CFlYOBlOLOCY
12,
309320
Biological
( 1975)
Interactions Between Cell and Glycerol or DMSO DONALD
Biology Departmnt,
B. PRIBOR
of Toledo,
University
When I oame to work in Luyet’s laboratory in 1962, he and Rapatz already had a great deal of evidence that cooling and rewarming rates were crucial for the surviva1 of red blood cells after freezing and thawing. The working hypothesis in the laboratory at that time was (I) an optimal cooling rate was necessary for maximum recovery of frozen erythrocytes; (2) at or beIow this rate only extraceIIular freezing occurred; (3) below the optimaI rate, the erythrocytes were “in some manner” damaged by the concentration of intra- and extracellular solutes; (4) above these rates the cells were damaged by the formation of intracellular ice; (5) if erythrocytes survived intracelluIar freezing at very high cooling rates, they would be subjected to further injury by a recrystallization of intracellular ice unless rewarming was equally rapid. In 1965, based on freezing studies with microorganisms (7) and by a derivation of a quantitative description of the rate of intraceIIuIar water Ioss during freezing (8), Mazur proposed the now, well known, two-factor theory of freeze-thaw damage (9). This theory was equivaIent to the working hypothesis used in Luyet’s laboratory. Mazur’s published formulation of the two-factor theory was an important contribution because it aIerted cryobiologists to the :importance of coobng rates and it provided a single framework for Received
April
7, 1975.
309 Copyright All rights
0 1975 by Academic Prm. Inc. of reproduction in any form reserved.
Membranes
Toledo,
Ohio 43606
interpreting diverse reports on freezing injury. Subsequent reports from Mazur’s group indicated that the theory could also be apphed to cells in tissue culture. This and the data from Luyet’s laboratory in addition to a number of other reports provided strong experimental evidence for an expIanation of cryoinjury. In this paper I want to acknowledge my debt for the information and the insights suggested by Luyet’s laboratory in the early sixties. At the same time I will present experimental evidence which directly contradicts some of this earlier work. The present data as well as that of some other workers, indicates that Mazur’s twofactor theory needs to be modified and extended. Freeze-thaw damage and the mechanism( s ) for cryoprotection seems to be more complex than any of us realized in the 1960s. Recent advances in membrane physiology implies that 1this must be so. The data to be reported in this communication is consistent with and supportive of the recent dynamic models of membrane structure and function, Thus, this paper is intended to extend the multifactor theory of cryoprotection, described elsewhere (ZO), to low molecular weight cryoprotective agents such as glycerol and dimethylsuIfoxide. MATERIALS
AND
METHODS
Blood. ToIedo Hospital which routineIy used EDTA as an anticoaguIant provided samples of whoIe blood. The blood ob-
310
DONALD
tained from healthy donors, was stored at 4°C and used within 4 days after collection for subsequent studies. Preparation of erythrocyte suspensions. Cryoprotective additives of the desired concentration were incorporated into a phosphate-buffered saline (PBS) which contained 0.154 M NaCl, 0.01 M Na2HP04, and 0.0016 M NaH2P04 in double distilled water. The final pH was adjusted to 7.5 with 1 N NaOH or 0,l N HCI. Erythrocytes were sedimented by centrifugation at 11,500 rpm in a Sorvall RC 2-B centrifuge for 15 min at 4°C. After removal of supernatant fluid and the white cell layer the erythrocytes were washed three times by suspending them in 1.5 vol of PBS and the intact cells removed by centrifugation. Cells, so collected, were then resuspended in sufficient PBS to give a final concentration of 2 x 10” erythrocyteslml. Aliquots were combined with equal voIumes of solutions containing dimethyl sulfoxide (DMSO) or glycerol to give the desired concentration of the protective agent. For example, a 40% (v/v) DMSO soIution was mixed with an equal volume of red cells suspended in PBS to give a 20% sohrtion of DMSO containing 1 x lo9 cells/ml. The cryoprotective concentration is expressed in percentages rather than molarity to facilitate a comparison with previous studies by other investigators. However, in order to make comparisons between glycerol and DMSO, the concentrations were adjusted so that corresponding solutions would have equivalent osmolarities. Thus, 2.5, 5.0, lO,O, and 15.0% DMSO solutions would be equivaIent in osmolarity to 2.95, 5.9, 11.8, and 17.17% glycerol solutions, respectively. Originally, the experiments were designed in this way, but much later it was discovered that the DMSO solutions were miscalculated by a factor of 2, As a result, the DMSO solution concentrations are actually 5, 10, 20, and 30%, respectively, and therefore only two of these
B. PRIBOR
have equivalent osmolarities with respect to the glycerol concentrations used. As the results indicate the effect of increasing the concentration of glycerol is much greater than with DMSO; so the overlap between the two sets of data is sufficient for a meaningful comparison. Freezing and thawing. The red cells suspended in the cryoprotective solutions were allowed to equilibrate for 1 hr at room temperature before freezing. Samples of the cell suspensions were then drawn into 50-lambda glass Micropets (Clay-Adams) so that the lower level of the celI suspension was approximately 6-7 mm above the bottom of the tube to allow room for cold Vaseline plugs. Samples to be frozen for 16 min were also sealed with cold Vaseline at the upper end of the tube, These 50 lambra samples were frozen and stored for 1 or 16 min in a weIlstirred alcohol bath at temperatures ranging from -10 to -80°C. Samples to be frozen for 1 min were immersed to a level sufficient to cover the entire column of the cell suspension; whereas those to be frozen for 16 mm were immersed so that the column was 34 cm beIow the bath level. The latter depth was required to maintain a constant temperature over the 16-min period. The specimens contained in the tubes were held in position with positive action forceps for the desired period commencing at the time of immersion, after which they were abruptly transferred into a water bath at 37°C and agitated gently to effect thawing. Freezing at -10 and -20°C was initiated by “seeding” as described in an earlier paper (19). In a previous article (20) based on the work of Rapatz ad Luyet (21) it was suggested that at freezing temperatures, with similar sized tubes, in baths from -20 to -80°C the cooling rates probably ranged between 15 and 83”C/sec (900 and 5000” C/min).
GLYCEROL
CONTRASTED TABLE
MEAN
PEXCENTAUEQF
WITH
DMSO
311
3
HEMOLYSISIN
Typeof
Degrees
PBS SOLUTION centigrade
expmment
1 min freezing” 16 min freezinga
-10
-20
-30
-40
-50
-60
-70
-80
89.4 90.9
95.8 93.2
94.3 90.9
85.3 81.8
69.1 36.7
57.4 44.1
43.9 37.2
43.2 28.5
These are means of 12 replications, i.e., 4 repliCatiOn8 for each of 3 different blood donors. a Difference between means required for significance at 5y0 level = 6.7 (Studentized Range Test). b Difference between means required for significance at 5% level = 7.0 (Studentized Range Test).
Processing after freezirzg and thawing.
After thawing, each tube was wiped dry, the Vaseline plug(s) removed, and the contents blown into 4 cc of the appropriate diluent. The tube was washed six times by drawing in and expelling the diluent to ensure complete removal of the cell suspension. The extent of hemolysis was determined colorometrically by measuring the amount of hemogIobin released from the cells of the treated specimens. Quadruplicate controls for zero and 100% hemolysis were prepared. In the case of the former, treatment was identical with each of the corresponding frozen and thawed samples with the exception that the specimens were maintained in a bath at room temperature. The latter were prepared by lysing cells in 4 m1 of a 2.5% solution of saponin. Design of Study. The total study was divided into eight experiments-four different concentrations of DMSO and four of gIycerol. Within each experiment blood from the same ‘three donors was used throughout with four replications; 1 and 18 min freezing were part of a singIe experiment. Blood donors varied between experiments, RESULTS
TabIe 1 and Fig. 1 illustrate the effect of cooIing rate on erythrocytes suspended in PBS in the absence of a protective agent and frozen for 1 and 16 min, respectively. The combined data for the 1 and 16 min freezing faiIed to pass Bartlett’s test for homogeneity of variance,
However, such an analysis was permissible for each of the two subclasses of the data. The difference between the means was statistically sign&ant at the 5% level, 6.7 after 1 min of freezing and 7.0 after 16 min freezing. The percentage of hemolysis began to level off at -70°C in those specimens frozen for 1 min; freezing at -70 to -80°C probably corresponds to the optimum cooling rates for red cells referred to in the papers of Rapatz and Luyet (21) and Mazur (10). Figure 1 indicates that in the range from -50 and -80°C a 16-min freezing exposure produces less hemolysis than 1 min. This is particularIy evident at -80°C where the hemolysis values are 28 and 43%, respectively. The mean, standard deviation and standard error are given in Tables 2 and 3 for the DMSO experiments and in Tables 4 and 5 for those with glycerol. The DMSO data is sufficiently homogeneous to permit an analysis of variance. This is not true for the results with gIycero1; however, a log transform of the data makes such an analysis possible. Two sets of experiments are reported in which red cells are frozen in 30% DMSO soIution. This concentration results in some hemolysis, particularly, to erythrocytes that have survived freezing stress. The results are inconsistent and cannot be accounted for systematicaNy. These effects are probably responsible for the increase in variation of the data at this concentration. In one set of experiments the DMSO present in the blanks probably gave a higher estimate
DONALD
312
B. PRIBOR
No Protective -I
mn.
---lbmin.
8
-2tF
-30”
-400
TEMPERATURE
-500
-60’
Agent freezing
freezIng
I -700
400
L’C)
FIG. 1. Percentage of hemolysis of red cell suspensions frozen without a protective agent in 50 lambda micropipettes (Micropets, Clay Adams) for 1 min or 18 min in baths at temperatures ranging from -10 to -80°C ,
of hemolysis; the others without DMSO gave lower values. DMSO experiments. The results of a l- or a 16-min exposure of erythrocytes, suspended in various concentrations of DMSO and frozen at various temperatures are given in Fig. 2. The vaIues are what one would expect on the basis of previous work reported by Luyet’s group (21) and predictions from Mazur’s two-factor theory of freezing injury: (1) a I6-min freezing exposure is more damaging than a 1-min low concentrations of exposure; (2) DMSO (5%) g ave high hemolysis values at freezing temperatures from -10 to -40°C. The extent of injury was considerably lower in the freezing range of -50 and -80°C; (3) concentrations of 20% DMSO affords good protection over
the entire freezing range regardless of the time of exposure to low temperatures; (4) higher DMSO concentrations (30%) result in an increase in hemoIysis at higher cooling rates. Glycerol experiments. Figure 3 compares the effects of a l- and a 16-min period of freezing on red cells suspended in various concentrations of glycerol. These rest&s are similar to those dbtained with DMSO but certain dserences are present. (1) A 16-min exposure to freezing conditions is more damaging than a l-mm exposure in suspensionscontaining 2.95 and 5.9% glycerol, but in the case of higher concentrations (11.8 and 17.7% ) the longer exposure, in particular, at temperatures below -40°C is less injurious. (2) As with DMSO, low concentrations of
GLYCEROL
CONTRASTED TABLE
PERCENT-4CE
OR HEYOLYSIS
IN
WITH
DMSO
313
2
DMSO
SOLUTION+-1
Degree8
min
FREEZING
centigrade
-10
-20
-30
-40
-50
-60
-70
-80
14.7
61.9
42.9
24.3
11.7 3.4
7.1 2.0
7.9
0.3
70.2 5.6 1.6
2.3
20.6 5.4 1.5
15.2 5.0 1.4
12.4 1.6 0.5
3.1
8.6
9.6
6.8
4.7
4.8
1.0
1.5
3.1
0.7
1.3
0.4
0.9
0.2
0.4
4.1 0.6 0.2
4.3 0.8
0.3
1.9 0.6
3.2 1.2 0.4
3.9
4.9
4.4
5.1
6.0
0.6
1.0
0.2
0.6 0.2
0.8 0.2
1.2 0.3
7.1 1.3 0.4
9.6 1.6 0.5
10.1
11.6 6.6 1.9
14.8 6.8 2.0
17.6 6.6
21.3 7.1 2.1
25.9
29.5 10.9 3.1
22.1 8.3
0.5
4.1 7.7 2.2
7.3 7.0 2.0
10.9
16.7 17.3
20.7
12.4
51% Mean SD SE IO:% Mean SD SE 2a:r, Mean SD SE 305Q Mean SD SE 30’7*~ Mean SD SE
1.1
7.4 2.2
-2.1 9.1 2.6
6.8 2.0
0.3
1.9
7.3 2.1
15.6 4.5
5.0
0.2
2.4
11.0
9.0
3.2
2.6
These are means of 12 replications, i.e., 4 replications for each of 3 different blood donors. QPercentage of hemolysis determined in usual way as described in Materials and Methods. b Percentage of hemolysis determined with respect to distilled water blanks.
gIycero1 (2,95 and 59% ) gave high hemolysis vaIues in the freezing range from -10 to -4O”C, however, considerably lower values were observed in the -50 and -80°C range. (3) Higher concentrations of glycerol ( 11.8 and 17,7% ) reduced, considerably, the injurious effects of freezing on specimens exposed for 1 min and 16 min in the temperature range -10 to -40°C. (4) At the higher cooling rates, obtained by freezing at lower temperatures, hemolysis begins to gradually increase, in particuIar in those specimens containing 17.7% glycerol. DISCUSSION
The resuIts presented are different in some respects from those reported in a similar study by Rapatz and Luyet (21). In their paper, red ceils in the presence of 2.5% glycerol show a large increase in the extent of hemoIysis after a 1-min
exposure to freezing in the temperature range -60 to -80°C. In the present paper, the increase in injury was not observed unIess the gIycero1 concentrations were increased to 12%. Likewise, Rapatz and Luyet (21) observed that DMSO, in 5% concentrations caused an increase in hemolysis over the same freezing range. Our resuhs show that such an increase in damage occurs only if concentrations are increased to 20%. These dii%erences can be partially explained by the fact that in the former study the cryoprotective agents were added directly to whole blood. In our experiments cells were exposed to an isotonic medium; in Rapatz and Luyet’s study the celIs were exposed to a slightly hypertonic medium, assuming that penetration of the cryoprotective agent across the cell membrane occurred during the equilibration period. Further, the plasma proteins, particularly albumin, exerts a
DONALD
314
B. PRIBOR TABLE
PERCENTAGE Concentration of dimethyl sulfoxide DMSO
OF
HEMOLYSIB
3
IN DMSO
sOLuTIONel6
Degrees
min
FREEzlNa
centigrade
-10
-20
-30
-40
-50
-60
-70
-80
5% Mean SD SE
20.2 5.1 1.5
79.9 3.8 1.1
84.3 2.0 0.6
54.0 12.4 3.6
16.6 2.0 0.6
10.6 1.8 0.5
8.0 1.5 0.4
8.1 2.6 0.8
10% Mean SD SE
4.0 1.1 0.3
27.4 4.0 1.2
55.8 5.9 1.7
15.x 2.9 0.8
5.4 1.3 0.4
4.6 1.7 0.5
4.4 0.6 0.2
6.2 3.0 0.9
Mean SD SE
2.6 1.1 0.3
2.4 2.0 0.6
5.0 1.9 0.6
4.1 0.6 0.2
4.0 0.9 0.3
5.2 1.5 0.4
9.0 2.3 0.7
7.6 2.1 0.6
3O%‘oe Mean SD SE
9.4 6.2 1.8
15.0 6.8 2.0
16.5 7.8 2.2
17.3 6.9 2.0
19.8 6.8 2.0
26.3 13.3 3.8
40.0 3.5 1.0
28.4 4.7 1.3
-0.6 6.1 1.8
4.3 6.6 1.9
6.0 8.2 2.4
6.9
9.4
17.2
7.0 2.0
7.3 2.1
13.6 3.9
32.5 3.1 0.9
19.5 4.8 1.4
20%
3O%:O”
Mean SD SE
These are means of 12 replications, i.e., 4 replications for each of 3 different a Percentage of hemolysis determined in usual way ag described in Materials b Percentage of hemolysis determined with respect to distilled water blanks.
ciyoprotective action at the lower cooling rates and is associated with an increase in damage at the higher cooling rates ( 14). This may expIain the fact that our results are more in accord with those of Morris and Farrant (14) who also used washed red cells in a buffered salt solution. However, while our experimental data shows that a 10 and 20% solution of DMSO gave better protection at the lower cooling rates than glycerol, in similar concentrations, our results differ from those of Morris and Farraut because DMSO gave better protection at higher cooling rates. Specifically, 1 min freezing in the presence of a 17.7% glycerol solution was considerably more damaging than one min freezing in 30% DMSO, On the other hand, the injury to red cells suspended in either DMSO or glycerol was nearly the same after a 16-min freezing exposure (see Tables 3
blood donors. and Methods.
and 5), which are similar to the results reported by Morris and Farrant. Glycerol contrasted with DMSO. The results indicate three similarities between glycerol and DMSO, (1) In low concentrations (5% or less), 16 ,min freezing is more damaging than 1 min freezing. (2) With increasing concentration of glycerol or DMSO there is a corresponding decrease in percentage of hemoIysis at the lower cooling rates (bath temperatures from -10 to -4O’C) but an increase in percentage of hemolysis at the higher cooling rates (temperatures from -50 to -80°C). (3) Between -10 and -40°C the hemolysis curve for red cells suspended in 1.28 M glycerol or DMSO (11.8 and 10% cryoprotectant, respectively) demonstrated a convex form when cells were exposed to freezing for 16 min and a straight line for a 1 min freezing. There
GLYCEROL
CONTRASTED
WITH
TABLE PERCENTAQE
OF HEMOLYSIS
IN
315
4
SOLUTIONS-~
GLYCEROL
Concentration
Of gl,oerol
DMSO
min FREEZINQ
Degrees cent&de
-10
-20
-30
-40
-50
-60
-70
-80
64.8 3.2 0.9
88.7 1.2 0.3
70.0 4.2 1.4
30.5 3.3 1.0
15.1 1.4 0.4
14.1 1.1 0.3
14.1 2.7 0.7
18.4 2.6 0.3
22.2 2.8 0.8
73.0 2.7 0.6
41.0 2.8 2.2
14.2 1.8 0.5
10.8 1.1 0.3
9.9 2.8 0.2
11.6 1.9 0.5
12.4 2.9 0.8
SD SE
1.7 0.8 0.2
5.5 1.2 0.3
5.9 1.3 0.4
7.2 1.8 0.5
13.0 5.8 1.7
19.7 8.1 2.4
25.8 9.7 2.8
33.9 12.9 3.7
17 70/o Mean SD SE
1.5 1.5 0.4
1.8 1.1 0.3
4.2 0.7 0.2
13.6 2.1 0.6
35.7 2.8 0.8
81.1 6.4 1.9
90.9 3.4 1.0
90.1 3.: 1.0
2.95yo Mean SD SEl 5.9%
Mean SD SE 11.sy*
Mean
These
are meanz
of 12 replications,
i.e., 4 replications
are, on the other hand, three differences between glycerol and DMSO. (1) A 16min freezing was equally or more damaging than a X-min freezing in DMSO, regardless of its concentrations; the conTABLE
PERCEKTAGEOF HEMOLYSIS
IN
far each of 3 different blood donors. verse was true in 11.8 and 17.7% glycero1
solutions. (2) 10 and 20% DMSO solutions gave better protection at the lower cooling rates than the glycerol soluticns in the same concentration range. (3) 5
GLYCEROL SOLUTION+-16
min FREEZIKG -~
Degrees
Conoentr&.ion of glycerol
centigrade
-10
-20
-30
-40
-50
-60
-70
-80
70.6
90.5
89.1
27.0 3.9 1.1
17.1 1.7 0.5
14.4
1.1
14.0 1.1
0.3
0.3
9.1
7.2
1.4 0.4
0.5
2 95%
Mean SD SE
1.6
0.9
2.2
0.5
0.3
0.6
67.5 3.0 0.9
54.2 21.3 6.2
83.5 1.5 0.4
65.8 24.2 7.0
30.6 2.7 0.8
10.7 1.4 0.4
9.5 0.5
2.6 1.0 0.3
21.3 3.5 1.0
21.1 2.2 0.6
8.0 1.3 0.4
7.9 2.0 0.6
9.2
2.2 1.5 0.4
4.0 0.4 0.1
4.7 0.5 0.2
9.0 1.3 0.4
19.5 5.3 1.5
5*9% Mean SD
SE ll,S% Mean SD
SE
0.1
1.8
0.3
10.8 4.0 1.2
9.7 2.1 0.6
27.3 4.3 1.3
37.4 2.7 0.8
37.8
1.1
17.7yo
Mean SD
SE These
are means
of 12 replications,
i.e., 4 replications
for
11.9 3.4
each of 3 ‘different blood donors.
DONALD
316
B. PRIBOR 100 c
10%
DMSO
00-
70-
.r g k&F
’
-30’
-40’
-500
-60’
-700
-80
z E IOOI s
go-
9020 % DMSO
m-
E!o-
m-
70-
60-
Eo-
30 % DMSO ~- 1 min. freezing I-_ 16 min. freezing
50-
-40’
-209
-300
-4o*
-500
-SG-%GO~
Temperature
FIG. 2. Percentage of hemolysis of red cells frozen in various wncentrations of DMSO fur 1 min or 16 min ,in 50 lambda micropipet,tes (Micropets, Clay Adams) in baths at temperaturesranging from -10 to -80°C.
In the 1Smin experiments, a higher homolysis was observed in the lI.8% glycerol at the higher cooling rates; 10% DMSO (the same osmolarity) did not produce a corresponding rise. In fact the increase in hemolysis at the higher cooling rates was
not observed in DMSO until its concenhation exceeded 20%. The simiIarities in the cryoprotection by glycerol and DMSO support Rapatz and Luyet’s (21) empirical classification of cryoprotective agents into three types:
GLYCEROL
2.95
CONTRASTED
WITH
DMSO
317
90
% Glycerol
5.9
% Glycerol
In\
60
’ tr
70
\I
:
‘j\ ?
: 60
\1
, ,’
50
40 30
4
20
Y) : -f $ s
10
10
0-100
-20
-30
-400
-50”
-600
-70”
-600
H -200
-30”
-400
8 -5OD
1 -60”
-70”
-80”
1001
100 90 !
-------*
+-+ 01. -100
Ii.8
%
Glycerol
90 ~ 80
17.7 % Glycerol 1 min freezanq
-~~ 16 min
freezing
I/
Temperature
FIG. 3. Percentage of hemolysis of red cells frozen in various concentrations of glycerol for 1 min or 16 min in 50 lambda micropipettes (Micropets, Clay Adams) in baths at temperatures ranging from -10 to -80°C.
( 1) “type glycerol”-small, penetrating molecules; (2) ‘type PVP”-1arge nonpenetrating molecules; (3) Yype dextrose” -protective agents with intermediate type properties, Likewise, all the DMSO experiments and the I-mm freezing experi-
ments with glycerol suggest the same inference Morris and Farrant drew from their data: that there are two processes of freezing injury; one occurs at Iower than optimum cooling rates and results from the concentration of solutes; the
318
DONALD
other occurs at higher than optimum cooling rates and relates to the formation of intracellular ice ( 14). The differences between glycero1 and DMSO suggest, however, that the mechanisms of freeze-thaw damage and cryoprotection are more complex than Mazur’s two-factor theory would indicate. Likeagents cannot be wise, cryoprotective consistently classified into two or three types. In the framework of the two-factor theory it is tempting to propose that those higher-than-optimal cooling rates at which there is a rise in percentage of hemolysis result in the development of intracellular ice in a portion of the ceils, at Ieast. For example, the I-min freezing in 11.8% glycerol drastically increased percent hemolysis at higher cooling rates; corresponding samples frozen for 16 min were, however, much less damaged. Had intracellular ice formed, it shouId have been equally damaging in both freezing experiments. Since intracellular ice concentrates intracellular solutes, a longer storage time at subzero temperatures (higher than dry ice temperatures) should, furthermore, have produced an even greater damage. Thus, at least with respect to glycerol, the increase in hemolysis at higher cooling rates is not necessarily solely due to the formation of intracellular ice. Other factors besides the concentration of cryoprotective agents in relation to optimum cooling and rewarming rates clearly contribute to red cel1 survival. Implications Df the results for theories of cryoprotection. In a previous paper (20) I proposed a muItifactor theory of cryoprotection which implicated biological interactions between cryoprotective agents and cell membranes as well as physical parameters. PureIy physica factors include colligative properties of protective agents, the permeability of cell membranes to water and other solutes, the cooIing and rewarming rates, the time and temperature of exposure to subzero temperatures, the rate of ice crystal growth,
B. PRIBOR
and the presence #andrate of recrystallization. Biological interaction refers to interactions with the cell membrane, which is a dynamic system existing in one of several possible steady states. While certain disturbances produce reversible membrane changes, others result in irreversible changes. The change would depend on the type of cell membrane involved as well as the physical factors causing the disruption. Thus, under some circumstances, and to a first approximation, the cell membrane may be considered as a passive, static, semipermeable barrier. In this framework Lovelock’s original theory and Mazur’s two-factor theory for cryoprotection and Meryman’s minimum volume theory for freeze-thaw damage (13) are suitable exp1anation.s. In other: circumstances more subtle changes in the membrane steady state must. be taken into ,account. For example, one possible interpretation of the results is that higher concentrations of glycerol in conjunction with higher cooling rates, affects in some way, the steady state of the red cell membrane. If these cells are thawed rapidIy they become susceptible to the damaging stress of a rapid rise in temperature and dilution effects. However, if they are allowed to reestablish a membrane steady state, during the 16-min storage period, the membranes would no longer be susceptible to the stressesof thawing. This interpretation is consistent with previous work in which glycerol was found to have a deterimenta1 effect on the membrane properties of frog sciatic nerves (18). Furthermore, the above interpretation is analogous to recent results reported by McGann and Farrant (6, 11, 12). In essence they found that Chinese hamster tissue cuhure ceIIs showed an increase in survival if they were frozen and held at subzero temperatures for lo100 min before subsequent cooIing to -196°C. Survival increased to a maximum after 10 min of storage and then decreased slightly over Ionger storage periods,
GLYCEROL
CONTRASTED
The recent studies by Farrant and Morris (1, 5, 15) suggest that a hypertonic environment in addition to thermal shock, both of which occur during freezing, is the primary cause of damage to the cell membrane, In a hypertonic environment the cell membrane becomes susceptibIe to thermal shock during the freezing process, and to an additional stress upon rewarming and thawing. This latter stress, according to Farrant and Morris (5), may account for the damaging effect of intraceIluIar ice. Intracellular ice concentrates intracellular solutes, this makes the membrane susceptible to the dilution stress which occurs during thawing. Thus, there are two more factors-thermal shock and dilution stress-to incorporate into theories relating to freeze-thaw damage and cryoprotection. However, even more intriguing is Farrant’s change of opinion on the damaging effect of concentrated solutes. In earlier papers (24) he reported that the predominant feature which caused damage to human red cell membranes was a severe level of cellular shrinkage. In more recent studies (15) he has shown that hypertonic solutions of sodium chloride or sucrose produced an equivalent amount of ceIJ. shrinkage but resulted in quite different time dependent susceptibility of red cells to thermal shock. Thus, the factors, thermal shock and hypertonicity, are damaging in relation to the bioIogica1 interaction of membrane constituents (probably membrane proteins) with sodium chloride or sucrose. In previous papers (16-20) I pointed out interactions between solutes and membrane constituents analogous to those described above. It is these interactions and more subtle membrane changes that cryobiologists must begin to recognize and characterize. This approach will give a better understanding, at the molecular level, of the mechanism of freeze-thaw damage and cryoprotection.
WITH
319
DMSO SUMMARY
Human erythrocytes washed with phosphate buffered saIine (PBS) were frozen for 1 or 16 min at temperatures ranging from -10 to -80°C. Red cell suspensions contained either no protective agent or concentrations of dimethylsulvarious foxide (DMSO) or glycerol. The similarities between cryoprotection by DMSO and glycerol reinforce Rapatz and Luyet’s classification of cryoprotective agents into three types and support Mazur’s twofactor theory of cryoprotection. However, there are important differences between the cryoprotective effects of DMSO and glycerol. The most noteworthy is that for a11 concentrations of DMSO a 16-min freezing exposure was equal to or more damaging than a 1-min exposure; the converse was true for 11.8 and 17.7% glycerol solutions. This and other differences suggest that the general mechanism of freezethaw damage and cryoprotection is more complex than described by Mazur’s twofactor theory, Likewise cryoprotective agents cannot be consistently classified into two or three types. A multifactor theory was suggested as a more extensive model for understanding freeze-thaw damage and cryoprotection. The major new contribution of this theory is the idea of biological interaction. This latter refers to solutes in conjunction with various factors which disturb the steady state of the cell membrane. The change in the membrane may be reversibIe or irreversible depending upon the circumstances. ACKNOWLEDGMENTS The author thanks Mrs. for her technical assistance. by Grant HE12114 (NlH).
Bonnie Work
Bright-Taylor was supported
REFERENCES 1. Daw, A., Farrant, J., and Morris, G. J. Membrane leakage of solutes after thermal shock or freezing. Cryobiology X0, 126133 (1973). 2. Farrant, J., and Wodgar, A. E. Human red ceils under hypertonic con&ions; a model
320
3.
4.
5.
6.
7.
8.
9. 10. Il.
DONALD system far investigating freezing damage. 1. Sodium GhIoride. Cryobiology 9, Q-15 (1972). Farrant, J., and Woolgar, A. E. Human red celIs under hypertonic conditions; a model system for investigating freezing damage. 2. Sucrose, CTyobioZogy 9, 16-U ( 1972). Farrant, J. Human red cells under hypertonic conditions; a model system far investigating freezing damage. 3. Dimethylsulfoxide. Cyobiohgy 9, 131-136 (1972). Farrant, J., and Morris, G. J. Thermal shock and dilution shock as the causes of freezing injury. CyobioEogy 10, 134-140 (1973). Farrant, J., McGann, L. E., and Knight, Stella C. Possible mechanisms of protection against freezing and thawing damage by subzero temperatures. Cryobiology 11, 548 (1974). Mazur, P. PhysicaI factors implicated in the death of microorganisms at subzero temperatures. Ann. N.Y. Acad. Sci. 85, 610629 (1960). Mazur, P. Kinetics of water loss from celIs at subzero temperatures and the Iikelihood of intracellular freezing. J. Gen. Physiol. 47, 347-369 (1963). Mazur, P. Causes of injury in frozen and #thawed cells. Fed. Proc. Amer. Sac. Exp. Bid. 24, 175-182 ( 1965). Mazur, P, Cryobiology: The freezing of biological systems. Science 168, 939-949 (1970). McGann, L. E., and Farrant, J. Cryoprotection of tissue culture cells by interrupting rapid cooling at subzero temperatures. Cyobkdogy 11, 547 (1974).
B. PRIBOR 12. McGann, L. E., and Farrant, J. Conditions affecting the cryoprotective effect of interrupting rapid cooling at subzero temperatures. Cryobiology II, 547 (1974). 13. Meryman, H. T. Freezing injury and its prevention in living .cells. APznual Rev. Biophy. Bioeng. 3, 341-363 ( 1974). 14. Morris, G. J., and Farrant, J. Interactions of cooling rate and protective additive on the survival of washed human erythrocytes frozen to -196°C. Cryobiology 9, 17%181 (1972). 15. Morris, G. J., and Farrant, J. Effects of cooling rate on thermal shack hemolysis. Cryobiology 10, 119-125 (1973). 16. Pribor, D. B. Osmotic hemolysis contrasted with freez.+thaw hemolysis. Cryobblogy 8, 14-24 ( 1971). 17. Pribor, D. B. Inner membrane protein: site of freezethaw dam’age. Cryobiology 9, 319 (1972). 18. P&or, D. B., and Nara, A. The effect of salt or various cryoprotective agents on frog sciatic nerves. CfyobioZogy IO, 33-44 (1973). 19. Pribor, D. B., and Pribor, H. C. Studies with dextran-40 in cryopreservation of blood. CryobioZogy 10, 93-103 ( 1973). 20. P&or, D. B. PVP contrasted with dextran and a multifactor theory of cryoprotecticm. CyobbEogy 11, 6&72 (1974). 21. Rapatz, G., and Luyet, B. Combined effeds of freezing rates and of various protective agents on the preservation of human erythrocytes. CryobioZogy 4, 215-222 (1968).
12,
309320
Biological
( 1975)
Interactions Between Cell and Glycerol or DMSO DONALD
Biology Departmnt,
B. PRIBOR
of Toledo,
University
When I oame to work in Luyet’s laboratory in 1962, he and Rapatz already had a great deal of evidence that cooling and rewarming rates were crucial for the surviva1 of red blood cells after freezing and thawing. The working hypothesis in the laboratory at that time was (I) an optimal cooling rate was necessary for maximum recovery of frozen erythrocytes; (2) at or beIow this rate only extraceIIular freezing occurred; (3) below the optimaI rate, the erythrocytes were “in some manner” damaged by the concentration of intra- and extracellular solutes; (4) above these rates the cells were damaged by the formation of intracellular ice; (5) if erythrocytes survived intracelluIar freezing at very high cooling rates, they would be subjected to further injury by a recrystallization of intracellular ice unless rewarming was equally rapid. In 1965, based on freezing studies with microorganisms (7) and by a derivation of a quantitative description of the rate of intraceIIuIar water Ioss during freezing (8), Mazur proposed the now, well known, two-factor theory of freeze-thaw damage (9). This theory was equivaIent to the working hypothesis used in Luyet’s laboratory. Mazur’s published formulation of the two-factor theory was an important contribution because it aIerted cryobiologists to the :importance of coobng rates and it provided a single framework for Received
April
7, 1975.
309 Copyright All rights
0 1975 by Academic Prm. Inc. of reproduction in any form reserved.
Membranes
Toledo,
Ohio 43606
interpreting diverse reports on freezing injury. Subsequent reports from Mazur’s group indicated that the theory could also be apphed to cells in tissue culture. This and the data from Luyet’s laboratory in addition to a number of other reports provided strong experimental evidence for an expIanation of cryoinjury. In this paper I want to acknowledge my debt for the information and the insights suggested by Luyet’s laboratory in the early sixties. At the same time I will present experimental evidence which directly contradicts some of this earlier work. The present data as well as that of some other workers, indicates that Mazur’s twofactor theory needs to be modified and extended. Freeze-thaw damage and the mechanism( s ) for cryoprotection seems to be more complex than any of us realized in the 1960s. Recent advances in membrane physiology implies that 1this must be so. The data to be reported in this communication is consistent with and supportive of the recent dynamic models of membrane structure and function, Thus, this paper is intended to extend the multifactor theory of cryoprotection, described elsewhere (ZO), to low molecular weight cryoprotective agents such as glycerol and dimethylsuIfoxide. MATERIALS
AND
METHODS
Blood. ToIedo Hospital which routineIy used EDTA as an anticoaguIant provided samples of whoIe blood. The blood ob-
310
DONALD
tained from healthy donors, was stored at 4°C and used within 4 days after collection for subsequent studies. Preparation of erythrocyte suspensions. Cryoprotective additives of the desired concentration were incorporated into a phosphate-buffered saline (PBS) which contained 0.154 M NaCl, 0.01 M Na2HP04, and 0.0016 M NaH2P04 in double distilled water. The final pH was adjusted to 7.5 with 1 N NaOH or 0,l N HCI. Erythrocytes were sedimented by centrifugation at 11,500 rpm in a Sorvall RC 2-B centrifuge for 15 min at 4°C. After removal of supernatant fluid and the white cell layer the erythrocytes were washed three times by suspending them in 1.5 vol of PBS and the intact cells removed by centrifugation. Cells, so collected, were then resuspended in sufficient PBS to give a final concentration of 2 x 10” erythrocyteslml. Aliquots were combined with equal voIumes of solutions containing dimethyl sulfoxide (DMSO) or glycerol to give the desired concentration of the protective agent. For example, a 40% (v/v) DMSO soIution was mixed with an equal volume of red cells suspended in PBS to give a 20% sohrtion of DMSO containing 1 x lo9 cells/ml. The cryoprotective concentration is expressed in percentages rather than molarity to facilitate a comparison with previous studies by other investigators. However, in order to make comparisons between glycerol and DMSO, the concentrations were adjusted so that corresponding solutions would have equivalent osmolarities. Thus, 2.5, 5.0, lO,O, and 15.0% DMSO solutions would be equivaIent in osmolarity to 2.95, 5.9, 11.8, and 17.17% glycerol solutions, respectively. Originally, the experiments were designed in this way, but much later it was discovered that the DMSO solutions were miscalculated by a factor of 2, As a result, the DMSO solution concentrations are actually 5, 10, 20, and 30%, respectively, and therefore only two of these
B. PRIBOR
have equivalent osmolarities with respect to the glycerol concentrations used. As the results indicate the effect of increasing the concentration of glycerol is much greater than with DMSO; so the overlap between the two sets of data is sufficient for a meaningful comparison. Freezing and thawing. The red cells suspended in the cryoprotective solutions were allowed to equilibrate for 1 hr at room temperature before freezing. Samples of the cell suspensions were then drawn into 50-lambda glass Micropets (Clay-Adams) so that the lower level of the celI suspension was approximately 6-7 mm above the bottom of the tube to allow room for cold Vaseline plugs. Samples to be frozen for 16 min were also sealed with cold Vaseline at the upper end of the tube, These 50 lambra samples were frozen and stored for 1 or 16 min in a weIlstirred alcohol bath at temperatures ranging from -10 to -80°C. Samples to be frozen for 1 min were immersed to a level sufficient to cover the entire column of the cell suspension; whereas those to be frozen for 16 mm were immersed so that the column was 34 cm beIow the bath level. The latter depth was required to maintain a constant temperature over the 16-min period. The specimens contained in the tubes were held in position with positive action forceps for the desired period commencing at the time of immersion, after which they were abruptly transferred into a water bath at 37°C and agitated gently to effect thawing. Freezing at -10 and -20°C was initiated by “seeding” as described in an earlier paper (19). In a previous article (20) based on the work of Rapatz ad Luyet (21) it was suggested that at freezing temperatures, with similar sized tubes, in baths from -20 to -80°C the cooling rates probably ranged between 15 and 83”C/sec (900 and 5000” C/min).
GLYCEROL
CONTRASTED TABLE
MEAN
PEXCENTAUEQF
WITH
DMSO
311
3
HEMOLYSISIN
Typeof
Degrees
PBS SOLUTION centigrade
expmment
1 min freezing” 16 min freezinga
-10
-20
-30
-40
-50
-60
-70
-80
89.4 90.9
95.8 93.2
94.3 90.9
85.3 81.8
69.1 36.7
57.4 44.1
43.9 37.2
43.2 28.5
These are means of 12 replications, i.e., 4 repliCatiOn8 for each of 3 different blood donors. a Difference between means required for significance at 5y0 level = 6.7 (Studentized Range Test). b Difference between means required for significance at 5% level = 7.0 (Studentized Range Test).
Processing after freezirzg and thawing.
After thawing, each tube was wiped dry, the Vaseline plug(s) removed, and the contents blown into 4 cc of the appropriate diluent. The tube was washed six times by drawing in and expelling the diluent to ensure complete removal of the cell suspension. The extent of hemolysis was determined colorometrically by measuring the amount of hemogIobin released from the cells of the treated specimens. Quadruplicate controls for zero and 100% hemolysis were prepared. In the case of the former, treatment was identical with each of the corresponding frozen and thawed samples with the exception that the specimens were maintained in a bath at room temperature. The latter were prepared by lysing cells in 4 m1 of a 2.5% solution of saponin. Design of Study. The total study was divided into eight experiments-four different concentrations of DMSO and four of gIycerol. Within each experiment blood from the same ‘three donors was used throughout with four replications; 1 and 18 min freezing were part of a singIe experiment. Blood donors varied between experiments, RESULTS
TabIe 1 and Fig. 1 illustrate the effect of cooIing rate on erythrocytes suspended in PBS in the absence of a protective agent and frozen for 1 and 16 min, respectively. The combined data for the 1 and 16 min freezing faiIed to pass Bartlett’s test for homogeneity of variance,
However, such an analysis was permissible for each of the two subclasses of the data. The difference between the means was statistically sign&ant at the 5% level, 6.7 after 1 min of freezing and 7.0 after 16 min freezing. The percentage of hemolysis began to level off at -70°C in those specimens frozen for 1 min; freezing at -70 to -80°C probably corresponds to the optimum cooling rates for red cells referred to in the papers of Rapatz and Luyet (21) and Mazur (10). Figure 1 indicates that in the range from -50 and -80°C a 16-min freezing exposure produces less hemolysis than 1 min. This is particularIy evident at -80°C where the hemolysis values are 28 and 43%, respectively. The mean, standard deviation and standard error are given in Tables 2 and 3 for the DMSO experiments and in Tables 4 and 5 for those with glycerol. The DMSO data is sufficiently homogeneous to permit an analysis of variance. This is not true for the results with gIycero1; however, a log transform of the data makes such an analysis possible. Two sets of experiments are reported in which red cells are frozen in 30% DMSO soIution. This concentration results in some hemolysis, particularly, to erythrocytes that have survived freezing stress. The results are inconsistent and cannot be accounted for systematicaNy. These effects are probably responsible for the increase in variation of the data at this concentration. In one set of experiments the DMSO present in the blanks probably gave a higher estimate
DONALD
312
B. PRIBOR
No Protective -I
mn.
---lbmin.
8
-2tF
-30”
-400
TEMPERATURE
-500
-60’
Agent freezing
freezIng
I -700
400
L’C)
FIG. 1. Percentage of hemolysis of red cell suspensions frozen without a protective agent in 50 lambda micropipettes (Micropets, Clay Adams) for 1 min or 18 min in baths at temperatures ranging from -10 to -80°C ,
of hemolysis; the others without DMSO gave lower values. DMSO experiments. The results of a l- or a 16-min exposure of erythrocytes, suspended in various concentrations of DMSO and frozen at various temperatures are given in Fig. 2. The vaIues are what one would expect on the basis of previous work reported by Luyet’s group (21) and predictions from Mazur’s two-factor theory of freezing injury: (1) a I6-min freezing exposure is more damaging than a 1-min low concentrations of exposure; (2) DMSO (5%) g ave high hemolysis values at freezing temperatures from -10 to -40°C. The extent of injury was considerably lower in the freezing range of -50 and -80°C; (3) concentrations of 20% DMSO affords good protection over
the entire freezing range regardless of the time of exposure to low temperatures; (4) higher DMSO concentrations (30%) result in an increase in hemoIysis at higher cooling rates. Glycerol experiments. Figure 3 compares the effects of a l- and a 16-min period of freezing on red cells suspended in various concentrations of glycerol. These rest&s are similar to those dbtained with DMSO but certain dserences are present. (1) A 16-min exposure to freezing conditions is more damaging than a l-mm exposure in suspensionscontaining 2.95 and 5.9% glycerol, but in the case of higher concentrations (11.8 and 17.7% ) the longer exposure, in particular, at temperatures below -40°C is less injurious. (2) As with DMSO, low concentrations of
GLYCEROL
CONTRASTED TABLE
PERCENT-4CE
OR HEYOLYSIS
IN
WITH
DMSO
313
2
DMSO
SOLUTION+-1
Degree8
min
FREEZING
centigrade
-10
-20
-30
-40
-50
-60
-70
-80
14.7
61.9
42.9
24.3
11.7 3.4
7.1 2.0
7.9
0.3
70.2 5.6 1.6
2.3
20.6 5.4 1.5
15.2 5.0 1.4
12.4 1.6 0.5
3.1
8.6
9.6
6.8
4.7
4.8
1.0
1.5
3.1
0.7
1.3
0.4
0.9
0.2
0.4
4.1 0.6 0.2
4.3 0.8
0.3
1.9 0.6
3.2 1.2 0.4
3.9
4.9
4.4
5.1
6.0
0.6
1.0
0.2
0.6 0.2
0.8 0.2
1.2 0.3
7.1 1.3 0.4
9.6 1.6 0.5
10.1
11.6 6.6 1.9
14.8 6.8 2.0
17.6 6.6
21.3 7.1 2.1
25.9
29.5 10.9 3.1
22.1 8.3
0.5
4.1 7.7 2.2
7.3 7.0 2.0
10.9
16.7 17.3
20.7
12.4
51% Mean SD SE IO:% Mean SD SE 2a:r, Mean SD SE 305Q Mean SD SE 30’7*~ Mean SD SE
1.1
7.4 2.2
-2.1 9.1 2.6
6.8 2.0
0.3
1.9
7.3 2.1
15.6 4.5
5.0
0.2
2.4
11.0
9.0
3.2
2.6
These are means of 12 replications, i.e., 4 replications for each of 3 different blood donors. QPercentage of hemolysis determined in usual way as described in Materials and Methods. b Percentage of hemolysis determined with respect to distilled water blanks.
gIycero1 (2,95 and 59% ) gave high hemolysis vaIues in the freezing range from -10 to -4O”C, however, considerably lower values were observed in the -50 and -80°C range. (3) Higher concentrations of glycerol ( 11.8 and 17,7% ) reduced, considerably, the injurious effects of freezing on specimens exposed for 1 min and 16 min in the temperature range -10 to -40°C. (4) At the higher cooling rates, obtained by freezing at lower temperatures, hemolysis begins to gradually increase, in particuIar in those specimens containing 17.7% glycerol. DISCUSSION
The resuIts presented are different in some respects from those reported in a similar study by Rapatz and Luyet (21). In their paper, red ceils in the presence of 2.5% glycerol show a large increase in the extent of hemoIysis after a 1-min
exposure to freezing in the temperature range -60 to -80°C. In the present paper, the increase in injury was not observed unIess the gIycero1 concentrations were increased to 12%. Likewise, Rapatz and Luyet (21) observed that DMSO, in 5% concentrations caused an increase in hemolysis over the same freezing range. Our resuhs show that such an increase in damage occurs only if concentrations are increased to 20%. These dii%erences can be partially explained by the fact that in the former study the cryoprotective agents were added directly to whole blood. In our experiments cells were exposed to an isotonic medium; in Rapatz and Luyet’s study the celIs were exposed to a slightly hypertonic medium, assuming that penetration of the cryoprotective agent across the cell membrane occurred during the equilibration period. Further, the plasma proteins, particularly albumin, exerts a
DONALD
314
B. PRIBOR TABLE
PERCENTAGE Concentration of dimethyl sulfoxide DMSO
OF
HEMOLYSIB
3
IN DMSO
sOLuTIONel6
Degrees
min
FREEzlNa
centigrade
-10
-20
-30
-40
-50
-60
-70
-80
5% Mean SD SE
20.2 5.1 1.5
79.9 3.8 1.1
84.3 2.0 0.6
54.0 12.4 3.6
16.6 2.0 0.6
10.6 1.8 0.5
8.0 1.5 0.4
8.1 2.6 0.8
10% Mean SD SE
4.0 1.1 0.3
27.4 4.0 1.2
55.8 5.9 1.7
15.x 2.9 0.8
5.4 1.3 0.4
4.6 1.7 0.5
4.4 0.6 0.2
6.2 3.0 0.9
Mean SD SE
2.6 1.1 0.3
2.4 2.0 0.6
5.0 1.9 0.6
4.1 0.6 0.2
4.0 0.9 0.3
5.2 1.5 0.4
9.0 2.3 0.7
7.6 2.1 0.6
3O%‘oe Mean SD SE
9.4 6.2 1.8
15.0 6.8 2.0
16.5 7.8 2.2
17.3 6.9 2.0
19.8 6.8 2.0
26.3 13.3 3.8
40.0 3.5 1.0
28.4 4.7 1.3
-0.6 6.1 1.8
4.3 6.6 1.9
6.0 8.2 2.4
6.9
9.4
17.2
7.0 2.0
7.3 2.1
13.6 3.9
32.5 3.1 0.9
19.5 4.8 1.4
20%
3O%:O”
Mean SD SE
These are means of 12 replications, i.e., 4 replications for each of 3 different a Percentage of hemolysis determined in usual way ag described in Materials b Percentage of hemolysis determined with respect to distilled water blanks.
ciyoprotective action at the lower cooling rates and is associated with an increase in damage at the higher cooling rates ( 14). This may expIain the fact that our results are more in accord with those of Morris and Farrant (14) who also used washed red cells in a buffered salt solution. However, while our experimental data shows that a 10 and 20% solution of DMSO gave better protection at the lower cooling rates than glycerol, in similar concentrations, our results differ from those of Morris and Farraut because DMSO gave better protection at higher cooling rates. Specifically, 1 min freezing in the presence of a 17.7% glycerol solution was considerably more damaging than one min freezing in 30% DMSO, On the other hand, the injury to red cells suspended in either DMSO or glycerol was nearly the same after a 16-min freezing exposure (see Tables 3
blood donors. and Methods.
and 5), which are similar to the results reported by Morris and Farrant. Glycerol contrasted with DMSO. The results indicate three similarities between glycerol and DMSO, (1) In low concentrations (5% or less), 16 ,min freezing is more damaging than 1 min freezing. (2) With increasing concentration of glycerol or DMSO there is a corresponding decrease in percentage of hemoIysis at the lower cooling rates (bath temperatures from -10 to -4O’C) but an increase in percentage of hemolysis at the higher cooling rates (temperatures from -50 to -80°C). (3) Between -10 and -40°C the hemolysis curve for red cells suspended in 1.28 M glycerol or DMSO (11.8 and 10% cryoprotectant, respectively) demonstrated a convex form when cells were exposed to freezing for 16 min and a straight line for a 1 min freezing. There
GLYCEROL
CONTRASTED
WITH
TABLE PERCENTAQE
OF HEMOLYSIS
IN
315
4
SOLUTIONS-~
GLYCEROL
Concentration
Of gl,oerol
DMSO
min FREEZINQ
Degrees cent&de
-10
-20
-30
-40
-50
-60
-70
-80
64.8 3.2 0.9
88.7 1.2 0.3
70.0 4.2 1.4
30.5 3.3 1.0
15.1 1.4 0.4
14.1 1.1 0.3
14.1 2.7 0.7
18.4 2.6 0.3
22.2 2.8 0.8
73.0 2.7 0.6
41.0 2.8 2.2
14.2 1.8 0.5
10.8 1.1 0.3
9.9 2.8 0.2
11.6 1.9 0.5
12.4 2.9 0.8
SD SE
1.7 0.8 0.2
5.5 1.2 0.3
5.9 1.3 0.4
7.2 1.8 0.5
13.0 5.8 1.7
19.7 8.1 2.4
25.8 9.7 2.8
33.9 12.9 3.7
17 70/o Mean SD SE
1.5 1.5 0.4
1.8 1.1 0.3
4.2 0.7 0.2
13.6 2.1 0.6
35.7 2.8 0.8
81.1 6.4 1.9
90.9 3.4 1.0
90.1 3.: 1.0
2.95yo Mean SD SEl 5.9%
Mean SD SE 11.sy*
Mean
These
are meanz
of 12 replications,
i.e., 4 replications
are, on the other hand, three differences between glycerol and DMSO. (1) A 16min freezing was equally or more damaging than a X-min freezing in DMSO, regardless of its concentrations; the conTABLE
PERCEKTAGEOF HEMOLYSIS
IN
far each of 3 different blood donors. verse was true in 11.8 and 17.7% glycero1
solutions. (2) 10 and 20% DMSO solutions gave better protection at the lower cooling rates than the glycerol soluticns in the same concentration range. (3) 5
GLYCEROL SOLUTION+-16
min FREEZIKG -~
Degrees
Conoentr&.ion of glycerol
centigrade
-10
-20
-30
-40
-50
-60
-70
-80
70.6
90.5
89.1
27.0 3.9 1.1
17.1 1.7 0.5
14.4
1.1
14.0 1.1
0.3
0.3
9.1
7.2
1.4 0.4
0.5
2 95%
Mean SD SE
1.6
0.9
2.2
0.5
0.3
0.6
67.5 3.0 0.9
54.2 21.3 6.2
83.5 1.5 0.4
65.8 24.2 7.0
30.6 2.7 0.8
10.7 1.4 0.4
9.5 0.5
2.6 1.0 0.3
21.3 3.5 1.0
21.1 2.2 0.6
8.0 1.3 0.4
7.9 2.0 0.6
9.2
2.2 1.5 0.4
4.0 0.4 0.1
4.7 0.5 0.2
9.0 1.3 0.4
19.5 5.3 1.5
5*9% Mean SD
SE ll,S% Mean SD
SE
0.1
1.8
0.3
10.8 4.0 1.2
9.7 2.1 0.6
27.3 4.3 1.3
37.4 2.7 0.8
37.8
1.1
17.7yo
Mean SD
SE These
are means
of 12 replications,
i.e., 4 replications
for
11.9 3.4
each of 3 ‘different blood donors.
DONALD
316
B. PRIBOR 100 c
10%
DMSO
00-
70-
.r g k&F
’
-30’
-40’
-500
-60’
-700
-80
z E IOOI s
go-
9020 % DMSO
m-
E!o-
m-
70-
60-
Eo-
30 % DMSO ~- 1 min. freezing I-_ 16 min. freezing
50-
-40’
-209
-300
-4o*
-500
-SG-%GO~
Temperature
FIG. 2. Percentage of hemolysis of red cells frozen in various wncentrations of DMSO fur 1 min or 16 min ,in 50 lambda micropipet,tes (Micropets, Clay Adams) in baths at temperaturesranging from -10 to -80°C.
In the 1Smin experiments, a higher homolysis was observed in the lI.8% glycerol at the higher cooling rates; 10% DMSO (the same osmolarity) did not produce a corresponding rise. In fact the increase in hemolysis at the higher cooling rates was
not observed in DMSO until its concenhation exceeded 20%. The simiIarities in the cryoprotection by glycerol and DMSO support Rapatz and Luyet’s (21) empirical classification of cryoprotective agents into three types:
GLYCEROL
2.95
CONTRASTED
WITH
DMSO
317
90
% Glycerol
5.9
% Glycerol
In\
60
’ tr
70
\I
:
‘j\ ?
: 60
\1
, ,’
50
40 30
4
20
Y) : -f $ s
10
10
0-100
-20
-30
-400
-50”
-600
-70”
-600
H -200
-30”
-400
8 -5OD
1 -60”
-70”
-80”
1001
100 90 !
-------*
+-+ 01. -100
Ii.8
%
Glycerol
90 ~ 80
17.7 % Glycerol 1 min freezanq
-~~ 16 min
freezing
I/
Temperature
FIG. 3. Percentage of hemolysis of red cells frozen in various concentrations of glycerol for 1 min or 16 min in 50 lambda micropipettes (Micropets, Clay Adams) in baths at temperatures ranging from -10 to -80°C.
( 1) “type glycerol”-small, penetrating molecules; (2) ‘type PVP”-1arge nonpenetrating molecules; (3) Yype dextrose” -protective agents with intermediate type properties, Likewise, all the DMSO experiments and the I-mm freezing experi-
ments with glycerol suggest the same inference Morris and Farrant drew from their data: that there are two processes of freezing injury; one occurs at Iower than optimum cooling rates and results from the concentration of solutes; the
318
DONALD
other occurs at higher than optimum cooling rates and relates to the formation of intracellular ice ( 14). The differences between glycero1 and DMSO suggest, however, that the mechanisms of freeze-thaw damage and cryoprotection are more complex than Mazur’s two-factor theory would indicate. Likeagents cannot be wise, cryoprotective consistently classified into two or three types. In the framework of the two-factor theory it is tempting to propose that those higher-than-optimal cooling rates at which there is a rise in percentage of hemolysis result in the development of intracellular ice in a portion of the ceils, at Ieast. For example, the I-min freezing in 11.8% glycerol drastically increased percent hemolysis at higher cooling rates; corresponding samples frozen for 16 min were, however, much less damaged. Had intracellular ice formed, it shouId have been equally damaging in both freezing experiments. Since intracellular ice concentrates intracellular solutes, a longer storage time at subzero temperatures (higher than dry ice temperatures) should, furthermore, have produced an even greater damage. Thus, at least with respect to glycerol, the increase in hemolysis at higher cooling rates is not necessarily solely due to the formation of intracellular ice. Other factors besides the concentration of cryoprotective agents in relation to optimum cooling and rewarming rates clearly contribute to red cel1 survival. Implications Df the results for theories of cryoprotection. In a previous paper (20) I proposed a muItifactor theory of cryoprotection which implicated biological interactions between cryoprotective agents and cell membranes as well as physical parameters. PureIy physica factors include colligative properties of protective agents, the permeability of cell membranes to water and other solutes, the cooIing and rewarming rates, the time and temperature of exposure to subzero temperatures, the rate of ice crystal growth,
B. PRIBOR
and the presence #andrate of recrystallization. Biological interaction refers to interactions with the cell membrane, which is a dynamic system existing in one of several possible steady states. While certain disturbances produce reversible membrane changes, others result in irreversible changes. The change would depend on the type of cell membrane involved as well as the physical factors causing the disruption. Thus, under some circumstances, and to a first approximation, the cell membrane may be considered as a passive, static, semipermeable barrier. In this framework Lovelock’s original theory and Mazur’s two-factor theory for cryoprotection and Meryman’s minimum volume theory for freeze-thaw damage (13) are suitable exp1anation.s. In other: circumstances more subtle changes in the membrane steady state must. be taken into ,account. For example, one possible interpretation of the results is that higher concentrations of glycerol in conjunction with higher cooling rates, affects in some way, the steady state of the red cell membrane. If these cells are thawed rapidIy they become susceptible to the damaging stress of a rapid rise in temperature and dilution effects. However, if they are allowed to reestablish a membrane steady state, during the 16-min storage period, the membranes would no longer be susceptible to the stressesof thawing. This interpretation is consistent with previous work in which glycerol was found to have a deterimenta1 effect on the membrane properties of frog sciatic nerves (18). Furthermore, the above interpretation is analogous to recent results reported by McGann and Farrant (6, 11, 12). In essence they found that Chinese hamster tissue cuhure ceIIs showed an increase in survival if they were frozen and held at subzero temperatures for lo100 min before subsequent cooIing to -196°C. Survival increased to a maximum after 10 min of storage and then decreased slightly over Ionger storage periods,
GLYCEROL
CONTRASTED
The recent studies by Farrant and Morris (1, 5, 15) suggest that a hypertonic environment in addition to thermal shock, both of which occur during freezing, is the primary cause of damage to the cell membrane, In a hypertonic environment the cell membrane becomes susceptibIe to thermal shock during the freezing process, and to an additional stress upon rewarming and thawing. This latter stress, according to Farrant and Morris (5), may account for the damaging effect of intraceIluIar ice. Intracellular ice concentrates intracellular solutes, this makes the membrane susceptible to the dilution stress which occurs during thawing. Thus, there are two more factors-thermal shock and dilution stress-to incorporate into theories relating to freeze-thaw damage and cryoprotection. However, even more intriguing is Farrant’s change of opinion on the damaging effect of concentrated solutes. In earlier papers (24) he reported that the predominant feature which caused damage to human red cell membranes was a severe level of cellular shrinkage. In more recent studies (15) he has shown that hypertonic solutions of sodium chloride or sucrose produced an equivalent amount of ceIJ. shrinkage but resulted in quite different time dependent susceptibility of red cells to thermal shock. Thus, the factors, thermal shock and hypertonicity, are damaging in relation to the bioIogica1 interaction of membrane constituents (probably membrane proteins) with sodium chloride or sucrose. In previous papers (16-20) I pointed out interactions between solutes and membrane constituents analogous to those described above. It is these interactions and more subtle membrane changes that cryobiologists must begin to recognize and characterize. This approach will give a better understanding, at the molecular level, of the mechanism of freeze-thaw damage and cryoprotection.
WITH
319
DMSO SUMMARY
Human erythrocytes washed with phosphate buffered saIine (PBS) were frozen for 1 or 16 min at temperatures ranging from -10 to -80°C. Red cell suspensions contained either no protective agent or concentrations of dimethylsulvarious foxide (DMSO) or glycerol. The similarities between cryoprotection by DMSO and glycerol reinforce Rapatz and Luyet’s classification of cryoprotective agents into three types and support Mazur’s twofactor theory of cryoprotection. However, there are important differences between the cryoprotective effects of DMSO and glycerol. The most noteworthy is that for a11 concentrations of DMSO a 16-min freezing exposure was equal to or more damaging than a 1-min exposure; the converse was true for 11.8 and 17.7% glycerol solutions. This and other differences suggest that the general mechanism of freezethaw damage and cryoprotection is more complex than described by Mazur’s twofactor theory, Likewise cryoprotective agents cannot be consistently classified into two or three types. A multifactor theory was suggested as a more extensive model for understanding freeze-thaw damage and cryoprotection. The major new contribution of this theory is the idea of biological interaction. This latter refers to solutes in conjunction with various factors which disturb the steady state of the cell membrane. The change in the membrane may be reversibIe or irreversible depending upon the circumstances. ACKNOWLEDGMENTS The author thanks Mrs. for her technical assistance. by Grant HE12114 (NlH).
Bonnie Work
Bright-Taylor was supported
REFERENCES 1. Daw, A., Farrant, J., and Morris, G. J. Membrane leakage of solutes after thermal shock or freezing. Cryobiology X0, 126133 (1973). 2. Farrant, J., and Wodgar, A. E. Human red ceils under hypertonic con&ions; a model
320
3.
4.
5.
6.
7.
8.
9. 10. Il.
DONALD system far investigating freezing damage. 1. Sodium GhIoride. Cryobiology 9, Q-15 (1972). Farrant, J., and Woolgar, A. E. Human red celIs under hypertonic conditions; a model system for investigating freezing damage. 2. Sucrose, CTyobioZogy 9, 16-U ( 1972). Farrant, J. Human red cells under hypertonic conditions; a model system far investigating freezing damage. 3. Dimethylsulfoxide. Cyobiohgy 9, 131-136 (1972). Farrant, J., and Morris, G. J. Thermal shock and dilution shock as the causes of freezing injury. CyobioEogy 10, 134-140 (1973). Farrant, J., McGann, L. E., and Knight, Stella C. Possible mechanisms of protection against freezing and thawing damage by subzero temperatures. Cryobiology 11, 548 (1974). Mazur, P. PhysicaI factors implicated in the death of microorganisms at subzero temperatures. Ann. N.Y. Acad. Sci. 85, 610629 (1960). Mazur, P. Kinetics of water loss from celIs at subzero temperatures and the Iikelihood of intracellular freezing. J. Gen. Physiol. 47, 347-369 (1963). Mazur, P. Causes of injury in frozen and #thawed cells. Fed. Proc. Amer. Sac. Exp. Bid. 24, 175-182 ( 1965). Mazur, P, Cryobiology: The freezing of biological systems. Science 168, 939-949 (1970). McGann, L. E., and Farrant, J. Cryoprotection of tissue culture cells by interrupting rapid cooling at subzero temperatures. Cyobkdogy 11, 547 (1974).
B. PRIBOR 12. McGann, L. E., and Farrant, J. Conditions affecting the cryoprotective effect of interrupting rapid cooling at subzero temperatures. Cryobiology II, 547 (1974). 13. Meryman, H. T. Freezing injury and its prevention in living .cells. APznual Rev. Biophy. Bioeng. 3, 341-363 ( 1974). 14. Morris, G. J., and Farrant, J. Interactions of cooling rate and protective additive on the survival of washed human erythrocytes frozen to -196°C. Cryobiology 9, 17%181 (1972). 15. Morris, G. J., and Farrant, J. Effects of cooling rate on thermal shack hemolysis. Cryobiology 10, 119-125 (1973). 16. Pribor, D. B. Osmotic hemolysis contrasted with freez.+thaw hemolysis. Cryobblogy 8, 14-24 ( 1971). 17. Pribor, D. B. Inner membrane protein: site of freezethaw dam’age. Cryobiology 9, 319 (1972). 18. P&or, D. B., and Nara, A. The effect of salt or various cryoprotective agents on frog sciatic nerves. CfyobioZogy IO, 33-44 (1973). 19. Pribor, D. B., and Pribor, H. C. Studies with dextran-40 in cryopreservation of blood. CryobioZogy 10, 93-103 ( 1973). 20. P&or, D. B. PVP contrasted with dextran and a multifactor theory of cryoprotecticm. CyobbEogy 11, 6&72 (1974). 21. Rapatz, G., and Luyet, B. Combined effeds of freezing rates and of various protective agents on the preservation of human erythrocytes. CryobioZogy 4, 215-222 (1968).
