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Influence of cooling rate on Saccharomyces cerevisiae destruction during freezing: unexpected viability at ultra-rapid cooling rates
Cryobiology 46 (2003) 33–42 www.elsevier.com/locate/ycryo
Influence of cooling rate on Saccharomyces cerevisiae destruction during freezing: unexpected viability at ultra-rapid cooling ratesq Frederic Dumont, Pierre-Andre Marechal, and Patrick Gervais* Laboratoire de G enie des Proc ed es Alimentaires et Biotechnologiques, ENSBANA, 1, Esplanade Erasme, 21000 Dijon, France Received 7 May 2002; accepted 12 November 2002
Abstract The purpose of this work was to study cell viability as a function of cooling rate during freezing. Cooling rate strongly influences the viability of cells during cold thermal stress. One of the particularities of this study was to investigate a large range of cooling rates and particularly very rapid cooling rates (i.e., faster than 20 000 C min1 ). Four distinct ranges of cooling rates were identified. The first range (A0 ) corresponds to very slow cooling rates (less than 5 C min1 ), and results in high cell mortality. The second range (A) corresponds to low cooling rates (5–100 C min1 ), at which cell water outflow occurs slowly and does not damage the cells. The third range (B) corresponds to rapid cooling rates (100–2000 C min1 ), at which there is competition between heat flow and water flow. In this case, massive water outflow, which is related to the increase in extracellular osmotic pressure and the membrane-lipid phase transition, can cause cell death. The fourth range (C) corresponds to very high cooling rates (more than 5000 C min1 ), at which the heat flow is very rapid and partially prevents water exit, which seems to preserve cell viability. 2002 Elsevier Science (USA). All rights reserved. Keywords: Ultra-rapid cooling rate; Saccharomyces cerevisiae; Cell viability; Osmotic pressure
The freeze-thawing process is a method that allows the preservation of microorganism specimens without the gain of genetic modifications or the loss of functional characteristics. However, despite many scientific studies, the mechanisms underlying cell freezing injury are not yet well understood. The optimal method chosen for cell cryoconservation is often decided on empirical q
This work was funded by institutional sources. Corresponding author. Fax: +33-03-80-39-66-11. E-mail address: [email protected] (P. Gervais). *
considerations. Many procedures, with or without cryoprotective agents, have been reported. Cell freezing, which is commonly used in the food and pharmaceutical industries, requires optimization of the process for each type of microorganism [2]. It is important that the mechanisms of freezing injury be understood if techniques that circumvent this damage are to be developed. The freeze-thawing process constitutes a double stress for the cell: thermal stress and hyperosmotic stress, which act simultaneously during cooling [18,19,21]. Because the cytoplasm is more
0011-2240/02/$ - see front matter 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S0011-2240(02)00161-X
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F. Dumont et al. / Cryobiology 46 (2003) 33–42
concentrated than the growth medium, the external water freezes before the cell contents. This increases the osmotic pressure of the medium. The extracellular solutes are concentrated in the remaining unfrozen extracellular water, and this dehydrates the cell by osmosis as water diffuses from the cytoplasm into the more concentrated external solution. The lower the temperature of the medium, the greater the concentration of the unfrozen solution and the greater the loss of intracellular water. This mass transfer is restricted by the water–membrane permeability value of the cell (Lp ). The osmotic water outflow that occurs from cells during freezing was considered by Muldrew and McGann [19] to be responsible for rupturing the plasma membrane, thus allowing extracellular ice to propagate into the cytoplasm. The hypothesis that osmotic flow leads to cell death [19] must be considered. In previous studies [5,8,9,11,12], the influence of the intensity and kinetics of osmotic pressure variations have been remarked and related to cell viability. An increase in the osmotic pressure gradient has dramatic effects on cell viability. Previous observations on yeast cells at 25 C [7,10] have shown that a too rapid exit of water (or rapid change in osmotic pressure) would damage the cells. Recent works have shown that for lower temperature (i.e., lower than 12 C) application of an osmotic shock was found to be less detrimental [5]. Complementary works on bacteria [21] and yeast cells (not published) have also shown that a too slow variation would also induced a high cell mortality for unknown reason by now. Two principal damage mechanisms associated with cooling rate have been suggested. For low cooling rates, the extracellular solutes concentrate in the remaining unfrozen extracellular water and cause cell dehydration by osmosis as water diffuses from the cytoplasm into the more concentrated external solution. In the second case, high cooling rates result in intracellular ice formation (IIF) during freezing; IIF is lethal for the cell [17]. There are two opinions regarding IIF. Toner and Cravalho [25] suggested that IIF occurs as a result of nucleation within the cell, but Muldrew and McGann [19] suggested that rupture of the plasma
membrane is due to the difference in osmotic pressure that develops across the plasma membrane during rapid cooling, which enables extracellular ice to propagate into the cytoplasm. The cooling rate is a parameter that strongly influences the behavior of cells during cold thermal stress. Ice first forms exclusively in the extracellular compartment because the concentration of ions and solutes is higher in the cell than in the extracellular medium due to the maintain of cell turgor pressure which has been shown in a previous work [13]. In the same way, thermodynamically, the compartment with the larger volume will nucleate first. The intracellular freezing point is slightly lower (about 0.3 C) [16]. The initiation of external freezing creates an osmotic gradient between the cell and its external environment due to the concentration of extracellular electrolytes. If a slow cooling rate is used, intracellular water has time to flow out of the cell, but if a very rapid cooling rate is used, water cannot flow out. In the literature, various cooling rates have been reported. Mazur [14] subjected Saccharomyces cerevisiae to various freeze-thawing procedures and observed a sharp decrease in survival when the cooling rate was increased from 1 C min1 to values in the order of 100 C min1 . Faster cooling rates were not investigated. The purpose of this work was to study cell viability during freezing and particularly at very rapid cooling rates (i.e., faster than 20 000 C min1 ). We based the study on recent research we have done on the relationship between cooling rates, osmotic pressure variations, and cell viability. A large range of cooling rates was investigated to study cell viability as a function of cooling rate.
Materials and methods Biological materials The yeast S. cerevisiae CBS 1171 was used in this study. Cultures were grown in 250-mL conical flasks containing 100 mL aerated modified Wickerham medium, at 250 rpm and 25 C. This medium is composed of 10 g glucose, 3 g pancreatic peptone, 3 g yeast extract, and 1.5 g Na2 HPO4 in
F. Dumont et al. / Cryobiology 46 (2003) 33–42
1 L distilled water. The pH of the medium was adjusted to 5.35 by adding orthophosphoric acid. Inoculation was carried out with a 0.1-mL yeast suspension from a 48-h sub-culture grown under analogous conditions. Cells were harvested after 65 h of growth (stationary phase) and used immediately for experiments. Thermodynamic characteristics of the freezing medium To maintain control of the osmotic pressure of the cell suspension, the cells were not frozen in growth medium. A 20 mL volume of yeast culture was centrifuged at 4000 rpm for 5 min. The pellet was resuspended in 20 mL of the appropriate binary solution. The osmotic disturbance thus provoked in the form of osmotic shock did not allow the cells to adapt physiologically [5]. Freezing were occurred 15 min after osmotic shock. Several binary media were used for freezing (Table 1). They were chosen for their water activity and the initial cellular volume they determined. The molar fraction of solute to be added to 1000 g of water is given by NorrishÕs equation [21]: 2
aw ¼ ð1 Xs Þ ekXs ;
ð1Þ
where aw is water activity, Xs is the molar fraction of the solute, and k is the Norrish coefficient related to the solute (1.16 for glycerol; 57.6 for PEG 600). Solutions were then checked using aqualab CX-2 water activity system (Decagon Devices, USA).
35
Water activity (aw ) can be expressed in terms of osmotic pressure (p) as follows: P¼
RT ln aw ; Vw
ð2Þ
where p is the osmotic pressure (Pa), R the gas constant (8.31 J mol1 K1 ), T the absolute temperature (K), and Vw the partial molar volume of water (18 106 m3 mol1 ). The nucleation temperature was not controlled but the freezing temperature in terms of the water activity of the solution can be calculated with Eq. (3) [4]: Lnðaw Þ ¼ 9:69 103 DTm 5:06106 DTm2 ð3Þ with DTm ¼ ðTo Tp Þ where To (K) is the freezing point of the solvent and Tp (K) is the freezing point of the solution. Cell volume and cell viability after osmotic shock When a cell is transferred from one solution to another of higher osmolality containing a permeating solute, it rapidly shrinks initially because of the loss in water and then slowly increases in volume as the solute permeates accompanied with water [20]. The final cell volume obtained was depending on the initial cell glycerol concentration and of the respective rate of water exit flow and glycerol entry flow which were functions of the osmotic pressure gradient. Cell shrinkage was measured by image analysis, by comparing cell volumes before and after
Table 1 Volumes and viabilities of Saccharomyces cerevisiae after different osmotic shocks Binary solutions
aw a
Osmotic pressure p (MPa)
Mass of solute added to 1000 g of water
Cell volume measured after osmotic shocks (% relative to initial volume)
Viability after osmotic shock
Water–glycerol Water–glycerol Water–glycerol Water–glycerol Water–PEG 600
0.992 0.900 0.800 0.750 0.900
1.38 14.5 30.0 40.0 14.5
51.1 513.8 1063.2 1363.7 1208.9
100% 63% 42.5% 32% 29.7%
99% 97% 96% 94% 93%
a
Water activity verified with a dew point osmometer.
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osmotic shock. Berner and Gervais [6] have already described the system. Measurements were made for more than 300 cells, for both the control yeast population and the stressed cells, at the maximum 15 min after the stress in order not to take into account the entry of the solute. The volume variations are expressed as a percentage of the initial volume of the control population.
Freezing and thawing methods Freezing was always carried out in liquid nitrogen at )196 C but two methods were used. First, various cooling rates were obtained with various containers: Cryotube Nunc (Vials, Germany); Cryotube (Sarstedt, Germany); metallic Micro-Syringe for HPLC (Exmire MS-C10, UK); and with various volumes of solutions placed in these containers (Table 2). For low cooling rates
(i.e., 5 and 6 C min1 ), samples in cryotubes were placed in 50-mL plastic tubes (PolyLabo, France) for centrifugation to protect them from the cold gradient with a gas layer. The second method was used to achieve the fastest cooling rates (more than 10 000 C min1 ). It was based on the principle of cells dropped directly into liquid nitrogen without any protection. Cells were fixed on different supports: Cryo-Billes plastic pellets (Laboratoire AES, France) with a mean diameter of 3.3 mm, or thermocouple T (TCSA Dardilly, France) with a diameter of 0.5 mm. These supports were retained for their natural property to fix the cells on their surface. For the fastest cooling rate (30 000 C min1 ) the support was constituted only by the temperature sensor which gave us effective measurement of 15 000 C min1 . Thanks to this measurement, the cooling rate in the first 20 lm, corresponding to the thickness of solute around the thermocouple, was calculated as being 30 000 C min1 .
Table 2 Cooling rates measured for several supports and conditions in different media Cooling rate (C min1 ) Volume of the sample (mL)
Water– glycerol 1.38 MPa
Cell in suspension Cryotube Sarstedt in plastic tube Cryotube Nunc in plastic tube Cryotube Nunc Cryotube Nunc Cryotube Sarstedt Cryotube Nunc Cryotube Nunc Cryotube Sarstedt Cryotube Sarstedt Cryotube Sarstedt Cylinder in glass Cylinder in glass Cylinder in glass Micro syringe
1
5
1 1.2 1 1 0.5 0.1 0.5 0.1 0.2 0.1 0.05 0.01 0.005
6
1800 4000 5000 17 000a
Immobilized cells Sphere in plastic Thermocouple in metal Thermocouple in metal
0.0025 0.0014 0.0014
12 000a 15 000 30 000a
a
Value calculated from FourierÕs second law.
120 180 250 270 650 700
Water– glycerol 14.5 MPa
Water– glycerol 30 MPa
Water– glycerol 40 MPa
Water– PEG600 14.5 MPa
10
26
20
160 240 140
80 100 160 120
100 110 350 120
115 260 170
210 700
280
290
520
320
280
1800
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The temperature was measured with a T thermocouple (copper/copper–nickel), which was connected to an ‘‘InstruNet 100’’ acquisition card (GWI, USA). A maximum of 60 000 measurements per second were made with this system, which could then be evaluated using a spreadsheet. The cooling rate was calculated from these measurements. During the experiments, the influence of thawing was not studied. Thawing was always carried out by dropping the frozen sample into a controlled-temperature water bath at 37 C. Indeed, Mazur [15] showed that it is necessary to thaw as rapidly as possible, to avoid conditions that could lead to recrystallization of the medium.
Viability measurement Viability measurements were made using the colony-forming unit (CFU) method, by plating cells in MW medium supplemented with 15 g L1 agar. Petri dishes were then incubated for 2 d at 25 C. Viability was determined by comparing the experimental and control microorganisms. We performed at least three repetitions for each experiment.
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Results Viability after cold stress relative to cooling rate Cold stress was applied first in a water–glycerol solution at 1.38 MPa which corresponds to the osmotic pressure of classical growth medium. The results obtained in this experiment can be broken up into three cooling-rate categories, as illustrated in Fig. 1. For cooling rates lower than 10 C min1 (i.e., 5 and 6 C min1 ), cells remained viable. The results were different for cooling rates from 100 to 1000 C min1 . Indeed, yeast viability fell to a low point of 2% for 180 C min1 , and remained stable at about 10%. For cooling rates greater than 1400 C min1 , yeast viability increased from 20% at 6000 C min1 to 50% at 17 000 C min1 (estimated value) until 80.5% at 30 000 C min1 (estimated value). The highest cooling rates were estimated values but confirmed the significant trend in increasing yeast viability. To limit the influence of the cooling rate on cell viability, we decreased the initial intracellular water volume using a preliminary hyperosmotic shock. Less water in the cell before freezing makes it possible to limit the possible effects of water efflux on cell viability. This has been confirmed with
Fig. 1. Viability of Saccharomyces cerevisiae relative to the cooling rate in a water–glycerol solution of 1.38 MPa. Bars represent confidence interval at 90% level.
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F. Dumont et al. / Cryobiology 46 (2003) 33–42
similar experiments using different initial osmotic pressures. Viability after cold stress as a function of the osmotic pressure of the medium used Preliminary results: cell volume and cell viability after an osmotic shock at 25 C Table 1 presents data on cell volume variations and cell viability after osmotic shock. The results show viabilities higher than 90% even for the highest osmotic level used (40 MPa). These results are in agreement with those of Beney et al. [5], who reported that S. cerevisiae can resist osmotic shock of up to about 50 MPa. When osmotic stresses were induced using a solution of water–glycerol at 40 MPa and water– polyethylene glycol (PEG) 600 at 14.5 MPa, minimum yeast volumes were 32% and 29.7%, respectively. These two volumes are different but the solutions are not comparable. Glycerol is permeant whereas PEG is non-permeant. During a glycerol-induced osmotic shock, there is an inflow of the permeant solute with an outflow of water, whereas during PEG-induced shock there is only water outflow and this leads to a fall in non-osmotic volume [11]. Obviously, glycerol permeates the cell following fickean diffusion. After a very rapid shrink of the cell due to only water exit (a few milliseconds) the fickean diffusion of glycerol accompanied by water occurred in about 25 s as demonstrated by Gervais and Beney [7]. So our measurements which were made after 15 min took into account this fickean entry and corresponded to the balanced state. Cells did not return to their initial volume because they initially contained glycerol (initial culture conditions is water/glycerol at aw ¼ 0:992) and because water exit flow rate and glycerol entry flow rate were different. The final cell volume obtained was depending on the control cell glycerol concentration and of the respective rate of water exit flow and glycerol entry flow which were functions of the osmotic pressure gradient. Non-osmotic volume corresponds to the minimum volume that a cell can reach after osmotic stress. It was reached at 14.5 MPa with the water– PEG 600 solution.
Viability results after cold stress To study the influence of cell volume before freezing, several water–glycerol media with osmotic pressures of 14.5, 30, or 40 MPa were used. The results for media at 14.5, 30, and 40 MPa are presented in Fig. 2. In experiments at osmotic pressures higher than 1.38 MPa, the same trend in yeast viability relative to cooling rate was found, but with higher mean viabilities. As described with water–glycerol medium at 1.38 MPa, the results can be divided into three ranges. With medium at 14.5 MPa (Fig. 2) and a cooling rate lower than 20 C min1 , cell viability remained significant (above 80%). With a cooling rate of 150–500 C min1 , viability decreased to less than 10%. With a cooling rate greater than 400 C min1 , cell viability increased and reached 60% at 1800 C min1 . For an osmotic pressure of 30 MPa (Fig. 2) and for cooling rates between 50 and 200 C min1 , the minimum cell viability was always higher than 55%. Below 5 C min1 and over 700 C min1 , cell viability was 100%. For cooling rates below 50 C min1 , cell viability was greater than 90%. At cooling rates from 50 C min1 to 500 C min1 , cell viability was below 90%. The minimum value for cell viability was 58% at a cooling rate of 100 C min1 . Above 500 C min1 , cell viability was again greater than 90%.
Fig. 2. Viability of Saccharomyces cerevisiae relative to the cooling rate in water–glycerol media (+, 1.38 MPa; r, 14.5 MPa; s, 30 MPa; , 40 MPa) and in PEG 600-water medium () with 14.5 MPa.
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In the presence of a water–glycerol medium at 40 MPa, there were still three ranges of cooling rates. As can be seen in Fig. 2 for cooling rates up to 75 C min1 , cell viability was above 90%. For cooling rates from 90 to 200 C min1 , cell viability was below 90%, with a minimum of 66% viability at a cooling rate of 110 C min1 . The use of PEG 600 allowed us to study the influence of glycerol as a cryoprotective agent. The water–PEG 600 solution at 14.5 MPa corresponds to a high cell volume decrease equivalent to the decrease obtained for water–glycerol medium at 40 MPa (see Table 1). The influence of the cooling rate on the cell viability obtained with these two solutions gives two curves relatively closed (Fig. 2) which exhibited three different viability compartments. At cooling rates below 110 C min1 , viability was 100%. For cooling rates from 110 to 320 C min1 , viability decreased, with 75% viability at a cooling rate of 260 C min1 . Compared with the results obtained for water– glycerol medium at 40 MPa, the zone of cell mortality in PEG 600 medium shifted towards higher cooling rates. This zone shifted from 50– 150 C min1 with glycerol to 150–300 C min1 with PEG 600.
Discussion The viability of S. cerevisiae relative to cooling rate has been the subject of numerous publications (e.g., [2,10,14,15,23]). It is interesting to compare the results described here with those of Mazur in (1961) [14], as in Fig. 3. From this figure, it is clear that at cooling rates below 1000 C min1 , our results are consistent with those of Mazur. However, our work is unique in having described the effects of cooling rates greater than 1000 C min1 on cell viability. Cell viability at very rapid cooling rates High levels of cell viability were observed after very rapid cooling. For a decrease in temperature corresponding to the difference in the freezing points of the intra- and extracellular media, the
39
Fig. 3. Comparison of data on the viability of Saccharomyces cerevisiae and cooling rate from Mazur 1961 (—) and from this paper (- - -).
variation in osmotic pressure due to external crystallization will cause intracellular water loss. However, the faster the cooling rate, the less water flows out. Medium at 1.38 MPa crystallizes at )0.8 C (Eq. (3)). If the turgor pressure of yeast is about 0.23 MPa [13], the difference in both freezing-point values will be about 0.3 C. Therefore, theoretically, water will flow from the cell during the time it takes to move between these two temperatures (i.e., )0.8 to )1.1 C). This time is a direct function of the cooling rate, as shown in Table 3. A value for the membrane hydraulic permeability of S. cerevisiae has recently been proposed by Martinez de Mara~ non and Gervais [12] using a mathematical model including the mixing time constant of 0.03 s: Lp ¼ 6:00 1011 m s1 Pa1 (i.e., 350 lm min1 atm1 ). The time constant s, corresponding to the membrane water transfer (assuming a first order system), is about 30 ms. Therefore, by comparing the value proposed in Table 3 with this value, we can deduce that at a cooling rate of 600 C min1 , the delay time is Table 3 Time necessary to reduce the temperature from the extracellular freezing point to the intracellular freezing point relative to cooling rate Cooling rate (C min1 )
Time for a decrease in temperature from )0.8 to )1.1 C (ms)
600 5000 10 000 20 000 30 000
30.0 3.6 1.8 0.9 0.6
40
F. Dumont et al. / Cryobiology 46 (2003) 33–42
comparable to the value of the time constant, so that most of water has flowed out of the cell. At 10 000 C min1 , the delay is 16 times smaller than the time constant and only about 6% of water would have flowed out of the cell. From 20 000 to 30 000 C min1 , the delay becomes shorter still and the water loss from the cell becomes almost zero. Therefore, we must consider the possibility that cell viability results from the lack of change in cell volume. For very rapid cooling rates, thermal flow is faster than the flow of water through the membrane, which then limits or eliminates the formation of an osmotic pressure gradient between the medium and the cell. Viability would therefore increase gradually with the increase in cooling rate. Furthermore, the time constant calculated by Martinez de Mara~ non and Gervais [12] was measured at 25 C and, as Lp has an activation energy, we can presume that the rate of outflow of cellular water at 25 C is higher than during freezing. Therefore, the previous calculation may have overestimated the rate of cell water outflow. Even if physical studies are subsequently undertaken, two hypotheses can explain these results: • formation of small intracellular crystals which do not damage the cell [15]; • vitrification of the intra- and extracellular media. Ice formation is related to the critical thermalcooling gradient when the transfer of thermal energy is more rapid than the transfer of mass [15]. Furthermore, the higher the cooling rate, the smaller the sizes of crystals. Therefore, the more rapid the increase, the less transmembrane water outflow and the more intracellular ice crystals will form, until optimal conditions for vitrification are reached. The vitreous state is an unstable thermodynamic state in which high viscosity and low temperature prevent the crystallization of molecules. Transition from the liquid state to the vitreous state does not produce a change in volume. High cooling rates and high concentrations promote vitrification [1]. The concentration that allows vitrification to occur is related to the kinetics of the temperature. These two hypotheses are neither exclusive nor incompatible.
Influence of slower cooling rates on yeast viability If the results of this study are combined with those of Mazur, the curve proposed in Fig. 3 indicates that the trend in viability relative to cooling rate can be divided into four ranges: Range A0 : very slow cooling rates and low viability; Range A: low cooling rate and high viability; Range B: intermediate cooling rate and low viability; Range C: very rapid cooling rate and high viability. For the three first ranges, A0 , A, and B, an analogy between freezing and osmotic pressure effects can be drawn in the light of previous results [5,8,9,11,12] which demonstrated that yeast viability is significantly influenced by osmotic pressure kinetics. For Range A0 , an analogy with very low osmotic pressure gradients can be drawn [22]. In this case, water outflow is slower than optimal water outflow and causes mortality in yeast cells. Even if this result is not yet understood completely, it underlines the analogy between cell viability after osmotic and cold stresses. During freezing of the extracellular medium, which occurs first, crystallization of the water causes an instantaneous increase in the concentration of the external medium, which immediately induces an osmotic response in the cell. The rate of osmotic pressure variation during freezing can be calculated using Eq. (3). For example, a cooling rate of 5 C min1 corresponds to a rate of increase in osmotic pressure of 6 MPa min1 (i.e., about 1 atm s1 ), and a cooling rate of 200 C min1 corresponds to a rate of change in osmotic pressure of 240 MPa min1 . In these examples, the first cooling rate caused no mortality but the second cooling rate caused cell viability to drop to 10%. The same results have been reported previously [11] with osmotic perturbation in S. cerevisiae at 25 C. At 25 C it was shown that a low rate of osmotic pressure variation, between 0.5 and 1.4 MPa min1 (or about 0.08 and 0.23 atm s1 ), caused less mortality (i.e., viability of 90% and 76%, respectively, relative to controls) than a high rate of osmotic pressure variation at 6000 MPa
F. Dumont et al. / Cryobiology 46 (2003) 33–42
min1 (viability fell to 20%). Recent works have shown that for lower temperature (i.e., lower than 12 C) application of an osmotic shock was found to be less detrimental [5]. Similar results have been found for the bacteria Lactobacillus plantarum [22]. Moreover, if dehydration is slower than this optimal osmotic gradient, mortality increases [22]. The rate at which water leaves the cell has direct consequences on the cell integrity. Indeed, a fast exit could induce a fast modification of the cell surface/volume ratio which could be detrimental for the cell due to membrane destabilisation as proposed by Steponkus in 1987 [24]. Such destabilisation, as vesiculation for example, would not necessarily occur for low water flow exit from the cell as the membrane could have time to physically react to such modifications. The mortality observed for ranges A and B can be explained by this hypothesis. Influence of yeast intracellular water volume and cooling rates on cell viability To highlight the influence of transmembrane water outflow on cold thermal stress, preliminary osmotic shocks were used to reduce the intracellular water volume. It appears that, under similar freezing conditions, the lower the cell volume before cold stress, the greater the cell viability. For cells that have reached their non-osmotic volume (i.e., about 30% of normal yeast volume), identical results for glycerol and PEG 600 media were observed. The intracellular quantity of water before freezing seems to determine the cellÕs resistance to shock. When the cell volume is reduced before freezing, there will be neither massive outflow of intracellular water nor crystallization during the decrease in temperature. The combined effects of water outflow and membrane-lipid phase shift [5] and the formation of lethal intracellular ice crystals [16] cannot occur. For glycerol-based solutions, vitrification may be involved in the protection of cells. High glycerol concentrations and rapid cooling rates promote the transition to a vitreous state. The higher the concentration of glycerol, the lower the cooling rate required for vitrification. A solution with 45% (w/w) glycerol vitrifies completely at cooling rates higher than
41
70 C min1 , whereas a solution with 50% glycerol vitrifies with cooling rates of 38 C min1 [3]. The combination of high glycerol concentrations and rapid cooling rates explains the maintenance of yeast viability.
Conclusions The study of cell viability as a function of cooling rate has allowed us to define four distinct ranges: the first range (A0 ) consists of very low cooling rates resulting in high cell mortality. The second range (A) corresponds to low cooling rates during which cell water outflow occurs slowly and does not damage the cells. The third range (B) is characterized by high cooling rates that induce competition between heat and water outflow from the cell. In this case, massive water outflow, which is related to the increase in extracellular osmotic pressure, causes cell death. The fourth range (C) corresponds to very high cooling rates that induce very high rates of heat flow. Vitrification or crystallization of the intracellular medium, before any water loss, is assumed to preserve high cell viability. We propose that there is a major link between cooling rate, the velocity of water outflow from the cell across the plasma membrane, and cell mortality. Further work will determine the effective state of the intracellular medium after freezing at very high cooling rates, to quantify the vitreous and crystallized distribution.
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[14] P. Mazur, Physical and temporal factors involved in the death of yeast at subzero temperatures, Biophys. J. 1 (1961) 247–264. [15] P. Mazur, Theoretical and experimental effects of cooling and warming velocity on the survival of frozen and thawed cells, Cryobiology 2 (4) (1966) 181–192. [16] P. Mazur, Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing, J. Gen. Physiol. 47 (1963) 347–369. [17] P. Mazur, Cryobiology: the freezing of biological systems, Science 168 (1970) 939–949. [18] G.J. Morris, G.E. Coulson, K.-J. Clarke, Freezing injury in Saccharomyces cerevisiae: the effect of growth conditions, Cryobiology 25 (1988) 471–482. [19] K. Muldrew, L.E. McGann, Mechanisms of intracellular ice formation, Biophys. J. 57 (1990) 525–532. [20] N. Niedermeyer, G.R. Parrish, H. Moor, Reaction of yeasts cells to glycerol treatment, Protoplasma 92 (1977) 177–193. [21] R.S. Norrish, An equation for the activity coefficients and equilibrium relative humidities of water in confectionery syrups, J. Food Technol. 1 (1966) 25–39. [22] I. Poirier, P.A. Marechal, P. Gervais, Effects of the kinetics of water potential variation on bacterial viability, J. Appl. Microbiol. 82 (1997) 101–106. [23] G.J. Schwartz, K.R. Diller, Osmotic response of individual cells during freezing, Cryobiology 20 (1983) 542–552. [24] P. Steponkus, Membrane destabilisation resulting from freeze-induced dehydration, Cryobiology 24 (1987) 555. [25] M. Toner, E.G. Cravalho, Thermodynamics and kinetics of intracellular ice formation during freezing of biological cells, J. Appl. Physiol. 67 (1990) 1582–1593.
Influence of cooling rate on Saccharomyces cerevisiae destruction during freezing: unexpected viability at ultra-rapid cooling ratesq Frederic Dumont, Pierre-Andre Marechal, and Patrick Gervais* Laboratoire de G enie des Proc ed es Alimentaires et Biotechnologiques, ENSBANA, 1, Esplanade Erasme, 21000 Dijon, France Received 7 May 2002; accepted 12 November 2002
Abstract The purpose of this work was to study cell viability as a function of cooling rate during freezing. Cooling rate strongly influences the viability of cells during cold thermal stress. One of the particularities of this study was to investigate a large range of cooling rates and particularly very rapid cooling rates (i.e., faster than 20 000 C min1 ). Four distinct ranges of cooling rates were identified. The first range (A0 ) corresponds to very slow cooling rates (less than 5 C min1 ), and results in high cell mortality. The second range (A) corresponds to low cooling rates (5–100 C min1 ), at which cell water outflow occurs slowly and does not damage the cells. The third range (B) corresponds to rapid cooling rates (100–2000 C min1 ), at which there is competition between heat flow and water flow. In this case, massive water outflow, which is related to the increase in extracellular osmotic pressure and the membrane-lipid phase transition, can cause cell death. The fourth range (C) corresponds to very high cooling rates (more than 5000 C min1 ), at which the heat flow is very rapid and partially prevents water exit, which seems to preserve cell viability. 2002 Elsevier Science (USA). All rights reserved. Keywords: Ultra-rapid cooling rate; Saccharomyces cerevisiae; Cell viability; Osmotic pressure
The freeze-thawing process is a method that allows the preservation of microorganism specimens without the gain of genetic modifications or the loss of functional characteristics. However, despite many scientific studies, the mechanisms underlying cell freezing injury are not yet well understood. The optimal method chosen for cell cryoconservation is often decided on empirical q
This work was funded by institutional sources. Corresponding author. Fax: +33-03-80-39-66-11. E-mail address: [email protected] (P. Gervais). *
considerations. Many procedures, with or without cryoprotective agents, have been reported. Cell freezing, which is commonly used in the food and pharmaceutical industries, requires optimization of the process for each type of microorganism [2]. It is important that the mechanisms of freezing injury be understood if techniques that circumvent this damage are to be developed. The freeze-thawing process constitutes a double stress for the cell: thermal stress and hyperosmotic stress, which act simultaneously during cooling [18,19,21]. Because the cytoplasm is more
0011-2240/02/$ - see front matter 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S0011-2240(02)00161-X
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F. Dumont et al. / Cryobiology 46 (2003) 33–42
concentrated than the growth medium, the external water freezes before the cell contents. This increases the osmotic pressure of the medium. The extracellular solutes are concentrated in the remaining unfrozen extracellular water, and this dehydrates the cell by osmosis as water diffuses from the cytoplasm into the more concentrated external solution. The lower the temperature of the medium, the greater the concentration of the unfrozen solution and the greater the loss of intracellular water. This mass transfer is restricted by the water–membrane permeability value of the cell (Lp ). The osmotic water outflow that occurs from cells during freezing was considered by Muldrew and McGann [19] to be responsible for rupturing the plasma membrane, thus allowing extracellular ice to propagate into the cytoplasm. The hypothesis that osmotic flow leads to cell death [19] must be considered. In previous studies [5,8,9,11,12], the influence of the intensity and kinetics of osmotic pressure variations have been remarked and related to cell viability. An increase in the osmotic pressure gradient has dramatic effects on cell viability. Previous observations on yeast cells at 25 C [7,10] have shown that a too rapid exit of water (or rapid change in osmotic pressure) would damage the cells. Recent works have shown that for lower temperature (i.e., lower than 12 C) application of an osmotic shock was found to be less detrimental [5]. Complementary works on bacteria [21] and yeast cells (not published) have also shown that a too slow variation would also induced a high cell mortality for unknown reason by now. Two principal damage mechanisms associated with cooling rate have been suggested. For low cooling rates, the extracellular solutes concentrate in the remaining unfrozen extracellular water and cause cell dehydration by osmosis as water diffuses from the cytoplasm into the more concentrated external solution. In the second case, high cooling rates result in intracellular ice formation (IIF) during freezing; IIF is lethal for the cell [17]. There are two opinions regarding IIF. Toner and Cravalho [25] suggested that IIF occurs as a result of nucleation within the cell, but Muldrew and McGann [19] suggested that rupture of the plasma
membrane is due to the difference in osmotic pressure that develops across the plasma membrane during rapid cooling, which enables extracellular ice to propagate into the cytoplasm. The cooling rate is a parameter that strongly influences the behavior of cells during cold thermal stress. Ice first forms exclusively in the extracellular compartment because the concentration of ions and solutes is higher in the cell than in the extracellular medium due to the maintain of cell turgor pressure which has been shown in a previous work [13]. In the same way, thermodynamically, the compartment with the larger volume will nucleate first. The intracellular freezing point is slightly lower (about 0.3 C) [16]. The initiation of external freezing creates an osmotic gradient between the cell and its external environment due to the concentration of extracellular electrolytes. If a slow cooling rate is used, intracellular water has time to flow out of the cell, but if a very rapid cooling rate is used, water cannot flow out. In the literature, various cooling rates have been reported. Mazur [14] subjected Saccharomyces cerevisiae to various freeze-thawing procedures and observed a sharp decrease in survival when the cooling rate was increased from 1 C min1 to values in the order of 100 C min1 . Faster cooling rates were not investigated. The purpose of this work was to study cell viability during freezing and particularly at very rapid cooling rates (i.e., faster than 20 000 C min1 ). We based the study on recent research we have done on the relationship between cooling rates, osmotic pressure variations, and cell viability. A large range of cooling rates was investigated to study cell viability as a function of cooling rate.
Materials and methods Biological materials The yeast S. cerevisiae CBS 1171 was used in this study. Cultures were grown in 250-mL conical flasks containing 100 mL aerated modified Wickerham medium, at 250 rpm and 25 C. This medium is composed of 10 g glucose, 3 g pancreatic peptone, 3 g yeast extract, and 1.5 g Na2 HPO4 in
F. Dumont et al. / Cryobiology 46 (2003) 33–42
1 L distilled water. The pH of the medium was adjusted to 5.35 by adding orthophosphoric acid. Inoculation was carried out with a 0.1-mL yeast suspension from a 48-h sub-culture grown under analogous conditions. Cells were harvested after 65 h of growth (stationary phase) and used immediately for experiments. Thermodynamic characteristics of the freezing medium To maintain control of the osmotic pressure of the cell suspension, the cells were not frozen in growth medium. A 20 mL volume of yeast culture was centrifuged at 4000 rpm for 5 min. The pellet was resuspended in 20 mL of the appropriate binary solution. The osmotic disturbance thus provoked in the form of osmotic shock did not allow the cells to adapt physiologically [5]. Freezing were occurred 15 min after osmotic shock. Several binary media were used for freezing (Table 1). They were chosen for their water activity and the initial cellular volume they determined. The molar fraction of solute to be added to 1000 g of water is given by NorrishÕs equation [21]: 2
aw ¼ ð1 Xs Þ ekXs ;
ð1Þ
where aw is water activity, Xs is the molar fraction of the solute, and k is the Norrish coefficient related to the solute (1.16 for glycerol; 57.6 for PEG 600). Solutions were then checked using aqualab CX-2 water activity system (Decagon Devices, USA).
35
Water activity (aw ) can be expressed in terms of osmotic pressure (p) as follows: P¼
RT ln aw ; Vw
ð2Þ
where p is the osmotic pressure (Pa), R the gas constant (8.31 J mol1 K1 ), T the absolute temperature (K), and Vw the partial molar volume of water (18 106 m3 mol1 ). The nucleation temperature was not controlled but the freezing temperature in terms of the water activity of the solution can be calculated with Eq. (3) [4]: Lnðaw Þ ¼ 9:69 103 DTm 5:06106 DTm2 ð3Þ with DTm ¼ ðTo Tp Þ where To (K) is the freezing point of the solvent and Tp (K) is the freezing point of the solution. Cell volume and cell viability after osmotic shock When a cell is transferred from one solution to another of higher osmolality containing a permeating solute, it rapidly shrinks initially because of the loss in water and then slowly increases in volume as the solute permeates accompanied with water [20]. The final cell volume obtained was depending on the initial cell glycerol concentration and of the respective rate of water exit flow and glycerol entry flow which were functions of the osmotic pressure gradient. Cell shrinkage was measured by image analysis, by comparing cell volumes before and after
Table 1 Volumes and viabilities of Saccharomyces cerevisiae after different osmotic shocks Binary solutions
aw a
Osmotic pressure p (MPa)
Mass of solute added to 1000 g of water
Cell volume measured after osmotic shocks (% relative to initial volume)
Viability after osmotic shock
Water–glycerol Water–glycerol Water–glycerol Water–glycerol Water–PEG 600
0.992 0.900 0.800 0.750 0.900
1.38 14.5 30.0 40.0 14.5
51.1 513.8 1063.2 1363.7 1208.9
100% 63% 42.5% 32% 29.7%
99% 97% 96% 94% 93%
a
Water activity verified with a dew point osmometer.
36
F. Dumont et al. / Cryobiology 46 (2003) 33–42
osmotic shock. Berner and Gervais [6] have already described the system. Measurements were made for more than 300 cells, for both the control yeast population and the stressed cells, at the maximum 15 min after the stress in order not to take into account the entry of the solute. The volume variations are expressed as a percentage of the initial volume of the control population.
Freezing and thawing methods Freezing was always carried out in liquid nitrogen at )196 C but two methods were used. First, various cooling rates were obtained with various containers: Cryotube Nunc (Vials, Germany); Cryotube (Sarstedt, Germany); metallic Micro-Syringe for HPLC (Exmire MS-C10, UK); and with various volumes of solutions placed in these containers (Table 2). For low cooling rates
(i.e., 5 and 6 C min1 ), samples in cryotubes were placed in 50-mL plastic tubes (PolyLabo, France) for centrifugation to protect them from the cold gradient with a gas layer. The second method was used to achieve the fastest cooling rates (more than 10 000 C min1 ). It was based on the principle of cells dropped directly into liquid nitrogen without any protection. Cells were fixed on different supports: Cryo-Billes plastic pellets (Laboratoire AES, France) with a mean diameter of 3.3 mm, or thermocouple T (TCSA Dardilly, France) with a diameter of 0.5 mm. These supports were retained for their natural property to fix the cells on their surface. For the fastest cooling rate (30 000 C min1 ) the support was constituted only by the temperature sensor which gave us effective measurement of 15 000 C min1 . Thanks to this measurement, the cooling rate in the first 20 lm, corresponding to the thickness of solute around the thermocouple, was calculated as being 30 000 C min1 .
Table 2 Cooling rates measured for several supports and conditions in different media Cooling rate (C min1 ) Volume of the sample (mL)
Water– glycerol 1.38 MPa
Cell in suspension Cryotube Sarstedt in plastic tube Cryotube Nunc in plastic tube Cryotube Nunc Cryotube Nunc Cryotube Sarstedt Cryotube Nunc Cryotube Nunc Cryotube Sarstedt Cryotube Sarstedt Cryotube Sarstedt Cylinder in glass Cylinder in glass Cylinder in glass Micro syringe
1
5
1 1.2 1 1 0.5 0.1 0.5 0.1 0.2 0.1 0.05 0.01 0.005
6
1800 4000 5000 17 000a
Immobilized cells Sphere in plastic Thermocouple in metal Thermocouple in metal
0.0025 0.0014 0.0014
12 000a 15 000 30 000a
a
Value calculated from FourierÕs second law.
120 180 250 270 650 700
Water– glycerol 14.5 MPa
Water– glycerol 30 MPa
Water– glycerol 40 MPa
Water– PEG600 14.5 MPa
10
26
20
160 240 140
80 100 160 120
100 110 350 120
115 260 170
210 700
280
290
520
320
280
1800
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The temperature was measured with a T thermocouple (copper/copper–nickel), which was connected to an ‘‘InstruNet 100’’ acquisition card (GWI, USA). A maximum of 60 000 measurements per second were made with this system, which could then be evaluated using a spreadsheet. The cooling rate was calculated from these measurements. During the experiments, the influence of thawing was not studied. Thawing was always carried out by dropping the frozen sample into a controlled-temperature water bath at 37 C. Indeed, Mazur [15] showed that it is necessary to thaw as rapidly as possible, to avoid conditions that could lead to recrystallization of the medium.
Viability measurement Viability measurements were made using the colony-forming unit (CFU) method, by plating cells in MW medium supplemented with 15 g L1 agar. Petri dishes were then incubated for 2 d at 25 C. Viability was determined by comparing the experimental and control microorganisms. We performed at least three repetitions for each experiment.
37
Results Viability after cold stress relative to cooling rate Cold stress was applied first in a water–glycerol solution at 1.38 MPa which corresponds to the osmotic pressure of classical growth medium. The results obtained in this experiment can be broken up into three cooling-rate categories, as illustrated in Fig. 1. For cooling rates lower than 10 C min1 (i.e., 5 and 6 C min1 ), cells remained viable. The results were different for cooling rates from 100 to 1000 C min1 . Indeed, yeast viability fell to a low point of 2% for 180 C min1 , and remained stable at about 10%. For cooling rates greater than 1400 C min1 , yeast viability increased from 20% at 6000 C min1 to 50% at 17 000 C min1 (estimated value) until 80.5% at 30 000 C min1 (estimated value). The highest cooling rates were estimated values but confirmed the significant trend in increasing yeast viability. To limit the influence of the cooling rate on cell viability, we decreased the initial intracellular water volume using a preliminary hyperosmotic shock. Less water in the cell before freezing makes it possible to limit the possible effects of water efflux on cell viability. This has been confirmed with
Fig. 1. Viability of Saccharomyces cerevisiae relative to the cooling rate in a water–glycerol solution of 1.38 MPa. Bars represent confidence interval at 90% level.
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F. Dumont et al. / Cryobiology 46 (2003) 33–42
similar experiments using different initial osmotic pressures. Viability after cold stress as a function of the osmotic pressure of the medium used Preliminary results: cell volume and cell viability after an osmotic shock at 25 C Table 1 presents data on cell volume variations and cell viability after osmotic shock. The results show viabilities higher than 90% even for the highest osmotic level used (40 MPa). These results are in agreement with those of Beney et al. [5], who reported that S. cerevisiae can resist osmotic shock of up to about 50 MPa. When osmotic stresses were induced using a solution of water–glycerol at 40 MPa and water– polyethylene glycol (PEG) 600 at 14.5 MPa, minimum yeast volumes were 32% and 29.7%, respectively. These two volumes are different but the solutions are not comparable. Glycerol is permeant whereas PEG is non-permeant. During a glycerol-induced osmotic shock, there is an inflow of the permeant solute with an outflow of water, whereas during PEG-induced shock there is only water outflow and this leads to a fall in non-osmotic volume [11]. Obviously, glycerol permeates the cell following fickean diffusion. After a very rapid shrink of the cell due to only water exit (a few milliseconds) the fickean diffusion of glycerol accompanied by water occurred in about 25 s as demonstrated by Gervais and Beney [7]. So our measurements which were made after 15 min took into account this fickean entry and corresponded to the balanced state. Cells did not return to their initial volume because they initially contained glycerol (initial culture conditions is water/glycerol at aw ¼ 0:992) and because water exit flow rate and glycerol entry flow rate were different. The final cell volume obtained was depending on the control cell glycerol concentration and of the respective rate of water exit flow and glycerol entry flow which were functions of the osmotic pressure gradient. Non-osmotic volume corresponds to the minimum volume that a cell can reach after osmotic stress. It was reached at 14.5 MPa with the water– PEG 600 solution.
Viability results after cold stress To study the influence of cell volume before freezing, several water–glycerol media with osmotic pressures of 14.5, 30, or 40 MPa were used. The results for media at 14.5, 30, and 40 MPa are presented in Fig. 2. In experiments at osmotic pressures higher than 1.38 MPa, the same trend in yeast viability relative to cooling rate was found, but with higher mean viabilities. As described with water–glycerol medium at 1.38 MPa, the results can be divided into three ranges. With medium at 14.5 MPa (Fig. 2) and a cooling rate lower than 20 C min1 , cell viability remained significant (above 80%). With a cooling rate of 150–500 C min1 , viability decreased to less than 10%. With a cooling rate greater than 400 C min1 , cell viability increased and reached 60% at 1800 C min1 . For an osmotic pressure of 30 MPa (Fig. 2) and for cooling rates between 50 and 200 C min1 , the minimum cell viability was always higher than 55%. Below 5 C min1 and over 700 C min1 , cell viability was 100%. For cooling rates below 50 C min1 , cell viability was greater than 90%. At cooling rates from 50 C min1 to 500 C min1 , cell viability was below 90%. The minimum value for cell viability was 58% at a cooling rate of 100 C min1 . Above 500 C min1 , cell viability was again greater than 90%.
Fig. 2. Viability of Saccharomyces cerevisiae relative to the cooling rate in water–glycerol media (+, 1.38 MPa; r, 14.5 MPa; s, 30 MPa; , 40 MPa) and in PEG 600-water medium () with 14.5 MPa.
F. Dumont et al. / Cryobiology 46 (2003) 33–42
In the presence of a water–glycerol medium at 40 MPa, there were still three ranges of cooling rates. As can be seen in Fig. 2 for cooling rates up to 75 C min1 , cell viability was above 90%. For cooling rates from 90 to 200 C min1 , cell viability was below 90%, with a minimum of 66% viability at a cooling rate of 110 C min1 . The use of PEG 600 allowed us to study the influence of glycerol as a cryoprotective agent. The water–PEG 600 solution at 14.5 MPa corresponds to a high cell volume decrease equivalent to the decrease obtained for water–glycerol medium at 40 MPa (see Table 1). The influence of the cooling rate on the cell viability obtained with these two solutions gives two curves relatively closed (Fig. 2) which exhibited three different viability compartments. At cooling rates below 110 C min1 , viability was 100%. For cooling rates from 110 to 320 C min1 , viability decreased, with 75% viability at a cooling rate of 260 C min1 . Compared with the results obtained for water– glycerol medium at 40 MPa, the zone of cell mortality in PEG 600 medium shifted towards higher cooling rates. This zone shifted from 50– 150 C min1 with glycerol to 150–300 C min1 with PEG 600.
Discussion The viability of S. cerevisiae relative to cooling rate has been the subject of numerous publications (e.g., [2,10,14,15,23]). It is interesting to compare the results described here with those of Mazur in (1961) [14], as in Fig. 3. From this figure, it is clear that at cooling rates below 1000 C min1 , our results are consistent with those of Mazur. However, our work is unique in having described the effects of cooling rates greater than 1000 C min1 on cell viability. Cell viability at very rapid cooling rates High levels of cell viability were observed after very rapid cooling. For a decrease in temperature corresponding to the difference in the freezing points of the intra- and extracellular media, the
39
Fig. 3. Comparison of data on the viability of Saccharomyces cerevisiae and cooling rate from Mazur 1961 (—) and from this paper (- - -).
variation in osmotic pressure due to external crystallization will cause intracellular water loss. However, the faster the cooling rate, the less water flows out. Medium at 1.38 MPa crystallizes at )0.8 C (Eq. (3)). If the turgor pressure of yeast is about 0.23 MPa [13], the difference in both freezing-point values will be about 0.3 C. Therefore, theoretically, water will flow from the cell during the time it takes to move between these two temperatures (i.e., )0.8 to )1.1 C). This time is a direct function of the cooling rate, as shown in Table 3. A value for the membrane hydraulic permeability of S. cerevisiae has recently been proposed by Martinez de Mara~ non and Gervais [12] using a mathematical model including the mixing time constant of 0.03 s: Lp ¼ 6:00 1011 m s1 Pa1 (i.e., 350 lm min1 atm1 ). The time constant s, corresponding to the membrane water transfer (assuming a first order system), is about 30 ms. Therefore, by comparing the value proposed in Table 3 with this value, we can deduce that at a cooling rate of 600 C min1 , the delay time is Table 3 Time necessary to reduce the temperature from the extracellular freezing point to the intracellular freezing point relative to cooling rate Cooling rate (C min1 )
Time for a decrease in temperature from )0.8 to )1.1 C (ms)
600 5000 10 000 20 000 30 000
30.0 3.6 1.8 0.9 0.6
40
F. Dumont et al. / Cryobiology 46 (2003) 33–42
comparable to the value of the time constant, so that most of water has flowed out of the cell. At 10 000 C min1 , the delay is 16 times smaller than the time constant and only about 6% of water would have flowed out of the cell. From 20 000 to 30 000 C min1 , the delay becomes shorter still and the water loss from the cell becomes almost zero. Therefore, we must consider the possibility that cell viability results from the lack of change in cell volume. For very rapid cooling rates, thermal flow is faster than the flow of water through the membrane, which then limits or eliminates the formation of an osmotic pressure gradient between the medium and the cell. Viability would therefore increase gradually with the increase in cooling rate. Furthermore, the time constant calculated by Martinez de Mara~ non and Gervais [12] was measured at 25 C and, as Lp has an activation energy, we can presume that the rate of outflow of cellular water at 25 C is higher than during freezing. Therefore, the previous calculation may have overestimated the rate of cell water outflow. Even if physical studies are subsequently undertaken, two hypotheses can explain these results: • formation of small intracellular crystals which do not damage the cell [15]; • vitrification of the intra- and extracellular media. Ice formation is related to the critical thermalcooling gradient when the transfer of thermal energy is more rapid than the transfer of mass [15]. Furthermore, the higher the cooling rate, the smaller the sizes of crystals. Therefore, the more rapid the increase, the less transmembrane water outflow and the more intracellular ice crystals will form, until optimal conditions for vitrification are reached. The vitreous state is an unstable thermodynamic state in which high viscosity and low temperature prevent the crystallization of molecules. Transition from the liquid state to the vitreous state does not produce a change in volume. High cooling rates and high concentrations promote vitrification [1]. The concentration that allows vitrification to occur is related to the kinetics of the temperature. These two hypotheses are neither exclusive nor incompatible.
Influence of slower cooling rates on yeast viability If the results of this study are combined with those of Mazur, the curve proposed in Fig. 3 indicates that the trend in viability relative to cooling rate can be divided into four ranges: Range A0 : very slow cooling rates and low viability; Range A: low cooling rate and high viability; Range B: intermediate cooling rate and low viability; Range C: very rapid cooling rate and high viability. For the three first ranges, A0 , A, and B, an analogy between freezing and osmotic pressure effects can be drawn in the light of previous results [5,8,9,11,12] which demonstrated that yeast viability is significantly influenced by osmotic pressure kinetics. For Range A0 , an analogy with very low osmotic pressure gradients can be drawn [22]. In this case, water outflow is slower than optimal water outflow and causes mortality in yeast cells. Even if this result is not yet understood completely, it underlines the analogy between cell viability after osmotic and cold stresses. During freezing of the extracellular medium, which occurs first, crystallization of the water causes an instantaneous increase in the concentration of the external medium, which immediately induces an osmotic response in the cell. The rate of osmotic pressure variation during freezing can be calculated using Eq. (3). For example, a cooling rate of 5 C min1 corresponds to a rate of increase in osmotic pressure of 6 MPa min1 (i.e., about 1 atm s1 ), and a cooling rate of 200 C min1 corresponds to a rate of change in osmotic pressure of 240 MPa min1 . In these examples, the first cooling rate caused no mortality but the second cooling rate caused cell viability to drop to 10%. The same results have been reported previously [11] with osmotic perturbation in S. cerevisiae at 25 C. At 25 C it was shown that a low rate of osmotic pressure variation, between 0.5 and 1.4 MPa min1 (or about 0.08 and 0.23 atm s1 ), caused less mortality (i.e., viability of 90% and 76%, respectively, relative to controls) than a high rate of osmotic pressure variation at 6000 MPa
F. Dumont et al. / Cryobiology 46 (2003) 33–42
min1 (viability fell to 20%). Recent works have shown that for lower temperature (i.e., lower than 12 C) application of an osmotic shock was found to be less detrimental [5]. Similar results have been found for the bacteria Lactobacillus plantarum [22]. Moreover, if dehydration is slower than this optimal osmotic gradient, mortality increases [22]. The rate at which water leaves the cell has direct consequences on the cell integrity. Indeed, a fast exit could induce a fast modification of the cell surface/volume ratio which could be detrimental for the cell due to membrane destabilisation as proposed by Steponkus in 1987 [24]. Such destabilisation, as vesiculation for example, would not necessarily occur for low water flow exit from the cell as the membrane could have time to physically react to such modifications. The mortality observed for ranges A and B can be explained by this hypothesis. Influence of yeast intracellular water volume and cooling rates on cell viability To highlight the influence of transmembrane water outflow on cold thermal stress, preliminary osmotic shocks were used to reduce the intracellular water volume. It appears that, under similar freezing conditions, the lower the cell volume before cold stress, the greater the cell viability. For cells that have reached their non-osmotic volume (i.e., about 30% of normal yeast volume), identical results for glycerol and PEG 600 media were observed. The intracellular quantity of water before freezing seems to determine the cellÕs resistance to shock. When the cell volume is reduced before freezing, there will be neither massive outflow of intracellular water nor crystallization during the decrease in temperature. The combined effects of water outflow and membrane-lipid phase shift [5] and the formation of lethal intracellular ice crystals [16] cannot occur. For glycerol-based solutions, vitrification may be involved in the protection of cells. High glycerol concentrations and rapid cooling rates promote the transition to a vitreous state. The higher the concentration of glycerol, the lower the cooling rate required for vitrification. A solution with 45% (w/w) glycerol vitrifies completely at cooling rates higher than
41
70 C min1 , whereas a solution with 50% glycerol vitrifies with cooling rates of 38 C min1 [3]. The combination of high glycerol concentrations and rapid cooling rates explains the maintenance of yeast viability.
Conclusions The study of cell viability as a function of cooling rate has allowed us to define four distinct ranges: the first range (A0 ) consists of very low cooling rates resulting in high cell mortality. The second range (A) corresponds to low cooling rates during which cell water outflow occurs slowly and does not damage the cells. The third range (B) is characterized by high cooling rates that induce competition between heat and water outflow from the cell. In this case, massive water outflow, which is related to the increase in extracellular osmotic pressure, causes cell death. The fourth range (C) corresponds to very high cooling rates that induce very high rates of heat flow. Vitrification or crystallization of the intracellular medium, before any water loss, is assumed to preserve high cell viability. We propose that there is a major link between cooling rate, the velocity of water outflow from the cell across the plasma membrane, and cell mortality. Further work will determine the effective state of the intracellular medium after freezing at very high cooling rates, to quantify the vitreous and crystallized distribution.
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