i n f o

Article history: Received 23 June 2014 Accepted 16 December 2014 Available online 24 December 2014 Keywords: Freezing Supercooling Osmotic pressure Membrane phase Membrane permeabilization Membrane depolarization Escherichia coli

a b s t r a c t 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 5 h 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. Ó 2015 Elsevier Inc. All rights reserved.

Introduction Because glycerol’s ability to serve as a cryoprotectant was first discovered in 1949 [26], considerable progress has been made in the field of cryopreservation. Today, cryopreservation is commonly used for the long-term preservation of the biological functions of a variety of cells, including spermatozoa or frozen cell stocks. However, despite ongoing research to improve our understanding of cryopreservation and to develop new cryopreservation techniques, numerous bottlenecks persist: the cryopreservation of composite tissues remains impossible [2]; numerous plant germplasms cannot be cryopreserved [10]; oocytes are also highly susceptible to physical stresses, and their cryopreservation is still considered as experimental [29]; and even the oldest applications require improvement for optimal preservation of biological functions, as stated by Fonseca [6] for lactic acid bacteria.

⇑ Corresponding author. http://dx.doi.org/10.1016/j.cryobiol.2014.12.006 0011-2240/Ó 2015 Elsevier Inc. All rights reserved.

Several physico-chemical phenomena are involved in cell damage during the freeze-thaw process. The role of water crystallization has been extensively reported [5,17,36]. It is now commonly accepted that at low freezing rates, extracellular ice formation leads to cell dehydration due to cryoconcentration of the extracellular medium. All the solutes and materials in suspension, including cells, concentrate in unfrozen compartments. The cells are thus exposed to an increasingly concentrated solution while the temperature decreases. This process continues until the cryoconcentrated solution crystallizes as a eutectic system, forms glass, or persists as an unfrozen solution depending on the final temperature. If cells are cooled more rapidly (200–5000 °C min1), they will undergo lethal intracellular ice-formation because the intracellular water does not flow out fast enough to prevent extensive supercooling, and eventual nucleation of the supercooled water in situ. The cooling rates that cause extracellular or intracellular crystallization depend on the cell type and the cryoprotectant concentration. Finally, at very high cooling rates and high cryoprotectant concentrations, it is possible to prevent water freezing. This phenomenon is due to the vitrification of the whole system. Recent

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developments in vitrification protocols have significantly improved the cryopreservation of various cell types, particularly germplasms [9,29]. Whatever the cooling protocol, the thawing and storage conditions must also be strictly controlled to decrease the chances of ice nucleation or recrystallization [6,18,29]. However, in addition to reports of injuries occurring during freezing, some studies report additional phenomena that may result in cell damage [6,22]. These studies attribute unexpected cell injury to the osmotic imbalance occurring during cell thawing. In addition to mechanical and osmotic stresses due to freezing, cells are also subjected to a cold stress when cryopreserved [25], and cold stress can be very harmful for two reasons: first, cooling rates are often high, which according to our knowledge, can be considered as injurious cold shocks [4]; second, cells remain at subzero temperatures while the medium is in liquid state before ice formation. In fact, water and aqueous solutions tend to cool to a temperature significantly below their melting point before ice nucleation occurs. It has been shown that supercooling is a critical step in the cryopreservation process [21]. Thus, there is still a need to determine the role of freezing, dehydration and cold-induced injuries in cell damage that occurs during cryopreservation. In this way, we will improve not only our understanding of the mechanisms of cryopreservation but also improve cryopreservation protocols. The objective of this work was to distinguish the damage to Escherichia coli caused by freezing from the damage caused by the cold. For this purpose, bacterial suspensions were supercooled for several hours. Supercooling is a metastable liquid state below the freezing point that can theoretically be obtained down to 40 °C in pure water [7]. In practice, the extent of supercooling depends mainly on the sample volume, its composition, and the cooling rate applied. For example, Searles et al. reported supercooling ranging from 8 °C to 18 °C in 2 mL of 10% (w/v) starch solutions in a freeze-dryer [30]. In the present work, supercooling was maintained for up to 5 h at 10 °C and 20 °C. For the experiments at 20 °C, the cell suspensions were subjected to an isostatic pressure of up to 40 MPa. At 40 MPa, the supercooling state was more stable than at ambient pressure because the pressure induced a decrease in the freezing point of water by 3.2 °C. The supercooling range can also be extended using this technique (down to 92 °C at 200 MPa). In a previous study, this pressure level was shown to have no effect on E. coli viability, even at 20 °C [24]. Our team has been studying the cell membrane’s responses to physical perturbations for over a decade and has demonstrated in many studies that it is a key target of osmotic, mechanical and thermal stresses [4,8,14,32]. Cell membrane integrity, polarity, and phase changes were thus chosen to investigate the mechanisms of cell injury.

Freezing and supercooling at 10 °C Freezing and supercooling at 10 °C were achieved using the protocol described by Moussa et al. [23]. Aliquots (1 mL) of cell suspensions were transferred aseptically to cryotubes. The samples were then cooled by immersing the cryotubes in a cryostat that was kept at 10 °C. The temperature was measured using K-type thermocouples (NiCr/NiAl, response time 70 ms; Thermocoax, Suresnes, France) inserted through the plugs in the cryotubes and positioned in the centre of the cell suspensions. The mean cooling rate estimated from the temperature profiles was 8 °C/ min. For the frozen samples, freezing was triggered by tapping the tubes as soon as the temperature reached 5 °C. Samples were then maintained at 10 °C for times ranging from 10 min to 5 h. Before warming up the supercooled samples, the supercooled state was visually checked to ensure that the samples were liquid by turning the tubes upside down. Before analysis, the samples were warmed at a rate of 16 °C/min by immersion in a water-bath at 27 °C. Freezing and supercooling at 20 °C Supercooling at 20 °C was achieved using a high-pressure device as described by Moussa et al. [23]. Briefly, aliquots (0.5 mL) of cell suspensions were transferred aseptically to polyethylene bags (Sarstedt), which were heat-sealed after removal of air bubbles. Samples were then placed in a small high hydrostatic pressure vessel (Top Industrie, France; 80 mm external diameter, 10 mm internal diameter, 100 mm high, and 55 mm deep, 4 mL inner volume) connected to a hand-operated pump (Novaswiss, Cesson, France). The inner chamber was then filled with 97% ethanol, which acted as a pressure-transmitting fluid. For supercooled samples, the pressure was first increased to 10 MPa (samples maintained for 10 min or 1 h at 20 °C) or 40 MPa (samples maintained for 5 h at 20 °C). The temperature was then lowered first by immersing the high-pressure vessel in a cryostat (F81-HP, Julabo, Seelbach, Germany) maintained at 60 °C to accelerate heat transfer, and then by transferring it to a second cryostat (RC6CP, Lauda, Germany) containing ethanol at 20 °C to maintain a constant temperature during the holding time. The mean cooling rate was 7.5 °C/min. After the holding time, the vessel was warmed via immersion in a water bath maintained at 27 °C. As described previously [22], the small volume of the vessel and the static conditions ensured that no crystallization occurred in the liquid even though the temperature was below the liquid’s theoretical freezing point. Changes in pressure and temperature in the treatment vessel were used to ensure the onset of freezing. Frozen samples were first crystallized by immersing the high-pressure vessel in the cryostat before increasing the pressure to 10 or 40 MPa. Osmotic treatment in cold conditions

Material and methods Cell culture Escherichia coli K12TG1 (PAM, Agrosup Dijon, France) was grown statically in 20 mL test tubes containing 10 mL of Luria Bertani broth (LB, Sigma Aldrich) at 37 °C for 24 h. Liquid cultures were then prepared by injecting 0.2 mL of subculture into 30 mL test tubes containing 20 mL of LB broth. The cultures were grown statically at 37 °C for 24 h until the early stationary phase was reached. Final cell concentrations ranged from 2  108 to 5  108 CFU/mL. Each cell culture was centrifuged at 2880g for 5 min and washed twice in the same volume of a sterile water–glycerol solution with an aw (water activity) of 0.992 (4% w/w glycerol in water).

The cells were subjected to cold osmotic shocks to simulate the osmotic stress they experience during freezing at 10 °C or 20 °C: 0.1 mL aliquots of cell suspensions were transferred aseptically to cryotubes and cooled to 5 °C to mimic the supercooling before freezing. Samples were then mixed at a constant temperature with 0.9 mL of a water–glycerol solution to a final aw of 0.908 or 0.822, thermostated at 5 °C to mimic cryoconcentration due to freezing. The water was considered to freeze until the concentration of the unfrozen solution reached a thermodynamic equilibrium with an aw of 0.908 at 10 °C and 0.822 at 20 °C. The aw of the theoretical cryoconcentrated solution was deduced from the phase diagram of water–glycerol solutions [12]. Samples were then cooled to 10 °C or 20 °C and maintained at this temperature for times ranging from 10 min to 5 h. Finally, the samples

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were slowly rehydrated at 0 °C before determination of cell viability and membrane damage. For this purpose, 10.088 mL or 6.632 mL of distilled water were gently added, under stirring, to the cell suspensions with an aw of 0.822 or 0.908, respectively. This permitted the cell suspension to recover to an aw of 0.992. Determination of cell viability Cell viability was determined by counting colony-forming units (CFUs) on agar plates. Samples were serially diluted and plated onto agar plates in duplicate. The plates were incubated for 24 h at 37 °C. Viability was expressed as a percentage of the control treatment. Cell suspensions maintained at ambient temperature during the experiment were used as a control. Experiments were repeated at least three times. Assessment of cell membrane damage Propidium iodide (PI) was used to assess membrane integrity because it can enter permeabilized cells and stain their DNA. Bis((1,3-dibutylbarbituric acid) trimethine oxonol) (BOX) was used to evaluate changes in membrane potential as it can penetrate depolarized cells where it binds to intracellular proteins or membranes. It exhibits enhanced fluorescence and a spectral red shift. Stock solutions of each dye were prepared as follows: a 1.4 mM PI solution was prepared in distilled water and a 0.25 mM BOX solution in dimethylsulphoxide (DMSO). An aliquot (1 mL) of cell suspension (107 cells/mL) was labelled with 1.9 lL of PI and 2 lL of BOX stock solutions (final probe concentrations of 2.7 lM PI and 0.5 lM BOX) for 30 min in the presence of 4 mM EDTA to facilitate BOX staining. A cell suspension heated at 90 °C for 10 min was used as a PI-positive control. Cells incubated with 20 lg/mL of ionophore gramicidin S (Sigma), which was used to abolish the cell membrane potential, were used as a BOX-positive-control. The cell suspensions were analyzed using a FACSAria II cytometer (BD Biosciences) with excitation at 488 nm by a 13 mW Saphire™ 488-20 laser (Coherent). Discriminators were set on forward (FSC) and side (SSC) scatter signals to reduce electronic and small-particle noise. The optical filters were set so that PI fluorescence was measured at 610 nm and BOX fluorescence at 525 nm. In all cases, there was some spectral overlap between the fluorescence emitted by the stained mixtures. Therefore, the system compensation was set to eliminate interference. Positively and negatively stained controls were used to achieve compensation and exclude the contribution of signals from the cells. Typically, signals from 10,000 cells were acquired and analyzed for each sample using BD FACS Diva software. The flow cytometry results presented in this study are representative of at least five independent experiments. Cell membrane labelling and measurement of phase changes using Laurdan generalized polarization (GP) The probe Laurdan was chosen to investigate phase changes in the plasma membranes of E. coli because this probe displays similar spectral characteristics and environmental sensitivity (e.g., phase transitions) in the E. coli membrane as it does in model membranes [38]. Laurdan steady state emission spectra shift to longer wavelengths when a gel to liquid crystalline phase transition occurs in membranes. The GP function determines the relative amount of liquid crystalline and gel phases in membranes [1]. Thus, it is possible to detect membrane phase transitions by measuring the GP as a function of temperature. A 4 mM stock solution of the probe Laurdan (6-lauroyl-2-dimetylaminonaphtalene) was prepared in DMSO. This probe is known to label phospholipid bilayers where its quantum yield is greatly enhanced. A cell culture

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was centrifuged at 2880g for 5 min and washed twice in the same volume of a sterile glycerol solution with an aw of 0.992. The pellet was then suspended in the same volume of a sterile glycerol solution either at an aw of 0.992 (4% w/w), 0.908 (32% w/w), or 0.822 (48% w/w). An aliquot (3 mL) of cell suspension (2  108 cells/ mL) was labelled with 5 lL of the Laurdan stock solution (final probe concentration, 6.7 lM) for 20 min at 37 °C directly in the cuvette. Fluorescence was measured using a Fluorolog-3 spectrometer (Jobin–Yvon, Horiba Group, USA), equipped with a thermostated sample chamber and a magnetic stirrer. The temperature was measured continuously using a K-type thermocouple inserted directly in the cuvette. Laurdan fluorescence emission spectra were recorded between 420 and 550 nm with an excitation wavelength of 350 nm. Background spectra were obtained using unlabelled cells and were subtracted from the spectra of labelled cells. These backgrounds were always <10% of samples. The generalized polarization (GP) of the spectra was calculated using Eq. (1):

GP ¼ ðB  RÞ=ðB þ RÞ

ð1Þ

where B (blue) and R (red) are the fluorescence intensities measured at 440 and 490 nm, respectively, with an excitation wavelength of 350 nm after subtraction of the background. The temperature was decreased from 37 °C to 9.5 °C at a rate of approximately 1 °C/min using a Pelletier device. GP was not measured below 9.5 °C due to the limitations of the cooling device. The GP was measured every 4 °C. The experiment was repeated four times. Statistical analyses Analysis of variance (ANOVA) and calculation of the lowest significant difference (LSD) were performed using the software R 2.14.0. Results Viability of E. coli after freezing or supercooling Cells were first subjected to freezing or supercooling at 10 °C for times ranging from 10 min to 5 h. Cell viability was then assessed after samples were warmed up (Fig. 1A). For the two treatments, viability decreased slightly after 10 min to between 80% and 90 %. For the frozen samples, viability was stable. In fact, it did not decrease significantly or go below 75% after 5 h of treatment. Conversely, viability after supercooling decreased during the treatment and reached 57% after 5 h. At 20 °C, supercooling could not be maintained without applying a moderate isostatic pressure, ranging between 10 and 40 MPa. Therefore, the frozen samples were also subjected to the same pressure throughout the treatment. When applied at ambient temperature for over 5 h, a pressure treatment such as this had no effect on E. coli viability. At 20 °C, the results were similar to those obtained at 10 °C (Fig. 1B). For the frozen samples, a small decrease in viability to 83% was measured after 10 min; the viability then remained stable until the end of the treatment (after 5 h). After supercooling, as at 10 °C, viability decreased during the treatment. Viability was even lower at 20 °C than at 10 °C, as it reached 39% after 5 h. Cell membrane damage after freezing or supercooling At the same time, cell membrane damage was assessed using two probes, BOX and PI, which stain depolarized and permeabilized cells, respectively. Cells were stained following different freezing or supercooling times at 10 °C and 20 °C, just after the cells

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Fig. 1. Viability of E. coli K12 determined by CFU counts after freezing (____) or supercooling (- - - -) at -10 °C (A) and 20 °C (B). The data are means, and the error bars are standard deviation. The level of significance of the difference between viability after freezing and after supercooling is represented by ⁄ (p < 0.05), ⁄⁄ (p < 0.01), or ⁄⁄⁄ (p < 0.001).

had been warmed. Fluorescence was then measured using flow cytometry. Four populations of cells could be differentiated according to their staining: unstained cells were considered as undamaged; BOX-stained cells had a depolarized plasma membrane; PI/ BOX double-stained and PI stained cells were all presumed to have permeabilized membranes with different degrees of permeability. The percentage of cells with intact membranes corresponded approximately to the percentage of viable cells determined by the CFU counts at 10 °C and 20 °C (Fig. 1). The results obtained after treatment at 20 °C are presented in Fig. 2A and B. Again, supercooling could be maintained using the high-pressure device. Thus, two controls were performed at ambient temperature: one at ambient pressure and a second at the higher pressure of 40 MPa for 5 h. After different treatment times for the frozen samples, the cell membranes suffered little damage, as less than 20% of the cells were labelled with BOX and/or PI. The percentage of damaged cells was not significantly different from that of the two controls (p > 0.05), where less than 10% were depolarized and less than 10% were permeabilized. On the other hand, the percentage of cells with damaged membranes increased with increasing time at 20 °C in the liquid state. After 5 h, only 30% of the cells had an intact membrane. The percentage of depolarized cells first increased to 27% after 1 h; then the percentage of permeabilized cells increased to 36%, of which 28% were only labelled with PI, probably reflecting a high degree of membrane permeabilization. Cell viability and membrane damage after a cold hyperosmotic shock simulating freezing Cells were then subjected to cold osmotic stresses to simulate the cryoconcentration of unfrozen media that occurs as water freezes at 10 °C or 20 °C. Glycerol was chosen as a solute

Fig. 2. Variations in the percentage of cells with intact membranes (unstained cells h), depolarized membranes (BOX-stained cells ) and permeabilized membranes (PI/BOX double-stained cells and PI-stained cells j) measured using flow cytometry after different holding times for freezing (A) or undercooling (B) at 20 °C. The ‘‘pressure control’’ was maintained at a 400 bar pressure for 5 h at ambient temperature. Data are means of at least three replicates. The level of significance of difference between unstained cells after freezing and after supercooling is represented by ⁄⁄ (p < 0.01), or ⁄⁄⁄ (p < 0.001) (B).

because cells had been suspended in an isotonic water–glycerol solution before freezing. Cells were subjected to osmotic shocks at aw 0.908 or 0.822 to mimic cryoconcentration at 10 °C or 20 °C, respectively. Osmotic shocks were induced at 5 °C, the temperature at which freezing was triggered, before subsequent cooling to 10 °C or 20 °C. Viability was determined by CFU counts after the cell suspensions had been slowly rehydrated at 0 °C and warmed up. Thus, the hyperosmotic shocks and the rehydration step led to a dilution of the cell suspensions in comparison to the freezing treatments. The results are presented in Fig. 3. For the two conditions tested, 10 °C/aw 0.908 and 20 °C/aw 0.822, and independent of the dilution, the viability remained high after rehydration and warming up and was stable throughout the treatment. There was no significant difference between the viabilities measured after different treatment times (p > 0.05). In fact, the percentage of viable cells were between 96% and 71% of the control. At 10 °C/aw 0.908, the viability measured after 1 or 5 h was very close to 80%. At 20 °C/aw 0.822, viability reached 72% after 5 h. The same trends were observed for cell membrane damage. Fig. 4 shows the changes in the percentage of cells with intact, depolarized, and permeabilized membranes according to the time spent at 20 °C/aw 0.822. The percentage of permeabilized cells (PI+) remained stable at 10%, and the percentage of depolarized cells was between 15% and 21%. There was no significant effect of the treatment duration on the membrane state (p > 0.05). The same trend was observed after treatments at 10 °C/aw 0.908 (results not shown).

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100

0.6

90

0.55

80

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0.45

50 40

GP

Viability (%)

70

0.4

30

0.35

20 10

0.3

0 0

60

120

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240

0.25

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Holding me (min) Fig. 3. Viability of E. coli K12 determined by CFU counts after a cold osmotic shock at 5 °C and holding at 10 °C at aw 0.908 (____) or a cold osmotic shock at 5 °C and holding at 20 °C at aw 0.822 (- - - -) and a slow rehydration at 0 °C. Error bars are standard deviations.

100

-10

-5

0.2

0

5

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15

20

25

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35

40

Temperature (°C) Fig. 5. Laurdan GP values as a function of temperature, measured at an excitation wavelength of 350 nm for aw 0.992 (j), 0.908 (N) and 0.822 (d). The data shown are representative of three replicates.

The effect of cold stress during supercooling

90 80 70

%

60 50 40 30 20 10 0 Control

10 min

60 min

300 min

Fig. 4. Variations in the percentage of cells with intact membranes (unstained cells h), depolarized membranes (BOX-stained cells ) and permeabilized membranes (PI/BOX double-stained cells and PI-stained cells j) measured using flow cytometry after a cold osmotic shock at 5 °C (aw 0.822), holding at 20 °C and a slow rehydration at 0 °C. The data are means of at least three replicates.

Estimation of the phase transition temperature range at different aw The temperature dependence of the Laurdan GP was employed to estimate the physical state of the lipid membrane. Fig. 5 shows the Laurdan GP as a function of temperature for the three aw tested: 0.992, 0.908 and 0.822. In isotonic conditions (aw 0.992), the GP could not be measured below 4 °C because of sample freezing. The GP gradually increased as the temperature decreased from 37 °C to 10 °C for the three aw tested. There was no significant difference between the GP values measured at the three aw tested between 37 °C and 16 °C. Below 16 °C, the GP values increased significantly with increasing aw (p < 0.05). In the three conditions tested, the GP values displayed a clear transition with a higher slope in a specific temperature range. The phase transition temperature range shifted from 16 °C to 4 °C at aw 0.992, and from 18 to 12 °C at aw 0.908 and 0.822. Discussion Our results show that maintaining cell suspensions in a supercooled state and at a high aw has a detrimental effect on both viability and plasma membrane permeability. Conversely, when cells were subjected to cold stress with freezing, the survival rate remained high throughout the experiment and the cell membranes suffered little damage.

Water and aqueous solutions tend to cool to a temperature significantly below their melting point before ice nucleation occurs, i.e., they undercool. In any case, ice nucleation can be induced using mechanical or chemical methods to control the freezing conditions. Controlled ice nucleation of small-volume samples has been demonstrated to be beneficial for many cell types, including bacteria [21]. Until now, this improvement was explained by the link between nucleation temperature and the solidification process. In fact, extensive supercooling was shown to favor detrimental intracellular ice formation in large mammalian cells such as oocytes, embryos, hepatocytes or erythrocytes [21]. In other cell types, such as spermatozoa [22] and lactic acid bacteria [6], where intracellular ice formation could not be shown, it is believed that extensive supercooling before freezing leads to a deleterious osmotic shock due to ice crystallization in the extracellular medium occurring upon warming. In the present work, the supercooling effect was maintained for a long period (up to 5 h). In this way, the damaging effect of cooling at temperatures as low as 20 °C, could be evaluated and separated from injury caused by freezing. For the experiments at 20 °C, the cell suspensions were subjected to an isostatic pressure. The pressure levels used did not exceed 40 MPa, and at this level did not induce any loss in viability or cause any detectable membrane damage at ambient temperature, as was shown in the control experiment presented in Fig. 2. Furthermore, in a previous study, Moussa et al. [24] showed that there was no difference in the viability of E. coli subjected to a cold treatment at 20 °C at atmospheric pressure or at high pressure (up to 50 MPa). We thus assumed that the effect of the treatments observed in the present work can be mainly attributed to the supercooling effect. At 10 °C and 20 °C, it was clear that maintaining the cell suspension in a supercooled state had a detrimental effect on both viability and plasma membrane integrity. These effects were observed with (20 °C) or without (10 °C) pressure being applied and confirm that they can be attributed to the cold stress. In fact, several studies have demonstrated that chilling at very low temperatures above 0 °C can be lethal to cell survival of different bacterial species [4,9,13,28,37]. However, the resistance of cells to supercooling has rarely been studied [23,28]. In our experiments, the rate of cell death increased with increasing time spent in a supercooled state. The consequences of cold stress were even more serious at the lower temperature, as observed by Rasmussen et al. [28]. The evaluation of membrane damage showed a gradual degeneration of the cell membranes. Over time, more and more cells had a depolarized

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membrane. Then, more and more cells were permeabilized. Surprisingly, a large number of cells were only stained with PI after 5 h in a supercooled state. We hypothesized that these PI-stained cells were too permeabilized to retain BOX. BOX enters depolarized cells where it binds to intracellular proteins and exhibits enhanced fluorescence, whereas PI binds to DNA. Permeabilized cells, and even ghost cells, normally take up both BOX and PI [31]. The occurrence of cells taking up PI but not retaining BOX may reflect a very high degree of cell permeability. In fact, several studies have shown that a sudden cold shock can lead to the release of proteins into the external medium [19,27,33]. Loss of membrane polarization would be unfavorable to maintaining homeostasis. As a result, it is likely that maintaining the cell membrane in a depolarized state caused uncontrolled mass transfer, leading to permeabilization and cell death over time. Freezing of the extracellular medium protects cells against cold damage due to cell dehydration When cells were subjected to a cold stress due to freezing, survival remained high throughout the experiment. Accordingly, cell membranes suffered little damage from the cold stress due to freezing. A few minutes after water freezing, a cell mortality of less than 20% was observed; it was not significantly different from the mortality measured after 10 min of supercooling at the same temperature. The viability was then stable throughout the experiment. Moussa et al. [23] measured a higher mortality after 10 min of freezing than after 10 min of supercooling with the same experimental conditions. These authors ascribed the mortality observed in the first minutes of freezing to the osmotic shock due to crystallization of the extracellular water. In fact, the cooling rate (8 °C/ min) and the temperatures used in this work only allowed the extracellular water to freeze. In cooling conditions such as these, cells are brought together in the cryoconcentrated solution, as observed by Fonseca et al. [6]. Cells are thus subjected to a cold osmotic shock as the water freezes [16]. Furthermore, the concentration of the unfrozen solution increases with decreasing temperature according to the colligative properties of water. In the present work, cells were suspended in an isotonic glycerol solution (aw 0.992) before freezing. The relationship between the water activity of the freeze-concentrated solution and the freezing temperature was thus deduced from the phase diagram of the glycerol solutions [12]. At 10 °C and 20 °C, the aw should have decreased to 0.908 and 0.822, respectively. Cells were thus submitted to an equivalent osmotic stress at 5 °C to simulate water crystallization and then maintained at 10 °C or 20 °C for 5 h. Interestingly, at these aw values and despite a higher cell dilution in comparison to freezing treatments, the cells were protected against cold damage and 70% to 80% of the cells were viable at the end of the experiments at the temperatures tested. These results corroborate those obtained previously with E. coli and glycerol [23], sucrose [19], or NaCl [13] where the cells were protected against cold shock by the addition of sucrose, NaCl or glycerol. In particular, the presence of some of these solutes seems to come from the leakage of intracellular compounds. In fact, after a cold osmotic shock at aw 0.822 and after 5 h at 20 °C, only 20% of the cells were depolarized. Is the protection against cold stress due to a lipid phase transition? Sucrose, NaCl, or glycerol all act by increasing the osmolarity or by decreasing the aw of the medium. The exposure of the cells to a hyperosmotic medium has the immediate effect of dehydrating the cells and inducing shrinkage [20]. Likewise, the cell membranes are dehydrated, and such dehydration can lead to a lipid phase transition. Changes in plasma membrane permeability have been largely linked to the occurrence of lipid phase transitions [3,20,35]. The

phase state of the bacterial membranes was thus investigated as a function of temperature for the three aw: 0.992, 0.908, and 0.822. At physiological temperatures and levels of hydration, the phospholipid bilayer of E. coli membranes is in a fluid, lamellar, liquid–crystalline phase [38]. The phospholipid phase transition temperature (Tm) varies according to the osmotic pressure of the surrounding medium. When the cells are dehydrated, a membrane phase transition from liquid-crystalline to gel occurs at a higher temperature than for fully hydrated cells. In fact, the phase transition shifted from 16 °C to 4 °C at aw 0.992, and from 18 °C to 12 °C at aw 0.908 and 0.822. So, at 5 °C and below, cell membranes should be in the gel state whatever the aw. If we say that freezing at 10 °C or 20 °C is equivalent to an osmotic stress at aw 0.908 or 0.822, respectively, we must concede that the membrane lipids should have been in the gel state throughout the supercooling treatment as they would be upon freezing at the same temperature. This means that membranes composed of lipids in the gel state may be insufficient to protect cells membranes against damage. However, the GP values measured below 16 °C were significantly higher at low aw than at aw 0.992. This result shows that cell membranes are not exactly in the same state upon freezing and supercooling. A higher GP value may reflect an increase in the abundance of ordered domains in the cell membranes [1], so that the membrane may have nanoscale differences in state between freezing and undercooling. This could partly explain the better cell survival upon freezing. In fact, membranes are distorted by the contraction in volume when dehydrated and solvation of lipids and proteins is different at lower aw. The mechanisms of cell protection against cold stress should thus be further investigated using more accurate biophysical methods to assess membrane states. Proteins are also susceptible to cold stress. This phenomenon, called cold denaturation, particularly affects globular proteins [11,15,34]. The cold denaturation process is thought to be closely linked to the change in affinity between non-polar groups and water at low temperatures. Thus, the depolarization and permeabilization of membranes upon supercooling could also be attributed to membrane protein denaturation. Glycerol and sucrose have been shown to have a protective effect against the cold denaturation of proteins [11]. The strong interaction between polyols and free water molecules may help to reduce the movement of the free water molecules. We thus believe that dehydration protects cells against cold stress also due to the protection of cell proteins. In any case, glycerol cannot protect cells against cold stress during long-term storage. In fact, Moussa et al. [23] showed that the number of permeabilized cells increases after several days at 20 °C after freezing. For long-term preservation, the liquid part of the cell suspensions, which is cryoconcentrated, should be allowed to vitrify via more extreme cooling and lower storage temperatures.

Conclusions This work confirms the known deleterious effects of supercooling in cryopreservation protocols and provides new knowledge regarding the mechanisms involved in damage resulting from supercooling. Indeed, we propose that extreme cold stress without dehydration, resulting from extensive supercooling, is a major cause of E. coli cell damage resulting in cell membrane depolarization followed by permeabilization. The dehydration of gram negative bacterial cells caused by freezing of the extracellular medium would be beneficial thanks to the protection of cells against cold stress. This protection could not be attributed to the plasma membrane main phase transition to a gel state alone, but could be

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