Yeast freeze-thaw survival rates as a function of different stages in the cell cycle

CRYOBIOLOGY

18, 506-510

(1981)

Yeast Freeze-Thaw

Survival Rates as a Function Stages in the Cell Cycle STEPHEN

Department

of Biology,

Brooklyn

College

F. COTTRELL

of the City

University

December

24, 1980; accepted

March

Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

York,

Brooklyn,

AND

New

York

11210

METHODS

Strain and culture conditions. A diploid strain of S. cerevisiae designated iso N (ATCC 42029) was used in this study. Cells were synchronized according to a selection and induction procedure (24) with minor modifications (5). Synchronous cultures were inoculated at an initial cell density of lo6 cells/ml and grown in a semisynthetic medium (21) modified to contain 2% glucose and 0.3% yeast extract. The extent of cell synchrony was monitored by the analysis of total cell DNA content (5) and by viable cell counts performed according to the methods described below. Freeze -thaw protocol and viability determinations. At 20-min intervals throughout three consecutive synchronous cell generations lo-ml samples of culture were withdrawn, washed once by centrifugation in sterile distilled HzO, and resuspended in the same volume of sterile 10% glycerol (v/v). Gel clumps were effectively removed by sonication for 5 set at a power setting of 5.5 with a Model W185 sonifier equipped with a microtip (Heat Systems-Ultrasonics, Inc., Plainview, N .Y .). Suitable aliquots of each cell suspension were removed, serially diluted in sterile H,O, and plated on solid medium containing 2% nutrient agar, 2% glucose, and 1% yeast extract. The remainder of each cell suspension was placed in a 20-ml plastic scintillation vial and frozen in a -80°C freezer (Revco, Inc., West Columbia, S.C.). Under these conditions the overall freezing rate was approximately l”C/min although considerable nonlinearity

12,

506 0011-22401811050506-05502.0010

of New

MATERIALS

In the yeast, Saccharomyces cerevisiae, freeze-thaw survival rates have been shown to be dependent upon not only the freezing and thawing protocol employed (15) but also on a wide variety of diverse biological factors including the strain of yeast (1) and numerous biochemical parameters such as the lipid composition of the yeast cell membranes (14). Comparable variations in both physiological state and biochemical composition are known to occur during the yeast cell cycle and are thought to be at least in part due to the differential synthesis of various biochemical components at discrete intervals throughout the cell cycle (for review see 18, 19). Such biochemical alterations during the cell cycle of S. cerevisiae are believed to be responsible for the observed cell cycledependent vulnerability to a wide variety of external insults including elevated temperature (20), ultraviolet radiation (3), and ethidium bromide-induced cytoplasmic mutagenesis (4). As part of a continuing investigation of different cell cycle events in S. cerevisiae, we demonstrate here that under uniform conditions of freezing and thawing the specific stage in the cell cycle also influences the rate of freeze-thaw survival in this organism. Moreover, we have correlated the timing of maximum and minimum survival rates during the cell cycle with major biochemical changes in membrane composition. Received 1981.

of Different

YEAST

FREEZE-THAW

occurred around 0°C where the rate was approximately O.TC/min. After a period of at least 7 days, the cell samples were rapidly thawed by immersion in a 30°C water bath (thawing rate approximately 45”CYmin) and plated as described above. All cell samples (both frozen and unfrozen) were serially diluted to yield approximately 200 colony forming units per plate and incubated at 30°C for 2 days before counting. The percentage of viable cells was determined by comparing plate counts of both frozen and unfrozen cell samples. All values reported here were derived from the means of at least three different plate counts for both the frozen and the unfrozen samples at each time point. Biochemical analyses. Determinations of total phospholipid phosphorous levels were performed according to the procedures of Getz et al. (9) with the exception that the cells were broken in a Braun MSK cell homogenizer (B. Braun, Melsungen, West Germany), Malate dehydrogenase activity was assayed spectrophotometrically according to the oxaloacetate method of Ferguson et al. (8). RESULTS

A measure of the quality of cell synchrony during the sampling period is shown in Fig. 1. The distinct step increases in both

FIG. 1. Total cell DNA

content (closed

circles) and viable cell counts (open circles) are plotted during synchronous growth. Bars designate the initiation of each new cell cycle as evidenced by synchronous bud formation.

507

SURVIVAL

total cell DNA content and viable cell counts indicated a high degree of cell synchrony throughout the three consecutive synchronous cell cycles examined for freeze-thaw viability. Since the parent-daughter complex remains attached until the initiation of the next generation in this budding yeast, no increases in viable cell counts were observed until the start of the second generation. Under the conditions employed in this study each synchronous cell cycle was approximately 90 min in duration. At appropriate times during synchronous growth, cell samples were removed and subjected to the -80°C freeze-thaw protocol outlined in the preceding section. Figure 2 depicts a typical determination and shows a distinct cyclic change in the percentage of viable cells in response to this freeze-thaw protocol during each of the three consecutive synchronous cell generations examined. Maximum rates of survival were observed during the initial phase of each cell cycle and corresponded in time to the initiation of total DNA synthesis and cell budding. Minimum rates of survival occurred approximately 30 min prior to the initiation of each new cell cycle. Replicate runs yielded essentially identical results, Control cultures in which asynchronously dividing cells were subjected to identical

Cell Cycka

FIG. 2. Percentage of survival as a function of freeze-thaw damage during three consecutive synchronous cell cycles. All values represent the means of at least three plate counts *SD.

508

STEPHEN

F. COTTRELL

culture and freeze- thaw conditions yielded relatively constant survival rates ranging from between 17.7 to 22% with no evidence for any cyclic variations (data not shown). The survival values for both asynchronous and synchronous cells are in sharp contrast to those values for stationary-phase cells of the same strain which consistently exhibited over 75% survival under the same conditions of freezing and thawing. To determine if cell lysis occurred as a result of the freeze-thaw treatment, cell samples so treated were spun down at 4000g for 10 min and the resulting supernatant fractions analyzed for the release of intracellular malate dehydrogenase activity. In cell mixes containing known percentages of mechanically lysed cells this procedure was shown to be capable of detecting less than 1% cell lysis by measuring the release of this soluble intracellular enzyme into the cell-free supernatant fraction. The absence of detectable malate dehydrogenase activity in all of the samples obtained during the three consecutive cell cycles examined suggested that periodic changes in cell lysis did not occur and could not account for the cyclic changes in cell viability observed during synchronous growth. Under our conditions of cell synchrony concentrations of total phospholipid phosphorous were observed to increase in stepwise fashion (Fig. 3). All step increases rep-

FIG. 3. Quantitative phosphorous content chronous cell cycles.

estimates of total phospholipid during three consecutive syn-

resented nearly a doubling in total phospholipid phosphorous content from the previous increment. Similar results were obtained in replicate experiments and indicated the discontinuous synthesis of cellular phospholipid content at or near the initiation of each new cell cycle. DISCUSSION

Previous studies have shown that specific stages in the yeast cell cycle exert profound influences on the degree of celluIar vulnerability to a wide variety of factors (3,4, 7, 12, 20). The data reported in the present study extend these findings and show that the rate of freeze-thaw survival is also influenced significantly by specific stages in the yeast cell cycle. A comparable dependency of freeze-thaw survival rates on certain stages in the cell cycle also has been observed in cultured sycamore cells (21), Chinese hamster cells (13, 16), and human HeLa cells (17, 22). Although distinct cyclic increases in freeze-thaw survival rates do occur at the initiation of each new cell cycle in our culture system, it is not clear why there was a decreasing rate of survival during the three synchronous cell generations studied. One possible explanation for this phenomenon might be related to possible changes in the physiological state of the cells during culture growth. We have shown that stationary-phase cells exhibit a much higher rate of freeze-thaw survival than asynchronously growing exponential-phase cells. Under our system of cell synchrony the initial population of synchronous cells was derived directly from a late stationary-phase culture (24) and thus might be expected to exhibit a higher freeze-thaw survival rate than cells which have undergone three synchronous generations of growth and are more comparable to exponential phase cells. That the particular method for inducing cell synchrony is responsibIe for the declining rates of freeze-thaw survival was supported by the

YEAST

following

FREEZE-THAW

preliminary experiment. Syncultures induced by an alternate procedure in which exponential-phase populations of cells at specific stages in the cell cycle were selected from an asynchronous cell population (11) showed similar cyclic patterns of survival in response to our standard freeze-thaw protocol, but in this case the peaks of survival ranged around 20% with no evidence for any decline during successive cell generations. These data support our hypothesis that the decline in survival rates reported in Fig. 2 is the result of the particular method used to induce synchrony, and, at the same time, suggest that the pattern of survival shown in Fig. 2 was not an artifact due to the particular method of synchrony since two independent methods both show the same cyclic patterns of freeze-thaw survival. The exact mechanism(s) responsible for the cyclic variations in freeze- thaw survival reported here remains unclear although it seems likely that some event occurring during the cell cycle may be responsible. Our results indicate that such an obvious cause as a differential rate of cell lysis during the cell cycle in response to our freeze-thaw protocol is unlikely since the absence of soluble malate dehydrogenase release indicates no detectable cell lysis at any time during the three cell generations examined. Because changes in membrane lipid content have been reported to influence the freeze-thaw survival rate in this yeast (14), we have examined the phospholipid content during the three consecutive cell cycles used in the freeze -thaw analysis. The stepwise increases in phospholipid content reported during this period probably represent the synthesis and rapid incorporation of this biochemical component into cellular membranes since nearly all detectable phospholipids have been reported to be associated with membrane fractions (2). This observation, in conjunction with the finding that all cell membranes with particular reference to mitochondrial membranes inC~~OIIOUS

SURVIVAL

509

crease at a continuous rate during the yeast cell cycle (6, IO), suggests that considerable fluctuations in the phospholipid content of cell membranes occur during the yeast cell cycle. By reference to Figs. 2 and 3, such a discontinuous rate of phosphohpid synthesis against a background of continuous membrane formation would produce a pattern in which the highest phospholipid to membrane ratio occurs at the time of maximal freeze-thaw survival with the lowest survival rate coinciding with the lowest phospholipid to membrane ratio during the cell cycle. The suggestion that a causal relationship may exist between membrane alterations and freeze- thaw survival rates during the yeast cell cycle is now undergoing further investigation. SUMMARY

In the present study we demonstrate that the -80°C freeze-thaw survival rate in the yeast, Saccharomyces cerevisiae, is dependent upon specific stages in the cell cycle. Samples removed from synchronous cultures at appropriate intervals during the first three consecutive synchronous cell cycles were subjected to a -80°C freezethaw protocol employing 10% glycerol as a cryoprotectant. Distinct cyclic changes in the percentage of viable cells in response to our freeze-thaw protocol were observed during each of the three consecutive synchronous cell generations examined. Maximum rates of survival occurred at the initiation of each new cell cycle and minimum rates of survival occurred approximately 30 min prior to each new cell cycle. These maximum and minimum rates of survival were shown to be correlated in time with maximum and minimum ratios of cellular phospholipid to membrane during each individual cell cycle. ACKNOWLEDGMENTS This study was supported by Public Health Service Grant GM25901 from the National Institute of General Medical Sciences and a PSC-BHE grant from the City University of New York.

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F. COTTRELL

REFERENCES

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