CHYOLIIOLOGY
Lethal
16, 132-140
(1979)
and Chromosomal Effects of Freezing, Thawing, Storage and X-Irradiation on Mammalian Cells Preserved at -196” in Dimethyl Sulfoxide M. 1 The
J. ASHWOOD-SMITH Departments
1* 3,
4 AND
of Biology and 2Physics, University Victoria, B.C. V8W 2Y2, Canada
INTRODUCTION
The DNA double helix, in vitro, is stable to freezing and thawing (16) and freeze-thaw procedures, in themselves, are not mutagenic in procaryotic cells (2). Little or no evidence, however, is available on the genetic effects of freezing and thawing vertebrate cells stored at low temperatures in the presence of standard cryoprotective agents such as dimethyl sulfoxide (DMSO) or glycerol. Recently, Ashwood-Smith and Grant (7) reported that Chinese hamster cells frozen in 10% DMSO showed a transient increase in chromosome abnormalities (as measured by an increase in micronuclei) 30 hr after thawing, and, when in tissue culture, after 2 hr or 3 months storage at -196°C. At the time of their communication to the London meeting of the Ciba Foundation in January 1977 on the occasion of the “Symposium on the Freezing of MamReceived February 17, 1978; accepted September 27, 1978. 3 Present address: Ecole Nationale Superieure de Biologie Appliqke B la Nutrition et B l’Alimentation, Universitk de Dijon, DIJON, 21000, France. 4 Supported by the National Research Council of Canada and, in part, by a N.A.T.O. Senior Research Fellowship. 5 Supported in part by a University of Victoria Research grant. 132 OOll-2240/79/020132-09$02.00/0 Conyright All rights
0.1975 by Academic Press, of reproduction in any form
G. B. FRIEDMANN
kc. reserved.
Time,
2~5 of Victoria,
malian Embryos” it was suggested by the authors that the results might have a trivial explanation and, that in any event, more experiments were under way, the results of which would be known within 6 months. This present paper, is in part a presentation of the comprehensive results of these studies and will report that gross chromosome damage of the type that is easily revealed by the micronucleus test, is in all probability, not produced by freeze-thaw procedures or storage for periods of up to 6 months in any of the standard cryoprotective additives. The second part of this paper describes an attempt to assessthe radiation damage to cells preserved in DMSO at -196°C both in terms of lethality (see Refs. 4 and 17 for earlier work in this area) and chromosome damage. Radiation from both intrinsic and extrinsic factors accumulates in frozen cells at -196°C and this radiation varies in different parts of the world but, on average, background radiation is probably of the order of about 0.1 rad per year (8, 13) of which, perhaps, 70% is associated with gamma rays and electrons ( 8). All vertebrate cells, with the exception of certain strains deficient in genetic repair processes,possessvery efficient cellular repair systems which operate on DNA damaged by radiation and certain chemicals.
GENETIC
STABILITY
IN
However, these repair systems are enzymatic and cannot operate at low temperatures in the frozen state. At the moment of cellular resurrection, during the rehydration and thawing process and immediately thereafter the radiation damage will become manifest. Clearly an interesting theoretical question is then posed: “To what extent is the accumulation of radiation damage detrimental to preserved cells.” In practical terms lethality is not a worry as very long periods of time would be required before radiation damage would become serious (see results and discussion for details) and this long time interval is extended even further as both DMSO and glycerol have radioprotective (1, 4, 9) as well as cryoprotective properties (4). Lethal effects and gross chromosomal effects produced by x-irradation are usually closely related. However, genetic damage in cells preserved at low temperatures and subjected to ionizing radiation has not been adequately studied. This paper presents information on the extent to which both the lethal and chromosomal effects of ionizing radiation are modified by DMSO at -196°C. With this information and with certain assumptions, estimates of storage times at -196°C can be made which will relate to the production of known amounts of both lethal and chromosomal damage. METHODS
Tissue culture methods. Eagle’s minimum essential medium was used in conjunction with Eagle’s balanced salt solution buffered with 0.05 M Tricine and 0.09% sodium bicarbonate at pH 7.2. The medium was supplemented with fetal calf serum ( 10% ), 2 mM L-glutamine and 50 Cells of kanamycin sulphate. dml (Chinese hamster ovary fibroblasts. Pucks clone A) were incubated at 37°C and grown as monolayers in disposable plastic tissue culture flasks. Two days before they had grown to confluence they were trypsinized with 0.25% trypsin in Ca2+ and
THE
FROZEN
STATE
133
Mg?’ free salt solution for 5 min before the trypsin was inactivated by the addition of an equal volume of tissue culture medium containing sedum. The cells were collected by gentle centrifugation (75g for 3 min) and the pellet of cells was then resuspended in fresh tissue culture medium; the cell density of this solution was then approximately 1 X lo6 cells/ml. The survival of cells after freezing and thawing or after x-irradiation was measured by their ability to form macroscopically visible colonies 10 days after incubation. The cloning efficiency of the cells used in this study was between 50 and 60%. Freezing and thawing procedures. These procedures have been described previously (7). The cryoprotective solutions were prepared as 20% solutions in complete tissue culture medium and were slowly added to the cell suspension 10 to 15 min before the freezing experiments were started. Polyvinylpyrrolidone (PVP) was the Plasdone C preparation of the General Aniline and Dye Corporation (GAF, Inc., New York, U.S.A.). This material is claimed to have a molecular weight of 30,000 daltons (however, see Ref. 3); it was dialysed to remove the low molecular weight fractions which account for nearly 14% of the sample and which increase toxicity in some instances during freezing and thawing. X-Irradiation. Irradiation of cells at 22°C was done with the cells attached as a monolayer at a “treating” distance of 16 cm and an effective open diameter of 5 cm using a Picker Zephyr therapy unit at 10 mA and a nominal 120 kV. The beam was filtered with additional 2 mm Al and 0.25 mm Cu and had a 0.39 mm Cu H.V.L. An ‘output correction for voltage variations was applied at 2.2% per volt and a conversion factor of 0.94 rad roentgen was used. The dose rate was 127.12 radlmin, backscatter being provided by a 30 X 30 x 5 cm deep wax base. Irradiation of cells at -196°C was performed with cells in
ASfiWOOD-SMITH
134
frozen suspension in tissue culture fluid and DMSO contained in small plastic vials and surrounded with liquid nitrogen. Micronucleus test for chromosomal aberrations. After telophase micronuclei form in the cytoplasm from acentric chromosome fragments that are left behind at anaphase. The number of these micronuclei has been used as a simple, rapid and effective means of screening for chromosomal damage produced by mutagenic agents (10, 14). Cells either control or after thawing (with or without radiation treatment) were plated in dishes (approximately 5 x lo5 viable cells) for the detection of chromosome damage. Samples were taken at a number of times after the freezing and thawing treatment and/ or the x-irradiation procedures in order that the damage would be expressed during segregation. Cells were trypsinized in the normal way and resuspended in 5 ml of 1% trisodium citrate and left at room temperature before centrifugation and resuspension in 1 ml of freshly prepared Carnoy’s fixative (ethanol: glacial acetic acid 3: 1); when the cells were dispersed the volume of the fixative was adjusted to 5 ml. After 5 min the cells were again centrifuged and this final pellet was then resuspended in 0.1 or 0.3 ml of fixative depending on its size. Four slides for each treatment point were prepared immediately and allow to dry in air for several hours before staining with Gurr’s new improved Giemsa for 5 min (the Giemsa solution was diluted 1 in 10 with dilute phosphate buffer, 0.07 M, pH 7.0). The slides were rinsed in distilled water to decolourise the cytoplasm and once more dried in air. Not less than 2000 cells were scored for each micronuclei treatment point and expression time; scoring was randomised and the identity of the shdes not discIosed until the scoring was finished. RESULTS
AND
DISCUSSION
Effects of freezing and thawing and of storage on the production of chromosomal
AND FRIEDMANN
damage. The survival of Chinese hamster cells after freezing and thawing and storage at -196°C (cooling rate of 5”C/min from +22” to -45°C and then rapid as the cells, in ampoules, were plunged into liquid nitrogen; thawing rate of approximately lSO”C/min) varied with the nature of the cryoprotective but essentially remained unchanged as a function of the 6 month storage time at -196°C. The figures, with standard deviations in parenthesis were as follows: 10% DMSO, 91% survival (‘6.2); 10% glycerol, 65% survival (*5.0), and 10% PVP, 17% survival ( 22.5). Survival of cells frozen and thawed in the absence of cryoprotective agents was about 1%. These values are similar to those obtained over a number of years in our laboratory. The better survival of cells frozen in the presence of DMSO rather than glycerol is probably associated with the procedures used for the removal of the cryoprotective agents by a ten-fold dilution at room temperature into fresh tissue culture fluid. No doubt a slower “step-wise” dilution would improve the glycerol survival values. However the point is, that survival values of between 65 and 90% are fairly typical of the results that are routinely attainable in many laboratories throughout the world and the key question is whether chromosome damage is present in the surviving population of cells. In a large first experiment which was reported to the Ciba Foundation Meeting in London, in 1977 (7) a significant number (P= 0.01) of micronuclei were seen in cells preserved with DMSO after 20 hr of culture following thawing of samples stored at -196°C for either 2 hr or 3 months. Examination of celIs cultured for longer times or preserved with either glycerol or PVP failed to show any abnormalities (see Tables 1 and 4 for the results with DMSO and Tables 2 and 3 for results with glycerol and PVP respectively). Because of the importance of these
GENETIC
Micronuclei
in Frozen Storage
STABILITY
Hamster time
and
Cells
Stored
IN
THE
TABLE
1
FROZEN
for Different
Periods
treatment
of Time
in 10%
NIicronuclei ( f standard
time
20
Control
cells not
Cells
a Cell
frozen
with
frozen DMSO
Cells frozen 3 months
and stored at --196”0
in DMSO
for
Cells frozen 6 months
and stored at -196Q
in DMSO
for
and
thawing
constant
2.1 (0.3)
1.2 (0.4)
3.9 (1.2)
Hamster
time
treatment
and
Cells
Stored
9.9 (2.1)
few cells survived adequate analysis)
for
8.2 (2.7)
4.0 (0.6)
3.6 (0.3)
4.6 (1.2)
3.5 (0.9)
4.8 (0.6)
12.5 (3.6)
time
period
of experiment
at 91y’.
nuclei in cells treated but not frozen with DMSO was higher than the untreated controls (5.2 per 100 cells compared with 2.3 per 160 cells) but the standard deviations were 1.1 and 1.2 respectively. No evidence of increased chromosome damage was seen with any of the freezethaw regimes ‘and the general increase in micronuclei seen in all samples as a 2
for Different
Periods
of Time
Micronuclei ( f standard
20
in 107,
Glycerol
per 100 cell3 deviation)
Expression
time
(hr)
30
44
1.3 (0.05)
2.1 (0.3)
10.4 (1.9)
1.4 (0.6)
1.9 (1.5)
7.1 (2.7)
Cells frozen” and stored in 10% glycerol for 2 hr at -196°C
4.6 (2.2)
3.6 (1.0)
6.5 (1.8)
Cells frozen” and stored glycerol for 3 months
in 10% at - 196°C
1.5 (1.2)
3.2 (1.5)
2.6 (1.0)
Cells frozena and stored glycerol for 6 months
in 10% at -196°C
1.0 (0.3)
2.9 (0.4)
2.9 (1.4)
Control
cells not frozen
Control cells 10% removed
a Cell
(1.9)
2.8 (1.3)
TABLE in Frozen Storage
10.4
3.2 (0.5)
over
results several more experiments with DMSO were commenced and the results of the experiments are given in Table 4. Both repeat experiments revealed no difference between cells frozen and thawed in DMSO and stored for 2 hr at -196°C and cells treated with DMSO and not frozen and thawed. Admittedly in the second experiment the number of micro-
Micronuclei
44
1.3 (0.05)
(Too for
freezing
(hr)
30
no additives in DlblSO
after
Sulfoxide
added
Cells frozen and stored 2 hr at - 196°C”
survival
Dimethyl
per 100 cells deviation)
Expression
Control cells 10% and removed
135
STATE
survival
after
glycerol
freezing
added
and thawing
and
constant
over
the time
period
of the experiment
at 657&
136
ASHWOOD-SMITH
AND TABLE
Micronuclei
in Frozen
Storage
time
and
Hamster
Cells
Stored
FRIEDMANN 3
for Different
Periods
treatment
of Time
Micronuclei (& standard
Control
cells not frozen
Control cells 10% removed
PVP
Cells frozen and stored for 2 hr at -196°C’~
G Cell
added
PVP
Cells frozen and stored in 10% for 3 months at - 196”Ca
PVP
Cells frozen and stored in 10% for 6 months at - 196°C”
PVP
after
freezing
and thawing
constant
Experiment
Experiments
44
1.3 (0.05)
2.1 (0.3)
10.4 (1.9)
1.5 (0.2)
2.0 (0.4)
10.0
1.4 (0.5)
4.2 (0.8)
7.0 (1.8)
2.5 (0.2)
5.4 (2.0)
1.4 (0.5)
2.0 (0.3)
2.7
5.3 (1.0)
on Micronuclei
the time
number
Control cells (no DMSO)
standard
Cells Micronuclei deviation)
(1 For
differences
B
1.3 (0.05) table
Frozen
1.2 (0.4)
1)
in 10%
per 100 cells expression time
Control cells (DMSO added, removed)
A
1 from
of experiment
at 18%.
4
in Hamster
(f
(Results
period
(0.7)
Dimethyl
Cells frozen and stored in DMSO, 2 hr -196°C C
3.2 (0.5)” 3 months at - 196°C = 4.0 (0.6)~
2.3
(1.2)
5.2 (1.1)
4.2 (1.35)
3
1.3 (1.0)
1.4 (0.6)
0.8 (0.2)
C and B the Student
t test was 0.01
(P).
Sulfoxide
= 20 hr
2
between
(2.0)
ture, a situation very different from that seen with X-rays or uv, both agents which cause chromosomal damage. It is possible that some of the cells or the cultures containing the thawed cells, in those early studies, contained viruses which were activated by the DMSO and freeze-thaw procedures. DMSO has recently been shown by Lyman and his colleagues (12) to induce the differentiation of Friend leukemic cells and it was suggested (15) that this effect was at the level of DNA transcription.
TABLE of Three
(hr)
30
over
function of expression time irrespective of the treatment is often observed with this test system. Cells, preserved at -196°C in 10% DMSO for 2 years have very recently been thawed and examined for micronuclei and no evidence of chromosomal damage was seen. It is difficult to explain the first results of chromosome damage in cells frozen in DMSO although it is important to note that this damage did not persist but rapidly disappeared from the population such that it was not demonstrable after 30 hr of cul-
Summary
time
and
in 10%
survival
PVP
per 100 cells deviation)
Expression 20
in 10%
GENETIC TABLE
STABILITY
IN
5
Modification of X-Ray Induced Lethality in Hamster Cells Irradiated at - 196’C in the Presence of 10% DMSO Compared With the Induced Lethality at Room Temperature (f22”) Percent survival
Irradiation dose in rads +22Y?
10.0
1.0 0.1 Data in Ref.
from 7.
600 1000 1290 complete
DOW reduction factor (D.R.F.)
-196°C
2125 3400 4320 survival
3.54 3.40 s.35 curves
published
None of these studies, however, exclude the possibility of genetic damage at the molecular level, as base pair substitutions, transitions, and frame shift mutations would not be detected at the level of chromosomal structural damage. Preliminary experiments in our laboratory in which drug resistance to ouabain was studied failed to reveal any changes after various freezing and thawing procedures. In the knowledge of the negative results with bacterial and yeast systems this was reassuring, if hardly surprising. Effects of x-irradiation on lethality and chromosomal damaged cells irradiated whilst preserved in the frozen state at -196°C in the presence of 10% DMSO. Both glycerol and DMSO are radioprotective chemicals as well as possessing cryoprotective properties and from the work of Vos and Kaalen (17) some protection against radiation damage at low temperatures was to be anticipated; irradiation of cells in the presence of DMSO at -t22”C was not performed asthe interest in these present studies centred on the effects at low temperatures so that estimates of longe\Gty of stored cells might be made. The complete survival curves for x-irradiation of cells at room temperature and at -196°C in the presence of 10% DMSO have been published (7) but the relevant information in terms of the dose reduction factors observed are shown in
THE
FROZEN
137
STATE
Table 5. The overall dose reduction factor of the DMSO and temperature on cell survival from x-ray induced kill is approximately 3.5 over three log cycles. Thus low temperature storage of cells is clearly advantageous in reducing the lethal effects of x-rays. Values for temperatures below -196°C are not available for mammalian cells but may not be very different from values obtained at -196°C. The spores of Bacillus megaterium, for example, show no changes in radiosensitivity at temperatures between -195” and -268°C although there is a big increase in radioresistance between zero and -196°C (8). The changes in x-ray induced chromosomal damage as a function of radiation temperatures are shown in Figs. 1 (+22”C) in the presence of 10% and 2 (-196’C DMSO). The peak for the dose dependent chromosomal damage was observed at 30 hr for cells irradiated at +22”C. Cells frozen in 10% DMSO and irradiated at -196°C required larger doses of x-rays to produce chromosomal damage and al-
0
10
20 EXPRESSION
30 TIME
40
50
( hr.)
FIG. 1. Induction of micronuclei by x-rays in Chinese hamster cells at room temperature (+22”C). Ordinate: micronuclei per 100 cells f standard deviation. Abscissa: expression time in hours. n 42 rads; v 104 rads; l 257 rads; 0 515 rads. All irradiations were in air.
138
ASHWOOD-SMITH
lb
0
20 30 EXPRESSION
40 TIME
60
50 (HOURS)
160
AND
170
FIG. 2. X-ray-induced production of micronuclei in Chinese hamster celIs at -196°C in the presence of 10% DMSO. Ordinate; micronuclei per 100 cells * standard deviation. Abscissa: expression time in hours. n 540 rads; v 1620 rads; l 2700 rads; II] 3780 rads.
though, in general, the peak for the observed damage was after 30 hr, high doses of radiation (e.g., 2700 rads) resulted in biphasic responses. The damage produced at +22”C reached a peak and decreased sooner than damage of equivalent lethality produced by the larger doses of x-rays delivered at -196°C. Comparison of Fig. 1 with Fig. 2 indicates this difference (the time scales on the two graphs are different and should be noted). The biphasic nature, at higher doses, of the x-ray response
FRIEDMANN
at - 196°C is shown in Fig. 3. The reason for the long expression time of chromosomal damage after x-irradiation at low temperatures is not easily explained unless it is assumed that a qualitatively different type of damage is produced by low temperature irradiation but there is little to suggest this possibility. The formation of micronuclei per cell as a function of the fraction of cells surviving a given amount of x-irradiation was plotted and the data is summarized in Table 6. X-irradiation at +22”C resulted in values that are within the expected theoretical limits in that the number of micronuclei per lethal hit was between 0.1 and 0.2 (6). Irradiation at -196°C resulted in values that, in essence, fitted the same slope at least until survival values fell below 10%. Thus, although about 3.3 to 3.5 times as much radiation was necessary to kill cells at -196°C as at +22”C the same number of micronuclei per cell were produced. This result suggests that the same lethal mechanisms are responsibIe at the two widely differing temperatures and physical states even when a radio-protective chemical, DMSO, is present. With larger doses of x-irradiation at -196°C the number of micronuclei per surviving fracTABLE
6
l\Iicronuclei per Cell as a Function of X-Ray Survival; Cells Irradiated at +22”C (air) or at -196°C in the Presence of lO?i DMSO; the Expression Time at +22”C was 29 hr and at - 196°C was 30 hr Percent survival
X-ray dose (rads)
Micronuclei per cell
X-X%y dove bade)
+22”c
0
0:s
110
1.5 DOSE
io IN
23 KKILORADS
30
33
40
FIG. 3. X-ray-induced production of micronuclei in Chinese hamster cells at -196°C in the presence of 10% DMSO. Ordinate: micronuclei per 100 cells 2 standard deviation. Abscissa: x-ray dose in kilorads. n 20 hr expression time; l 30 hr expression time; V 44 hr expression time.
100.0 80.0 60.0 40.0 20.0 10.0 5.0 1.0 0.5 0.1
180 305 400 510 600 765 1000 1106 1290
Alicronuclei per cell -196X
0 0.05 0.12 0.20 0.33 0.47 0.60 0.89 1.00 1.32
590 1000 1350 1800 2125 2700 3400 3750 4320
0 0.05 0.12 0.22 0.40 O..% 0.32 O.RO 0.55 0.71
GENETIC
STABILITY
tion of cells is less (and the slope of the survival curve changes) suggesting that the mechanism(s) of chromosome damage may be different under those conditions. If large, unfractionated doses ‘of x-rays as used in these present studies have the same final effect as a number ‘of small, but cumulative doses of x-rays impinging on a frozen target and if the damage sustained by the frozen cells is not repairable until the cells are thawed then it is possible to arrive at estimates for storage times at -196°C and concomitant cumulative background radiation damage. About 70’70 ‘of the yearly background radiation dose of 0.1 rad/year is due to x- and y-rays and this means that the effective background dose of x-rays is about 0.07 rad/year. Neglecting any energy dependence (as between background radiation and our experimental x-ray beam), this latter value would be reduced both in terms of lethality and chromosomal damage by a factor of 3.5 to 0.02 rad/year at -196°C. The Di,, value for Chinese hamster cells is 600 rad at +22”C and 2125 rad at -196°C in the presence of 10% DMSO. Assuming no repair and no cell division these two values would not be reached for 8571 and 30,395 years! If both the lethal and the gross genetic effects of x-irradiation are modified in the same quantitative manner, as these present experiments strongly suggest, then tissue culture cells could be stored for about 30,060 years in the presence of DMSO at -196°C sustaining the before, on resurrection, equivalent of an acute Dlo dose of x-rays. Cells preserved in glycerol would probably behave in the same manner ‘as would most other vertebrate diploid cells preserved at very low temperatures. The behavi.or of frozen embryos to increased background radiation has recently been directly tested by Lyon and her colleagues (13, 18). They found that, when the background radiation level to frozen mouse blastulae was stored (in DMSO)
IN
THE
FROZEN
STATE
139
increased by a factor of 84 for 10 to 12 months, the proportions of implanted embryos and live foetuses was slightly reduced. The authors concluded that “the hazard of background radiation during storage, of mouse embryos, appears negligible although the effect observed at the highest radiation dose still needs further examination during longer periods of storage.” SUMMARY
1. Tissue culture cells preserved at -196°C in 10% glycerol or 10% PVP for periods of up to 6 months showed no gross chromosomal aberrations as disclosed by the micronucleus test. In one experiment cells preserved in 10% DMSO, which offered the best protection (91% survival after freezing and thawing), showed a statistically (P = 0.05) greater number of micronuclei 20 hr after thawing. This was a transient effect and three more separate experiments failed to demonstrate any chromosomal damage ‘associated with freezing, thawing and storage at -196°C for periods of up to 2 years in 10% DMSO. 2. Both the lethal and chromosomal effects of x-irradiating cells in 10% DMSO at -196°C were reduced by a factor of approximately 3.5 compared to irradiation at +22”C. 3. It is estimated that a period of approximately 30,000 years of storage of cells in 10% DMSO at -196°C would pass before an accumulated x-ray dose from background radiation would reach a level, which upon cellular resurrection, would result in the equivalent lethal and chromosomal damage of an accute Dlo dose. 4. Cells cannot be kept at -196°C ad infinitum as there is no escape from background radiation damage. Shielding would reduce the level but internal radiation from isotopic disintegration is inescapable. ACKNOWLEDGMENTS We would like and Miss Elizabeth
to thank Mrs. Alexa Kennedy Grant for the excellent tech-
140
ASHWOOD-SMITH
nical help with the tissue culture, freezing and micronuclei analyses and the Victoria Clinic, a Unit of the B.C. Cancer Control for the use of their irradiating facilities.
methods Cancer Agency,
REFERENCES 1. Ashwood-Smith, M. J. The radioprotective action of dimethyl sulphoxide and various other sulphoxides. Intern. J. Rad. Biol. 34, 41-48 (1961). 2. Ashwood-Smith, M. J. On the genetic stability of bacteria to freezing and thawing. Cryobiology 2, 39438 ( 1965). 3. Ashwood-Smith, M. J., and Warby. C. Studies on the molecular weight and cryoprotective properties of polyvinylpyrrolidone and dextran with bacteria and erythrocytes. Cryobiology 8, 453464 ( 1971). 4. Ashwood-Smith, M. J. Current concepts concerning radioprotective and cryoprotective properties of dimethyl sulphoxide in cellular systems. N.Y. Acad. Sciences, 243, 246-256 ( 1975). 5. Ashwood-Smith, M. Mutation induction drying. Cryobiology
J., and Grant, in bacteria 13, 206-213
Elizabeth. by freeze ( 1976).
6. Ashwood-Smith, M. J., Grant, E. L., HeddIe, G. B. Chromosome J. A., and Friedmann, damage in Chinese hamster cells sensitized to near-ultraviolet light by psoralen and angelicin. Mutation Res. 43, 377-385 (1977). 7. Ashwood-Smith, M. J., and Grant, Elizabeth. Genetic stability in cellular systems stored in the frozen state. In “The freezing of mammalian embryos” ( CIBA Foundation Symposium No. 52, new series, 251-272. Elsevier/Excerpta Medics/North Holland (1977). 8. Bacq, Z. M., and of radiobiology. and New York
Alexander, Pergamon (1961).
P. Fundamentals Press, London
AND
FRIEDMANN
9. Bridges, B. A. The chemical protection of Pseudomonas species against ionizing radiation. Radiation Res. 17, 801-806 ( 1962). 10. Heddle, J. A. A rapid in viva test for chromosome damage. Mutation Res. 18, 187190 ( 1973). 11. Hieda, K. Genetic changes induced by freezing and drying. Abstract. Low Temperature Medicine 3, 13 ( 1977). 12. Lyman, G. H., Preisler, H. D., and Papahadjopoulos, D. Membrane action of DMSO and other chemical inducers of Friend leukaemic cell differentiation. Nature (London) 262, 360-363 ( 1976). 13. Lyon, M. F. Implications of freezing for the preservation of genetic stocks. In Basic aspects of Freeze Preservation of Mouse Strains (ed. 0. Muhlbock), pp. 57-65. Stuttgart: Bustav-Fischer Verlag ( 1975). 14. Matter, B. E., and Grauwiler, J. Micronuclei in mouse bone marrow cells. A simple in civo model for the evaluation of drug induced chromosomal aberrations. Mutation Res. 23, 239-249 (1974). 15. Preisler, H. D., Houseman, D., Scher, W., and Friend, C. Effects of 5-bromo-e-deoxyuridine on production of globin mRNA in dimethyl sulfoxide stimulated Friend leukemic cells. PTOC. Nat. Acad. Sci. USA 70, 2956-2959 ( 1973). 16. Shikama, K. Thermal and freezing denaturation of deoxyribonucleic acid. Sci. Rep. Tohoku Univ. 4th Ser.. Biol. 30, 133-141 (1964). 17. Vos, 0.. Kaalen, M. C. A., and Budke, L. Radiation protection by a number of substances preventing freezing damage. 1. Protection of mammalian cells in vitro. Int. 1. Rad. Biol. 9, 3341 (1965). 18. Whittingham, D. G., Lyon, Mary F., and Glenister, P. H. Long-term storage of mouse embryos at -196°C: the effect of background radiation. Genet. Res. Camb. 29, 171-181 (1977).
Lethal
16, 132-140
(1979)
and Chromosomal Effects of Freezing, Thawing, Storage and X-Irradiation on Mammalian Cells Preserved at -196” in Dimethyl Sulfoxide M. 1 The
J. ASHWOOD-SMITH Departments
1* 3,
4 AND
of Biology and 2Physics, University Victoria, B.C. V8W 2Y2, Canada
INTRODUCTION
The DNA double helix, in vitro, is stable to freezing and thawing (16) and freeze-thaw procedures, in themselves, are not mutagenic in procaryotic cells (2). Little or no evidence, however, is available on the genetic effects of freezing and thawing vertebrate cells stored at low temperatures in the presence of standard cryoprotective agents such as dimethyl sulfoxide (DMSO) or glycerol. Recently, Ashwood-Smith and Grant (7) reported that Chinese hamster cells frozen in 10% DMSO showed a transient increase in chromosome abnormalities (as measured by an increase in micronuclei) 30 hr after thawing, and, when in tissue culture, after 2 hr or 3 months storage at -196°C. At the time of their communication to the London meeting of the Ciba Foundation in January 1977 on the occasion of the “Symposium on the Freezing of MamReceived February 17, 1978; accepted September 27, 1978. 3 Present address: Ecole Nationale Superieure de Biologie Appliqke B la Nutrition et B l’Alimentation, Universitk de Dijon, DIJON, 21000, France. 4 Supported by the National Research Council of Canada and, in part, by a N.A.T.O. Senior Research Fellowship. 5 Supported in part by a University of Victoria Research grant. 132 OOll-2240/79/020132-09$02.00/0 Conyright All rights
0.1975 by Academic Press, of reproduction in any form
G. B. FRIEDMANN
kc. reserved.
Time,
2~5 of Victoria,
malian Embryos” it was suggested by the authors that the results might have a trivial explanation and, that in any event, more experiments were under way, the results of which would be known within 6 months. This present paper, is in part a presentation of the comprehensive results of these studies and will report that gross chromosome damage of the type that is easily revealed by the micronucleus test, is in all probability, not produced by freeze-thaw procedures or storage for periods of up to 6 months in any of the standard cryoprotective additives. The second part of this paper describes an attempt to assessthe radiation damage to cells preserved in DMSO at -196°C both in terms of lethality (see Refs. 4 and 17 for earlier work in this area) and chromosome damage. Radiation from both intrinsic and extrinsic factors accumulates in frozen cells at -196°C and this radiation varies in different parts of the world but, on average, background radiation is probably of the order of about 0.1 rad per year (8, 13) of which, perhaps, 70% is associated with gamma rays and electrons ( 8). All vertebrate cells, with the exception of certain strains deficient in genetic repair processes,possessvery efficient cellular repair systems which operate on DNA damaged by radiation and certain chemicals.
GENETIC
STABILITY
IN
However, these repair systems are enzymatic and cannot operate at low temperatures in the frozen state. At the moment of cellular resurrection, during the rehydration and thawing process and immediately thereafter the radiation damage will become manifest. Clearly an interesting theoretical question is then posed: “To what extent is the accumulation of radiation damage detrimental to preserved cells.” In practical terms lethality is not a worry as very long periods of time would be required before radiation damage would become serious (see results and discussion for details) and this long time interval is extended even further as both DMSO and glycerol have radioprotective (1, 4, 9) as well as cryoprotective properties (4). Lethal effects and gross chromosomal effects produced by x-irradation are usually closely related. However, genetic damage in cells preserved at low temperatures and subjected to ionizing radiation has not been adequately studied. This paper presents information on the extent to which both the lethal and chromosomal effects of ionizing radiation are modified by DMSO at -196°C. With this information and with certain assumptions, estimates of storage times at -196°C can be made which will relate to the production of known amounts of both lethal and chromosomal damage. METHODS
Tissue culture methods. Eagle’s minimum essential medium was used in conjunction with Eagle’s balanced salt solution buffered with 0.05 M Tricine and 0.09% sodium bicarbonate at pH 7.2. The medium was supplemented with fetal calf serum ( 10% ), 2 mM L-glutamine and 50 Cells of kanamycin sulphate. dml (Chinese hamster ovary fibroblasts. Pucks clone A) were incubated at 37°C and grown as monolayers in disposable plastic tissue culture flasks. Two days before they had grown to confluence they were trypsinized with 0.25% trypsin in Ca2+ and
THE
FROZEN
STATE
133
Mg?’ free salt solution for 5 min before the trypsin was inactivated by the addition of an equal volume of tissue culture medium containing sedum. The cells were collected by gentle centrifugation (75g for 3 min) and the pellet of cells was then resuspended in fresh tissue culture medium; the cell density of this solution was then approximately 1 X lo6 cells/ml. The survival of cells after freezing and thawing or after x-irradiation was measured by their ability to form macroscopically visible colonies 10 days after incubation. The cloning efficiency of the cells used in this study was between 50 and 60%. Freezing and thawing procedures. These procedures have been described previously (7). The cryoprotective solutions were prepared as 20% solutions in complete tissue culture medium and were slowly added to the cell suspension 10 to 15 min before the freezing experiments were started. Polyvinylpyrrolidone (PVP) was the Plasdone C preparation of the General Aniline and Dye Corporation (GAF, Inc., New York, U.S.A.). This material is claimed to have a molecular weight of 30,000 daltons (however, see Ref. 3); it was dialysed to remove the low molecular weight fractions which account for nearly 14% of the sample and which increase toxicity in some instances during freezing and thawing. X-Irradiation. Irradiation of cells at 22°C was done with the cells attached as a monolayer at a “treating” distance of 16 cm and an effective open diameter of 5 cm using a Picker Zephyr therapy unit at 10 mA and a nominal 120 kV. The beam was filtered with additional 2 mm Al and 0.25 mm Cu and had a 0.39 mm Cu H.V.L. An ‘output correction for voltage variations was applied at 2.2% per volt and a conversion factor of 0.94 rad roentgen was used. The dose rate was 127.12 radlmin, backscatter being provided by a 30 X 30 x 5 cm deep wax base. Irradiation of cells at -196°C was performed with cells in
ASfiWOOD-SMITH
134
frozen suspension in tissue culture fluid and DMSO contained in small plastic vials and surrounded with liquid nitrogen. Micronucleus test for chromosomal aberrations. After telophase micronuclei form in the cytoplasm from acentric chromosome fragments that are left behind at anaphase. The number of these micronuclei has been used as a simple, rapid and effective means of screening for chromosomal damage produced by mutagenic agents (10, 14). Cells either control or after thawing (with or without radiation treatment) were plated in dishes (approximately 5 x lo5 viable cells) for the detection of chromosome damage. Samples were taken at a number of times after the freezing and thawing treatment and/ or the x-irradiation procedures in order that the damage would be expressed during segregation. Cells were trypsinized in the normal way and resuspended in 5 ml of 1% trisodium citrate and left at room temperature before centrifugation and resuspension in 1 ml of freshly prepared Carnoy’s fixative (ethanol: glacial acetic acid 3: 1); when the cells were dispersed the volume of the fixative was adjusted to 5 ml. After 5 min the cells were again centrifuged and this final pellet was then resuspended in 0.1 or 0.3 ml of fixative depending on its size. Four slides for each treatment point were prepared immediately and allow to dry in air for several hours before staining with Gurr’s new improved Giemsa for 5 min (the Giemsa solution was diluted 1 in 10 with dilute phosphate buffer, 0.07 M, pH 7.0). The slides were rinsed in distilled water to decolourise the cytoplasm and once more dried in air. Not less than 2000 cells were scored for each micronuclei treatment point and expression time; scoring was randomised and the identity of the shdes not discIosed until the scoring was finished. RESULTS
AND
DISCUSSION
Effects of freezing and thawing and of storage on the production of chromosomal
AND FRIEDMANN
damage. The survival of Chinese hamster cells after freezing and thawing and storage at -196°C (cooling rate of 5”C/min from +22” to -45°C and then rapid as the cells, in ampoules, were plunged into liquid nitrogen; thawing rate of approximately lSO”C/min) varied with the nature of the cryoprotective but essentially remained unchanged as a function of the 6 month storage time at -196°C. The figures, with standard deviations in parenthesis were as follows: 10% DMSO, 91% survival (‘6.2); 10% glycerol, 65% survival (*5.0), and 10% PVP, 17% survival ( 22.5). Survival of cells frozen and thawed in the absence of cryoprotective agents was about 1%. These values are similar to those obtained over a number of years in our laboratory. The better survival of cells frozen in the presence of DMSO rather than glycerol is probably associated with the procedures used for the removal of the cryoprotective agents by a ten-fold dilution at room temperature into fresh tissue culture fluid. No doubt a slower “step-wise” dilution would improve the glycerol survival values. However the point is, that survival values of between 65 and 90% are fairly typical of the results that are routinely attainable in many laboratories throughout the world and the key question is whether chromosome damage is present in the surviving population of cells. In a large first experiment which was reported to the Ciba Foundation Meeting in London, in 1977 (7) a significant number (P= 0.01) of micronuclei were seen in cells preserved with DMSO after 20 hr of culture following thawing of samples stored at -196°C for either 2 hr or 3 months. Examination of celIs cultured for longer times or preserved with either glycerol or PVP failed to show any abnormalities (see Tables 1 and 4 for the results with DMSO and Tables 2 and 3 for results with glycerol and PVP respectively). Because of the importance of these
GENETIC
Micronuclei
in Frozen Storage
STABILITY
Hamster time
and
Cells
Stored
IN
THE
TABLE
1
FROZEN
for Different
Periods
treatment
of Time
in 10%
NIicronuclei ( f standard
time
20
Control
cells not
Cells
a Cell
frozen
with
frozen DMSO
Cells frozen 3 months
and stored at --196”0
in DMSO
for
Cells frozen 6 months
and stored at -196Q
in DMSO
for
and
thawing
constant
2.1 (0.3)
1.2 (0.4)
3.9 (1.2)
Hamster
time
treatment
and
Cells
Stored
9.9 (2.1)
few cells survived adequate analysis)
for
8.2 (2.7)
4.0 (0.6)
3.6 (0.3)
4.6 (1.2)
3.5 (0.9)
4.8 (0.6)
12.5 (3.6)
time
period
of experiment
at 91y’.
nuclei in cells treated but not frozen with DMSO was higher than the untreated controls (5.2 per 100 cells compared with 2.3 per 160 cells) but the standard deviations were 1.1 and 1.2 respectively. No evidence of increased chromosome damage was seen with any of the freezethaw regimes ‘and the general increase in micronuclei seen in all samples as a 2
for Different
Periods
of Time
Micronuclei ( f standard
20
in 107,
Glycerol
per 100 cell3 deviation)
Expression
time
(hr)
30
44
1.3 (0.05)
2.1 (0.3)
10.4 (1.9)
1.4 (0.6)
1.9 (1.5)
7.1 (2.7)
Cells frozen” and stored in 10% glycerol for 2 hr at -196°C
4.6 (2.2)
3.6 (1.0)
6.5 (1.8)
Cells frozen” and stored glycerol for 3 months
in 10% at - 196°C
1.5 (1.2)
3.2 (1.5)
2.6 (1.0)
Cells frozena and stored glycerol for 6 months
in 10% at -196°C
1.0 (0.3)
2.9 (0.4)
2.9 (1.4)
Control
cells not frozen
Control cells 10% removed
a Cell
(1.9)
2.8 (1.3)
TABLE in Frozen Storage
10.4
3.2 (0.5)
over
results several more experiments with DMSO were commenced and the results of the experiments are given in Table 4. Both repeat experiments revealed no difference between cells frozen and thawed in DMSO and stored for 2 hr at -196°C and cells treated with DMSO and not frozen and thawed. Admittedly in the second experiment the number of micro-
Micronuclei
44
1.3 (0.05)
(Too for
freezing
(hr)
30
no additives in DlblSO
after
Sulfoxide
added
Cells frozen and stored 2 hr at - 196°C”
survival
Dimethyl
per 100 cells deviation)
Expression
Control cells 10% and removed
135
STATE
survival
after
glycerol
freezing
added
and thawing
and
constant
over
the time
period
of the experiment
at 657&
136
ASHWOOD-SMITH
AND TABLE
Micronuclei
in Frozen
Storage
time
and
Hamster
Cells
Stored
FRIEDMANN 3
for Different
Periods
treatment
of Time
Micronuclei (& standard
Control
cells not frozen
Control cells 10% removed
PVP
Cells frozen and stored for 2 hr at -196°C’~
G Cell
added
PVP
Cells frozen and stored in 10% for 3 months at - 196”Ca
PVP
Cells frozen and stored in 10% for 6 months at - 196°C”
PVP
after
freezing
and thawing
constant
Experiment
Experiments
44
1.3 (0.05)
2.1 (0.3)
10.4 (1.9)
1.5 (0.2)
2.0 (0.4)
10.0
1.4 (0.5)
4.2 (0.8)
7.0 (1.8)
2.5 (0.2)
5.4 (2.0)
1.4 (0.5)
2.0 (0.3)
2.7
5.3 (1.0)
on Micronuclei
the time
number
Control cells (no DMSO)
standard
Cells Micronuclei deviation)
(1 For
differences
B
1.3 (0.05) table
Frozen
1.2 (0.4)
1)
in 10%
per 100 cells expression time
Control cells (DMSO added, removed)
A
1 from
of experiment
at 18%.
4
in Hamster
(f
(Results
period
(0.7)
Dimethyl
Cells frozen and stored in DMSO, 2 hr -196°C C
3.2 (0.5)” 3 months at - 196°C = 4.0 (0.6)~
2.3
(1.2)
5.2 (1.1)
4.2 (1.35)
3
1.3 (1.0)
1.4 (0.6)
0.8 (0.2)
C and B the Student
t test was 0.01
(P).
Sulfoxide
= 20 hr
2
between
(2.0)
ture, a situation very different from that seen with X-rays or uv, both agents which cause chromosomal damage. It is possible that some of the cells or the cultures containing the thawed cells, in those early studies, contained viruses which were activated by the DMSO and freeze-thaw procedures. DMSO has recently been shown by Lyman and his colleagues (12) to induce the differentiation of Friend leukemic cells and it was suggested (15) that this effect was at the level of DNA transcription.
TABLE of Three
(hr)
30
over
function of expression time irrespective of the treatment is often observed with this test system. Cells, preserved at -196°C in 10% DMSO for 2 years have very recently been thawed and examined for micronuclei and no evidence of chromosomal damage was seen. It is difficult to explain the first results of chromosome damage in cells frozen in DMSO although it is important to note that this damage did not persist but rapidly disappeared from the population such that it was not demonstrable after 30 hr of cul-
Summary
time
and
in 10%
survival
PVP
per 100 cells deviation)
Expression 20
in 10%
GENETIC TABLE
STABILITY
IN
5
Modification of X-Ray Induced Lethality in Hamster Cells Irradiated at - 196’C in the Presence of 10% DMSO Compared With the Induced Lethality at Room Temperature (f22”) Percent survival
Irradiation dose in rads +22Y?
10.0
1.0 0.1 Data in Ref.
from 7.
600 1000 1290 complete
DOW reduction factor (D.R.F.)
-196°C
2125 3400 4320 survival
3.54 3.40 s.35 curves
published
None of these studies, however, exclude the possibility of genetic damage at the molecular level, as base pair substitutions, transitions, and frame shift mutations would not be detected at the level of chromosomal structural damage. Preliminary experiments in our laboratory in which drug resistance to ouabain was studied failed to reveal any changes after various freezing and thawing procedures. In the knowledge of the negative results with bacterial and yeast systems this was reassuring, if hardly surprising. Effects of x-irradiation on lethality and chromosomal damaged cells irradiated whilst preserved in the frozen state at -196°C in the presence of 10% DMSO. Both glycerol and DMSO are radioprotective chemicals as well as possessing cryoprotective properties and from the work of Vos and Kaalen (17) some protection against radiation damage at low temperatures was to be anticipated; irradiation of cells in the presence of DMSO at -t22”C was not performed asthe interest in these present studies centred on the effects at low temperatures so that estimates of longe\Gty of stored cells might be made. The complete survival curves for x-irradiation of cells at room temperature and at -196°C in the presence of 10% DMSO have been published (7) but the relevant information in terms of the dose reduction factors observed are shown in
THE
FROZEN
137
STATE
Table 5. The overall dose reduction factor of the DMSO and temperature on cell survival from x-ray induced kill is approximately 3.5 over three log cycles. Thus low temperature storage of cells is clearly advantageous in reducing the lethal effects of x-rays. Values for temperatures below -196°C are not available for mammalian cells but may not be very different from values obtained at -196°C. The spores of Bacillus megaterium, for example, show no changes in radiosensitivity at temperatures between -195” and -268°C although there is a big increase in radioresistance between zero and -196°C (8). The changes in x-ray induced chromosomal damage as a function of radiation temperatures are shown in Figs. 1 (+22”C) in the presence of 10% and 2 (-196’C DMSO). The peak for the dose dependent chromosomal damage was observed at 30 hr for cells irradiated at +22”C. Cells frozen in 10% DMSO and irradiated at -196°C required larger doses of x-rays to produce chromosomal damage and al-
0
10
20 EXPRESSION
30 TIME
40
50
( hr.)
FIG. 1. Induction of micronuclei by x-rays in Chinese hamster cells at room temperature (+22”C). Ordinate: micronuclei per 100 cells f standard deviation. Abscissa: expression time in hours. n 42 rads; v 104 rads; l 257 rads; 0 515 rads. All irradiations were in air.
138
ASHWOOD-SMITH
lb
0
20 30 EXPRESSION
40 TIME
60
50 (HOURS)
160
AND
170
FIG. 2. X-ray-induced production of micronuclei in Chinese hamster celIs at -196°C in the presence of 10% DMSO. Ordinate; micronuclei per 100 cells * standard deviation. Abscissa: expression time in hours. n 540 rads; v 1620 rads; l 2700 rads; II] 3780 rads.
though, in general, the peak for the observed damage was after 30 hr, high doses of radiation (e.g., 2700 rads) resulted in biphasic responses. The damage produced at +22”C reached a peak and decreased sooner than damage of equivalent lethality produced by the larger doses of x-rays delivered at -196°C. Comparison of Fig. 1 with Fig. 2 indicates this difference (the time scales on the two graphs are different and should be noted). The biphasic nature, at higher doses, of the x-ray response
FRIEDMANN
at - 196°C is shown in Fig. 3. The reason for the long expression time of chromosomal damage after x-irradiation at low temperatures is not easily explained unless it is assumed that a qualitatively different type of damage is produced by low temperature irradiation but there is little to suggest this possibility. The formation of micronuclei per cell as a function of the fraction of cells surviving a given amount of x-irradiation was plotted and the data is summarized in Table 6. X-irradiation at +22”C resulted in values that are within the expected theoretical limits in that the number of micronuclei per lethal hit was between 0.1 and 0.2 (6). Irradiation at -196°C resulted in values that, in essence, fitted the same slope at least until survival values fell below 10%. Thus, although about 3.3 to 3.5 times as much radiation was necessary to kill cells at -196°C as at +22”C the same number of micronuclei per cell were produced. This result suggests that the same lethal mechanisms are responsibIe at the two widely differing temperatures and physical states even when a radio-protective chemical, DMSO, is present. With larger doses of x-irradiation at -196°C the number of micronuclei per surviving fracTABLE
6
l\Iicronuclei per Cell as a Function of X-Ray Survival; Cells Irradiated at +22”C (air) or at -196°C in the Presence of lO?i DMSO; the Expression Time at +22”C was 29 hr and at - 196°C was 30 hr Percent survival
X-ray dose (rads)
Micronuclei per cell
X-X%y dove bade)
+22”c
0
0:s
110
1.5 DOSE
io IN
23 KKILORADS
30
33
40
FIG. 3. X-ray-induced production of micronuclei in Chinese hamster cells at -196°C in the presence of 10% DMSO. Ordinate: micronuclei per 100 cells 2 standard deviation. Abscissa: x-ray dose in kilorads. n 20 hr expression time; l 30 hr expression time; V 44 hr expression time.
100.0 80.0 60.0 40.0 20.0 10.0 5.0 1.0 0.5 0.1
180 305 400 510 600 765 1000 1106 1290
Alicronuclei per cell -196X
0 0.05 0.12 0.20 0.33 0.47 0.60 0.89 1.00 1.32
590 1000 1350 1800 2125 2700 3400 3750 4320
0 0.05 0.12 0.22 0.40 O..% 0.32 O.RO 0.55 0.71
GENETIC
STABILITY
tion of cells is less (and the slope of the survival curve changes) suggesting that the mechanism(s) of chromosome damage may be different under those conditions. If large, unfractionated doses ‘of x-rays as used in these present studies have the same final effect as a number ‘of small, but cumulative doses of x-rays impinging on a frozen target and if the damage sustained by the frozen cells is not repairable until the cells are thawed then it is possible to arrive at estimates for storage times at -196°C and concomitant cumulative background radiation damage. About 70’70 ‘of the yearly background radiation dose of 0.1 rad/year is due to x- and y-rays and this means that the effective background dose of x-rays is about 0.07 rad/year. Neglecting any energy dependence (as between background radiation and our experimental x-ray beam), this latter value would be reduced both in terms of lethality and chromosomal damage by a factor of 3.5 to 0.02 rad/year at -196°C. The Di,, value for Chinese hamster cells is 600 rad at +22”C and 2125 rad at -196°C in the presence of 10% DMSO. Assuming no repair and no cell division these two values would not be reached for 8571 and 30,395 years! If both the lethal and the gross genetic effects of x-irradiation are modified in the same quantitative manner, as these present experiments strongly suggest, then tissue culture cells could be stored for about 30,060 years in the presence of DMSO at -196°C sustaining the before, on resurrection, equivalent of an acute Dlo dose of x-rays. Cells preserved in glycerol would probably behave in the same manner ‘as would most other vertebrate diploid cells preserved at very low temperatures. The behavi.or of frozen embryos to increased background radiation has recently been directly tested by Lyon and her colleagues (13, 18). They found that, when the background radiation level to frozen mouse blastulae was stored (in DMSO)
IN
THE
FROZEN
STATE
139
increased by a factor of 84 for 10 to 12 months, the proportions of implanted embryos and live foetuses was slightly reduced. The authors concluded that “the hazard of background radiation during storage, of mouse embryos, appears negligible although the effect observed at the highest radiation dose still needs further examination during longer periods of storage.” SUMMARY
1. Tissue culture cells preserved at -196°C in 10% glycerol or 10% PVP for periods of up to 6 months showed no gross chromosomal aberrations as disclosed by the micronucleus test. In one experiment cells preserved in 10% DMSO, which offered the best protection (91% survival after freezing and thawing), showed a statistically (P = 0.05) greater number of micronuclei 20 hr after thawing. This was a transient effect and three more separate experiments failed to demonstrate any chromosomal damage ‘associated with freezing, thawing and storage at -196°C for periods of up to 2 years in 10% DMSO. 2. Both the lethal and chromosomal effects of x-irradiating cells in 10% DMSO at -196°C were reduced by a factor of approximately 3.5 compared to irradiation at +22”C. 3. It is estimated that a period of approximately 30,000 years of storage of cells in 10% DMSO at -196°C would pass before an accumulated x-ray dose from background radiation would reach a level, which upon cellular resurrection, would result in the equivalent lethal and chromosomal damage of an accute Dlo dose. 4. Cells cannot be kept at -196°C ad infinitum as there is no escape from background radiation damage. Shielding would reduce the level but internal radiation from isotopic disintegration is inescapable. ACKNOWLEDGMENTS We would like and Miss Elizabeth
to thank Mrs. Alexa Kennedy Grant for the excellent tech-
140
ASHWOOD-SMITH
nical help with the tissue culture, freezing and micronuclei analyses and the Victoria Clinic, a Unit of the B.C. Cancer Control for the use of their irradiating facilities.
methods Cancer Agency,
REFERENCES 1. Ashwood-Smith, M. J. The radioprotective action of dimethyl sulphoxide and various other sulphoxides. Intern. J. Rad. Biol. 34, 41-48 (1961). 2. Ashwood-Smith, M. J. On the genetic stability of bacteria to freezing and thawing. Cryobiology 2, 39438 ( 1965). 3. Ashwood-Smith, M. J., and Warby. C. Studies on the molecular weight and cryoprotective properties of polyvinylpyrrolidone and dextran with bacteria and erythrocytes. Cryobiology 8, 453464 ( 1971). 4. Ashwood-Smith, M. J. Current concepts concerning radioprotective and cryoprotective properties of dimethyl sulphoxide in cellular systems. N.Y. Acad. Sciences, 243, 246-256 ( 1975). 5. Ashwood-Smith, M. Mutation induction drying. Cryobiology
J., and Grant, in bacteria 13, 206-213
Elizabeth. by freeze ( 1976).
6. Ashwood-Smith, M. J., Grant, E. L., HeddIe, G. B. Chromosome J. A., and Friedmann, damage in Chinese hamster cells sensitized to near-ultraviolet light by psoralen and angelicin. Mutation Res. 43, 377-385 (1977). 7. Ashwood-Smith, M. J., and Grant, Elizabeth. Genetic stability in cellular systems stored in the frozen state. In “The freezing of mammalian embryos” ( CIBA Foundation Symposium No. 52, new series, 251-272. Elsevier/Excerpta Medics/North Holland (1977). 8. Bacq, Z. M., and of radiobiology. and New York
Alexander, Pergamon (1961).
P. Fundamentals Press, London
AND
FRIEDMANN
9. Bridges, B. A. The chemical protection of Pseudomonas species against ionizing radiation. Radiation Res. 17, 801-806 ( 1962). 10. Heddle, J. A. A rapid in viva test for chromosome damage. Mutation Res. 18, 187190 ( 1973). 11. Hieda, K. Genetic changes induced by freezing and drying. Abstract. Low Temperature Medicine 3, 13 ( 1977). 12. Lyman, G. H., Preisler, H. D., and Papahadjopoulos, D. Membrane action of DMSO and other chemical inducers of Friend leukaemic cell differentiation. Nature (London) 262, 360-363 ( 1976). 13. Lyon, M. F. Implications of freezing for the preservation of genetic stocks. In Basic aspects of Freeze Preservation of Mouse Strains (ed. 0. Muhlbock), pp. 57-65. Stuttgart: Bustav-Fischer Verlag ( 1975). 14. Matter, B. E., and Grauwiler, J. Micronuclei in mouse bone marrow cells. A simple in civo model for the evaluation of drug induced chromosomal aberrations. Mutation Res. 23, 239-249 (1974). 15. Preisler, H. D., Houseman, D., Scher, W., and Friend, C. Effects of 5-bromo-e-deoxyuridine on production of globin mRNA in dimethyl sulfoxide stimulated Friend leukemic cells. PTOC. Nat. Acad. Sci. USA 70, 2956-2959 ( 1973). 16. Shikama, K. Thermal and freezing denaturation of deoxyribonucleic acid. Sci. Rep. Tohoku Univ. 4th Ser.. Biol. 30, 133-141 (1964). 17. Vos, 0.. Kaalen, M. C. A., and Budke, L. Radiation protection by a number of substances preventing freezing damage. 1. Protection of mammalian cells in vitro. Int. 1. Rad. Biol. 9, 3341 (1965). 18. Whittingham, D. G., Lyon, Mary F., and Glenister, P. H. Long-term storage of mouse embryos at -196°C: the effect of background radiation. Genet. Res. Camb. 29, 171-181 (1977).
