CRYOBIOLOGY
14, %%-2% (1977)
BRIEF Retention
COMMUNICATIONS
of K at Low Temperature in Mouse before and after Cold Adaptation J. S. WILLIS
Institute
1 AND
L Cells
E. HOLECKOVA
of Physiology, Czechoslovak Academy of Sciemes, Prague, Czechoslovakia
The possibility that loss of cytoplasmic K days (1, 3). Furthermore, they have found is an important, perhaps the primary, cause that the survival of aneuploid cells could be of damage and death in mammalian cells improved by repeated exposure to cold ( 1). at temperatures just above freezing (2, 4) In these studies the criteria of survival were has been difficult to test for two reasons. ability to exclude dye ( eosin B ) and ability First, one needs cells in which K can be in- to regrow at 36°C. dependently maintained at high or low We, therefore, undertook to discover concentrations. Second, one needs unam- whether mouse L cells exhibit an unusual bivalent criteria for cell damage or death. ability to regulate K at 5°C and whether In one attempt to fulfill these requirements this ability would be improved by repeated the effects of high and low K on protein cold exposure. synthesis and on K regulation itself were MATERIALS AND METHODS investigated in kidney slices of hamster stored at 5°C (4). (Cells of hamster tend Culture to maintain a high K, but this can be lowMouse L cells were cultivated in Eagle’s ered reversibly and without primary metaminimal medium with 10% inactivated calf bolic inhibition by ouabain and/or low K serum and antibiotics (penicillin, 100 pg/ml in the medium. ) nud streptomycin, 100 pg/ml of culture An alternative model preparation could medium). be mammalian cell cultures. Holeckova and For preliminary cold exposure Roux her co-workers have shown that mammalian flasks containing confluent cultures were aneupliod established cell lines survive cooling to 4°C far better than diploid cells put into the refrigerator without change of of mammals, even of hibernators. Thus, medium aud left there for 14 weeks. They with mouse L cells, only 20% of the cells were then returned to 36°C and after 2 or 3 days at that temperature the medium was die within 2 to 3 weeks, whereas the halftime of survival of fibroblasts of hamster changed. When the population had reand human embryos is between 2 and 3 grown the culture was transferred to new -__ flasks. Received April 22, 1976. 1 To whom reprint requests should be addressed. Permanent address: University of Illinois at Urbana-Champaign, Department of Physiology and Biophysics, 524 Burrill Hall, Urbana, Illinois 61801.
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GJ 1Y77 by Acadrmi~ l’trsb, In, of reproduction in any form reserved
Survivul Tests Miiller flasks were inoculated with a known numl,er of cells and put into the in-
1WEF COhlhlUNICrZTION
1 +
I +
2 +
:I +
4 (=i)
“37
2 (=fi,
cubator and allowed to grow until they formed a monolayer. The number of cells per flask was calculated before cold cxposure and then at weekly intervals after the flasks were placed in a refrigerator at 4-5°C. Usually, eight counts were performed in a hemocytometer for four flasks. The number of dead cells was estimated by their uptake of the dye eosin B. At the end of 4 weeks in the cold four flasks of each subline were returned to 36°C for 1 week. At the end of this time the number of living cells was estimated in each flask. All results are expressed as a percentage of the original cell number before exposure to cold. K Determinution To follow the loss of K in the cold, Miiller flasks of cells of several sublines were placed in a refrigerator at 5°C and K san~plcs were takct1 after various p(xriods. The monolayer of cells in Miiller flasks (usually four to six for each period) wx w:Lsl~c~l L11wc~ titlics with I~llosl)lr;ltc~-l)llfI’c~rcd isototlic salilw ( NuCl, 130 1111~1, lV:l-
phosphate bufIer, 3 mM, pH 7.4). The wash solution was ice-chilled and the vials were placed upon ice-cold metal during washing to minimize rapid loss of K. It was found that in some cultures cells detached from the glass and floated in the medium after a short interval in the cold. To be certain, therefore, that all cell K was recovered, the cells in the supernatant medium were centrifuged at 5°C and washed three times. The centrifuge was a Unipan microcentrifuge, Type 310, with rapid acceleration. Each ccntrifugation lasted 20 set and the whole procedure required about 3 min. After being washed, the cells were extracted with 10% trichloroacetic acid and the K content estimated by flame photometry. Ccl1 K is expressed as a percentage of the original K at 36°C as measured in sister flasks of the same culture. To ascertain that total cell number had not changed during storage,, ccl11 collnts wcrc made both of the ttrcdiulll a~rd of the cells still adhering to tlw glass ill flasks handled in exact prallel \\,illl tllosc, 111x)11\vhic~h K tl(~tc~l.lnitl:ltiot~s \\‘(‘I‘(‘ II Id(~.
238
BHIEF COX~lMUNICATION
FIG. 1. Loss of K in cold-adapted mouse L cells kept at 5°C for varying times. Circles, LC, (cell line cold-exposed repeatedly for years). Triangles, LA (control stock, not previously cold-exposed systematically). Points represent the means of three to six flasks, SE shown for five or more cases.
RESULTS
Two groups of L cells were used in this study. The first, LCS, was isolated in 1963 and has been cold-exposed intermittently since that time for 4-8 weeks at 4°C. Its superior survival in the cold, in comparison with its parent stock, LA, has been reported previously ( 1). In order to examine the effects of a shorter period of repeated cold exposure, a second series was begun on another line of mouse L cells, LAS. In Table 1 cell survival and regrowth in this second line are shown following two regimes of preliminary cold exposure and then a trial cold exposure of 4 weeks. In one subculture (L&) the cells were exposed to cold for an aggregate of 6 weeks in four short intervals (1, 1, 2, and 2 weeks), and in the other ( LCsB) the cells were exposed for 7 weeks in two longer periods (3 and 4 weeks). In neither case was there any marked improvement in survival over that of the control LAS in the cold. Flasks of LAS cells returned to the warm for 1 week recovered 57% of their original cell number, whereas L& regained only 20% and those of LC&, 88%. There was little change in K content in the first week in the cold in either LA 01 LC, cultures (Fig. 1). After that, however,
K content fell rapidly in LA and less so in LC, so that by 3 weeks LA cells had lost 53% of their K but LC2 only 34% (P = 0.03). In the short-term cold-exposed cell ( LC3) there was an early loss of K (Fig. 2), but even so after two weeks at 5°C the K content of both LCSA (80% ) and LCau (650/O ) was well above that of two subcultures of control LAS cells (44 and 23% ). By 3 weeks there was little or no difference between the LC3 and the LAS cultures. DISCUSSION
The results show that the features of survival of mouse L cells described in the introduction are also reflected in their K regulation. Thus, even the parent stock L cells showed an ability to retain K up to 4 days (LAS, Fig. 2) or a week (LA, Fig. 1) which was superior to that of cultured diploid cells from other mammals and equal or superior to that even of cells from hibernators (6, 7). In addition, repeated which ultimately yields cold exposure,
I
I 2 Weeks
3 at
I 4
5%~
FIG. 2. Loss of K in mouse L cells cold-adapted for short period. Circles, LC (cold-exposed intermittently ). Closed circles, LC:,,j ( see Table 1, text), Open circles, L&3. Triangles, LAS (previously unexposed control stock); results of two suhcultures of LAS are shown (open and closed triangles). Lines arc drawn to connect points for IX:,4 alld mitlpoint of two LAS cldtlucs. SE arc S~IOWII for points representing five of ruon tleterminations.
BKIEF
COhlhlUNICr\‘TION
L’S!) SCSl?\IARY
It is also of interest that in most casts the loss of K at low temperature precedes cell death as detected by dye exclusion (compare LAS, Fig. 2 with LAS, Table 1 and LA, Fig. 1 with Fig. 1 in Ref. 1). Such a result is in accord with the hypothesis that loss of K is a causal factor in cell injury and death in the cold. On the other hand, it should be borne in mind that dye exclusion and K content both reflect in different ways the same intact cellular property, membrane integrity. In LC, cells, loss of cell K coincided with and did not greatly precede cell death. 111 this case however, division of relative K at 2 weeks in the cold (Fig. 2) by relative cell number at that time (Table 1) yields the result that, if one assumes that dead cells contain negligible K, the K content of the living cells is undiminished. In contrast, the coutrol LAS cells would all have lost K at 2 weeks. The observation that intermittent cold exposure could result in better retention of K at 5°C before survival at 5°C was improved (e.g., LC&) is in accord with the proposition that K is a precondition of survival. But it also raises the question of how the improvement of K retention came about. There are two possibilities : either intermittent cold exposure triggered a latent cellular regulatory mechanism similar to that suspected in cold-stored kidney slices of hamsters and ground squirrels (3), or else a selection occurred for cells with greater ability to retain K. For selection to bc the explanation it would have to be assumed that those cells which lost less K at the cud of a short period of exposure to cold were better able to initiate growth and to dominate the culture when it was rewarmed. In any case, the results indicate the usefulness of this cell preparation for further investigation of the role and significance of K in cell survival in the cold.
\\T1~c~kcq)oscd to 5°C for periods of 3-4 weeks, mouse L cells, grown as monolayer cultures, lose K more slowly than do diploid mammalian cells. Subcultures of mouse L cells previously exposed to low temperature for intermittent periods lose K more slowly than subcultures not previously cold exposed. The superior retention of K in the cold may account in part for the better survival in the cold of mouse L cells than of 1, diploid cells, and of cold-conditioned cells than of unconditioned L cells. i\ChSO\VLEDG’\IENTS The LAS stock \vas kindly provided by Dr. B. ?\lanersl)erger, Academy of Sciences of the German Democratic Republic. WC are grateful to Dr. J. Cort of the Institute of Organic Chemistry for the 11se of the Hame photometer and to J. Cortova for help in K deterlninations. J. \V. was an eschnnge scientist of the U.S. National Academy of Sciences and tlw C:zdm~lovak Acnclclny of Sciencc~s. REFERESCES 1. Iloleckovn, E., Baudysova, ?\I., and Cinnerova, 0. Adaptation of mnmmnlian cells to cold. resistance to cold and multiplication of I, Detroit-G and IleLn cells ndnptcd to low temperatwc. I:‘xp. Ccl1 Hcs. 40, 396-401 (1905). 2. Leonard, C. I)., and Scribner, B. II. Effect of choline and hypothcrmin upon II production and K loss from isolated canine kidney. Crc~obiology 8, 290-292 ( 1971). 3. hliclrl, J., Rczncova, D., and I-Iolcckoq E. Adaptation of m,unmalian cells to cold. 117. Diploid cells. Erp. Cdl Rcs. 44, 680-683
(1966). 4. IVillis, J. S. Thr possible survival of cells at low
roles of cell temperature.
K for Cryo-
biozog~9, 351-366 (1972). 5. b?llis, J. S., Foster, R. F., and Bchrends, C. I,. Cold-stored kidney tissue of hibernators: Effects of brief wuming on K wgulation. CryobiologfJ 12, ‘55-“G5 (19%). 6. Zritller, R. B., and \Villis, J. S. Cultured cells from renal cortex of hibernators and nonhibernators: Regulation of cell K at low tcmper&we. Biochim. Bio&s. Acta, 4.76,628651 (1976). 7. Willis, J. S., and Baudyso~~a, bl. Retention of K’ in relation to cold resistance of cultured cells from hamster and human cmlxyos. CrrJobiolopy, in press.