Cell Size and Water Permeability as Determining Factors for Cell Viability after Freezing at Different Cooling Rates

This work studied the viabilities of five types of cells (twoyeast cells, Saccharomyces cerevisiae CBS 1171 and Candida utilis;two bacterial strains, Escherichia coli and Lactobacillus plantarum;and one human leukemia K562 cell) as a function of cooling rateduring freezing. The range of investigated cooling rates extendedfrom 5 to 30,000°C/min. Cell viability was classified intothree ranges: (i) high viability for low cooling rates (5 to180°C/min), which allow cell water outflow to occur completelyand do not allow any intracellular crystallization; (ii) lowviability for rapid cooling rates (180 to 5,000°C/min),which allow the heat flow to prevail over water outflow (inthis case, cell water crystallization would occur as water wasflowing out of the cell); (iii) high viability for very highcooling rates (>5,000°C/min), which allow the heat flowto be very rapid and induce intracellular crystallization and/orvitrification before any water outflow from the cell. Finally,an assumption relating cell death to the cell water crystallizationas water is flowing out of the cell is made. In addition, thisgeneral cell behavior is different for each type of cell andseems to be moderated by the cell size, the water permeabilityproperties, and the presence of a cell wall.

INTRODUCTION

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Introduction

The freeze-thawing process remains the principal method of cellpreservation to date, and the high survival rates achieved bythis method are of interest from both the biophysical and practicalpoints of view. This is to ensure that the recovery of entirecell populations is free from the risk of possible subsequentalteration of its genetic composition.

Cell cryopreservation, which is commonly used in the food andpharmaceutical industries, requires optimization for each typeof microorganism. Moreover, each type of cell has its own protocolfor freezing. Numerous researchers have attempted to developmethods that permit 100% preservation of freeze-thawing of diversecellular specimens (3, 6), but some microorganisms cannot yetbe preserved by freezing.

For a better cell preservation some cryoprotectants such asglycerol or dimethyl sulfoxide can be used (8). These moleculesimprove the cell preservation by minimizing the cell water content(6) and/or supporting the vitrification occurrence (1) and finallyby protecting the cell's constitutive macromolecules (2, 5).

The freeze-thawing process constitutes a double stress for thecell, i.e., thermal and hyperosmotic stresses, which act simultaneouslyduring cooling (15, 16). The scenario of cell evolution duringslow freezing is well known (13). The water surrounding thecell freezes before the cell contents, because the cytoplasmis more concentrated than the growth medium, and because thermodynamically,the component with the largest volume will nucleate first (6,16). This freezing increases the osmotic pressure of the medium,and the extracellular solutes become concentrated in the remainingliquid extracellular water. Consecutive osmosis will then dehydratecells as water diffuses from the cytoplasm into the more concentratedexternal solution. If the water outflow is faster than the heatoutflow, then progressively, as the temperature decreases, thewater contained in the medium will freeze and the osmotic pressureof the medium will continuously increase. The lower the temperatureof the medium, the greater the concentration of the unfrozensolution and the greater the loss of intracellular water. Thismass transfer is restricted by the hydraulic permeability (Lp)of the cell membrane.

In a previous study (6), we have shown that the cooling rateplays a major role in yeast cell preservation. Furthermore,it was shown that contrary to the generally proposed concept,low cooling rates are not the only ones that can be used forcell preservation; in fact, by using the yeast Saccharomycescerevisiae, we have shown that very rapid cooling rates makeit possible to obtain a cell viability equivalent to that obtainedwith low cooling rates.

The aim of this work was to study the viabilities of differenttypes of cells (e.g., yeast cells, bacterial cells, and humancells) as a function of applied cooling rate. A large rangeof cooling rates, between 5 and 30,000°C/min, were investigated.Then, the properties of the cells were used to explain the cellviability results.

MATERIALS AND METHODS

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Materials and Methods

(i) Biological materials.
Five types of cells were used in this study: two yeast cells(S. cerevisiae and Candida utilis), two bacterial strains (Escherichiacoli and Lactobacillus plantarum), and one human cell (humanmyelogenous leukemia cells).

Cultures of the yeast S. cerevisiae CBS 1171 were grown in 250-mlconical flasks containing 100 ml of aerated, modified Wickerhammedium and stirred at 250 rpm, while being maintained at 25°C.The medium was synthesized from 10 g of glucose, 3 g of pancreaticpeptone, 3 g of yeast extract, and 1.5 g of Na₂HPO₄ in 1 literof distilled water. The pH of the medium was adjusted to 5.35by adding orthophosphoric acid. Inoculation was carried outby using 0.1 ml of a yeast suspension from a 48-h subculturegrown under analogous conditions. Cells were harvested aftera 65-h growth period (stationary phase) and used immediatelyin the experiments.

Starter cultures of C. utilis (ATCC 9950) were grown in yeastextract-malt extract broth (YM broth) (Difco Laboratories, Elancourt,France) at 30°C on a rotary shaker operating at 250 rpmfor 24 h. The cultures were then used to inoculate 100 ml ofYM broth at a final concentration of 1% (vol/vol) and were incubatedat 30°C in a shaker operating at 250 rpm for 24 h (stationaryphase).

L. plantarum 103151 T (Institut Pasteur, Paris, France) wasmaintained on petri dishes with a de Man, Rogosa, Sharpe (MRS)medium, supplemented with 20 g of agar (VWR International, Fontenaysous Bois, France) per liter. L. plantarum was grown in 250-mlconical flasks containing 100 ml of the MRS medium and maintainedat 30°C for 18 h without shaking. One milliliter of culturewas transferred into a conical flask containing the same mediumand allowed to grow to the early stationary phase. The wateractivity of this medium was 0.992.

Escherichia coli K12 strain TG 1 [supE hsd ${Delta}$ 5thi ${Delta}$ (lac-proAB)F' (traD 36proA⁺ proB⁺ lacI^q lacZ ${Delta}$ M15)] was maintained on Luria-Bertani(LB) broth on agar plates (Sigma Chemical Co., St, Louis, Mo.)with a water activity value equal to 0.992. For the experiments,liquid cultures were prepared by inoculation with colonies intotest tubes containing 9 ml of LB broth maintained at 37°Cfor 12 h. A subculture was then prepared by injecting 0.1 mlof bacterial suspension into 9 ml of LB broth, and this wasgrown at the same temperatures as those described above. Cellswere harvested after a 24-h growth period (stationary phase)and used immediately in the experiments.

Human myelogenous leukemia K562 cells (etoposide-resistant cellline) were maintained at 37°C (in 5% CO₂) and cultured inan RPMI 1640 medium serum (VWR International) supplemented with1% (vol/vol) glutamine and 10% (vol/vol) fetal bovine serum.The cells were cultured for 48 h before use, which correspondsto the early stationary phase.

(ii) Freeze-thawing conditions.
Freezing was always carried out with liquid nitrogen at a temperature(T) of -196°C, and the nucleation temperature was not controlled.Cells were stored for 5 min at -196°C before thawing. Foreach experiment, cold stress was applied in a water-glycerolsolution at an osmotic pressure ( ${Pi}$ ) of 1.38 MPa (5.1 g of glycerolwas added to 100 g of water), which corresponds to the osmoticpressure of a classical growth medium. The different methodsthat were used to obtain the cooling rates under investigationare described in Table 1.

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TABLE 1. Observed cooling rates for supports and sample volumes

For low cooling rates (5°C/min), samples in Sarstedt cryotubeswere placed in 50-ml plastic tubes (PolyLabo, Paris, France)for centrifugation to protect them from the cold gradient byusing a gas layer. For the cooling rate of 180°C/min, aSarstedt cryotube containing 1 ml of the cell suspension wasused. For cooling rates of 250 and 650°C/min, a Nunc cryotubeand a Sarstedt cryotube containing 0.5 ml of the cell solutionwere used. For the cooling rate of 5,000°C/min, 10 µlof the sample was contained within a glass cylinder. For thefastest cooling rate (30,000°C/min), cells were fixed ona 0.5-mm-diameter type T thermocouple (TCSA, Dardilly, France)acting as the support. This thermocouple was used because cellscan be fixed onto its surface. It provided effective measurementof the cooling rate at 15,000°C/min. The cooling rate inthe first 20-µm layer, corresponding to the thicknessof the cell suspension layer around the thermocouple, was calculatedby using classical heat transfer equations as being 30,000°C/min.

The influence of the thawing period was not studied during theexperiments, and thawing was always carried out by droppingthe frozen sample into a temperature-controlled water bath maintainedat 37°C. Indeed, Mazur (12) has shown that it is necessaryto thaw samples as rapidly as possible to avoid conditions thatcould lead to recrystallization of the medium. In the case offreezing performed by use of the thermocouple, a water bathcould not be used. So, cells were taken from the thermocoupleby washing it three times with a controlled volume of 1 ml at37°C of the water-glycerol solution previously described.

(iii) Viability measurements.
Viability measurements for the microorganisms were performedby using the CFU method, in which plating cells in the correspondingmedium were supplemented with 15 g of agar per liter. The petridishes were then incubated at the temperature correspondingto the growth conditions of the microorganisms. The viabilitywas determined by comparing the experimental and control microorganisms.

For K562 cells, the viability measurements were followed bytrypan blue staining. There were at least three repetitionsfor each experiment, and average values were calculated as wellas confidence intervals (CI) at the 90% means level. The CIvalues were found to be inferior by 7% to the mean viability.

RESULTS

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Results

The viabilities of the five types of cells with regard to thefive cooling rates used (5, 180, 250, 5,000, and 30,000°C/min)are shown in Fig. 1.

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FIG. 1. Relation of cell viability to cooling rate. Symbols: ${diamondsuit}$ , C. utilis; *, K562; x, S. cerevisiae; ${blacktriangleup}$ , L. plantarum; ${blacksquare}$ , E. coli. The CI values at 90% were always less than 7% of the corresponding viability.

The cells of the yeast S. cerevisiae remained viable at a coolingrate of 5°C/min (i.e., >80% of the control). The resultswere different for cooling rates higher than 180°C/min andlower than 5,000°C/min. Indeed, the S. cerevisiae viabilityfell to a low point of 2% for a cooling rate of 180°C/minand then remained stable at about 3%. For higher cooling rates,the yeast viability increased from 20% at a cooling rate of5,000°C/min to 80% at a cooling rate of 30,000°C/min.

The other yeast, C. utilis, showed its highest viability of70% for a cooling rate of 5°C/min. Then, the viability fellgradually to 24% at a cooling rate of 250°C/min. For fastercooling rates, the viability was higher, with viabilities of30% at a cooling rate of 5,000°C/min and 35% at a coolingrate of 30,000°C/min.

The bacterium L. plantarum showed no significant change in itsviability between cooling rates of 5 and 30,000°C/min. Theviability of this bacterium remained quite stable consideringthe limits of its viability, which were 100% for a cooling rateof 5°C/min and 87% for a cooling rate of 250°C/min.

The results were quite different for the other bacterium, E.coli, which showed changes in its viability that were comparableto the U pattern obtained for S. cerevisiae. Indeed, the viabilityof E. coli was 100% for a cooling rate of 5°C/min. Then,the viability fell progressively to 38% for a cooling rate of250°C/min. For higher cooling rates, the viability increaseduntil reaching 100% for a cooling rate of 30,000°C/min.

Such levels of high cell viability were not obtained with thehuman leukemia K562 cells. Indeed, the maximum viability valuewas observed for a cooling rate of 5°C/min and only reached20% of the initial cell viability. For cooling rates of 180,250, and 5,000°C/min, the viability fell and remained stableat about 2%. For a cooling rate of 30,000°C/min, the cellviability returned to nearly the maximum cell viability value(about 18%).

A cell's cryopreservation depends on numerous factors that influencethe entire cell's viability. These results clearly show thatthe viabilities of the five microorganisms studied are influencedby the cooling rate. From an overall perspective, with the observedresults it is possible to extract three types of cell behaviorwith regard to the freezing rate conditions.

The first type of behavior is the U-pattern curve, as obtainedfor S. cerevisiae, as a function of increasing cooling rate.In this case, a high viability is observed for low cooling rates(5°C/min) but also for ultrarapid cooling rates (30,000°C/min).The yeast S. cerevisiae and the bacterium E. coli both showthis type of behavior.

The second type of behavior, as shown by C. utilis and K562cells, shows a low resistance to freezing. Cell mortality wasobserved even with very rapid cooling rates.

The third type of behavior, as shown by L. plantarum cells,shows the cells maintaining a high viability irrespective ofcooling rate.

These results illustrate that the cooling rate strongly influencescell viability. From a general point of view, in each case theminimal cell viability value was obtained for intermediate coolingrates (i.e., between 180 and 5,000°C/min). Lower coolingrates and very rapid cooling rates are less detrimental forthe cells. To explain these different behaviors, it is necessaryto focus on the factors that can influence the cell viabilityduring the freezing process.

DISCUSSION

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Discussion

From an overall perspective, the results showed that, for mostof the cells used and in the range of cooling rates studied,a U-pattern curve generally represented the evolution of thecell viability. Previous works have already shown that the coolingrate is one of the main factors (6, 12) that influence cellviability during the freeze-thawing process.

A major link between cooling rate, velocity of water outflowfrom the cell across the plasma membrane, and cell mortalityis proposed in this work, based on previous work (6) on thecorrelation of viability of S. cerevisiae with cooling rate.In that study, at lower cooling rates (less than 100°C/min)cell water outflow occurred slowly, and this did not damagethe yeast cells. In contrast, at intermediate cooling rates(between 100 and 1,000°C/min), cell viability was dramaticallyreduced. Then, for very rapid cooling rates (from 1,000 to 30,000°C/min),the recovery in cell viability was observed after thawing.

The understanding of these results is based on the hypothesisthat if the water flow is faster than the thermal flow, thenno intracellular crystallization will occur, and the cell survivalrate will be high. In contrast, as soon as the thermal flowis equal to the water flow, then the freezing rate will induceintracellular water crystallization during the osmotic exitof water, a process which seems to provoke cell death.

Cell death can be prevented in two cases: the first case correspondsto a slow cooling rate during which intracellular water canflow out of the cell, and the second case corresponds to veryrapid cooling rates, which involve a complete freezing of theintracellular water before any water transport occurs. In regardsto the three kinds of cell viability behavior, it is interestingto study the factors that can induce differences between allthe studied cells. Some determinant factors for cryopreservationutilizing heat and mass transfer are shown in Table 2. The firstfactor is cell size, which influences the surface-to-volume(S/V) ratio and directly defines the transfer surface for intracellularheat and mass. The second factor corresponds to the cell permeabilityproperties, which determine the resistance of the cell membraneto water transfer. The presence of a cell wall is the thirdfactor, which is of interest in terms of its mechanical protectiveproperties.

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TABLE 2. Characteristics of the cells used

The S/V ratio can be linked to both thermal and water flow.The larger the cell radius is, then the smaller the S/V ratiois, i.e., larger cells offer a smaller transfer surface comparedto their volume than smaller cells do, for both heat and masstransfer. Therefore, water and heat will flow out faster fromcells with a high S/V ratio (i.e., smaller cells). Moreover,the cylindrical shape of bacteria gives them a significant excesssurface area compared to their volume (the S/V ratio is aboutfive times larger for bacteria than for yeast cells [Table 2]).The minimum viability values obtained during freezing couldbe related to this factor, irrespective of the cooling rate.Indeed, as shown in Fig. 1, the bacterial cells, which havethe smallest size, were less destroyed by the freeze-thawingprocess (i.e., minimal viabilities of 87 and 37.8% for L. plantarumand E. coli, respectively) than were the yeast cells (i.e.,minimal viabilities of 2 and 24% for S. cerevisiae and C. utilis,respectively). The large human cell K562 was found to be themost fragile of the cells, with a minimum viability of 2% obtainedat cooling rates in the range of 250 to 5,000°C/min.

In the hypothesis discussed above, it was explained that cellmortality could result from the competition between water andheat efflux. Indeed, in the heat exchange occurring during freezing,a large part of the heat flow is taken up by the latent heatof crystallization, which is directly proportional to the massof water to be frozen. This parameter would strongly influencethe freezing time lag of high-volume cells, irrespective ofcooling rate. Then, for this reason, in high-volume cells, thewater would have more time to exit the cell before crystallization.Therefore, it could be proposed that for the same cooling rateof 30,000°C/min, the intracellular water of the K562 cellshad begun to flow out of the cells, leading to cellular death,in contrast to what occurred with S. cerevisiae, which crystallizedinstantaneously owing to its smaller size.

The existence of the latent heat of water crystallization allowsus to propose that the water mass transfer is more influencedby the S/V ratio than the heat transfer properties. Given thatit is lethal for a cell to freeze when it loses some of itsintracellular water and that the larger the cell, the more timeit will take to freeze, then it is easier to freeze smallercells without involving any loss of water and thus obtain ahigher survival rate.

Therefore, the smaller size of the bacterial cells determinestheir better preservation properties after freezing than thoseof larger cells, such as yeast cells and animal cells. If heatefflux is significantly faster than the water outflow for highfreezing rates, then cells can be frozen without any water lossand cell viability can be preserved.

Concurrent with the important role played by the cellular size,the cell water permeability can also be considered a factorthat effectively influences the mass transfer, because the timelags under consideration are very short. Since water outflowwill be the main phenomenon observed during the first stageof cell volume decrease (7), only the hydraulic permeabilityvalues of the cells need to be considered. The water permeabilityvalue is represented by the water permeability coefficient,Lp (in units of m · s^-1 · Pa^-1), and this varieswith each type of cell.

Considering that cells with the same water permeability valueare able to lose intracellular water for a given osmotic stressat the same rate whatever their sizes, then a comparison ofwater permeability values will allow for a prediction of a cell'scryopreservation. Therefore, looking at the data related toE. coli and S. cerevisiae (Table 2), one can observe that theLp values of these two cells are of the same magnitude. Consequently,water can flow out from both cells at the same rate. Moreover,the difference in their cell viability results (100 and 80%viability at a cooling rate of 30,000°C/min for E. coliand S. cerevisiae, respectively) can be attributed to theirdifferent cell volumes alone, as S. cerevisiae is 40 times largerthan E. coli. The major role of the S/V ratio is still to behighlighted in this case.

However, the considerations are different in regard to K562cells and S. cerevisiae cells. Indeed, for a mean volume thatis twice that of S. cerevisiae, the data in Table 2 show thatK562 has an Lp value that is 400 times smaller than that ofthe S. cerevisiae yeast.

With regard to the viability obtained with low cooling rates(i.e., between 20 and 100°C/min, as shown in Fig. 1), thedifference in the Lp values can explain why the viability ofthe yeast cells is higher than that of the human cells (about20% viability for K562 versus 70% for S. cerevisiae at a coolingrate of 5°C/min). Indeed, at this cooling rate, the morequickly water can flow out of a cell for a given osmotic perturbation,then the more the cell is protected from internal crystallization.Consequently, S. cerevisiae is less affected by the coolingrate than are the K562 cells.

Concerning the rapid cooling rates (i.e., between 100 and 5,000°C/min),the time lag permitting the cellular osmotic response beforecrystallization commences is shorter than for slow cooling rates.In this case, the K562 cell damage due to crystallization increasesand the viability decreases from 10 to 2%. For S. cerevisiae,this range of cooling rate sees the onset of intracellular crystallization,and the yeast viability dramatically decreases from 80 to 2%.

At the very rapid cooling rates (i.e., from 5,000 to 30,000°C/min),the K562 cells should be able to freeze without any change incell volume because of their low Lp value. In contrast, thehigh Lp value of S. cerevisiae should aid the outflow of cellwater, even at very rapid cooling rates, and therefore, cellviability should rapidly increase for K562 cells as the coolingrate increases. However, as shown in Fig. 1, the results donot verify this hypothesis. Indeed, even though a better preservationof the K562 cell is possible for very rapid cooling rates, theirviability did not exceed 10%. In comparison, S. cerevisiae,in spite of its higher water permeability value, showed a betterviability (80%).

Therefore, water permeability and size values alone cannot explainall the observed results. With regard to the proposed deathscenario, i.e., the combination of water outflow and water crystallization,such a combination provokes irreversible membrane damage andthen cell death. In the case of S. cerevisiae, the existenceof a cell wall could certainly provide additional support toexplain previous results. Indeed, a cell wall could preventor repair membrane damages, such as membrane vesiculations,induced by simultaneous crystallization and water outflow (10,14, 19).

Conclusions. This work has shown that a U-pattern behavior ofcell viability versus cooling rate occurs for cooling ratesbetween 5 and 30,000°C/min. For all types of cells investigated,the viability was found to be higher at low and very high coolingrates. This evolution of viability during the freeze-thawingprocess can be explained by a competition between the heat flowand the water outflow from the cell.

The S/V ratio plays a major role in the determination of theviability results, as it determines the time constant of theheat and mass transfer into and out of the cells. For slowercooling rates, the latent heat of cell water crystallizationallows time for water to exit the cell and so prevents internalcrystallization. For faster cooling rates, the latent heat delaysintracellular crystallization and so becomes a negative factorfor cell viability.

Cell death is proposed to occur from crystallization duringwater outflow from the cell, which involves lethal membranedamage. Thus, the S/V ratio, the latent heat of water crystallization,and the Lp value play a role in a cell's survival after freezing.High S/V values (i.e., small cell sizes), high Lp values, andhigh water content seem to influence positively the survivalrate for low cooling rates. In contrast, low Lp values shouldbe positive for high cooling rates.

FOOTNOTES

* Corresponding author. Mailing address: Laboratoire de Génie des Procédés Alimentaires et Biotechnologiques, ENSBANA, 1, Esplanade Erasme, 21000 Dijon, France. Phone: 33-3-80-39-66-99. Fax: 33-3-80-39-68-98. E-mail: gervais{at}u-bourgogne.fr.

REFERENCES

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