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The effect of cooling rate and of dimethyl sulfoxide concentration on the ultrastructure of neonatal rat heart cells after freezing and thawing
CRYOBIOLOGY
13, 305316
(1976)
The Effect of Cooling Rate and of Dimethyl Sulfoxide Concentration on the Ultrastructure of Neonatal Rat Heart Cells after Freezing and Thawing1 G. M. ALINK, D#epartment Netherlands
J. AGTERBERG,
A. W. HELDER,
F. G. J. OFFERIJNS
Physiology and Cryobiology, Central Laboratory of the Red Cross Blood Transfusion Service, Amsterdam, The Netherlands
Neonatal rat heart cells can be preserved in the frozen state (1, 7, 21). Their survival after freezing and thawing ,depends on cooling rate, on ,dimethyl sulfoxide (DMSO) concentration ( 1 ), and probably also on other f,actors. In order to obtain a better understanding of differences in survival we investigated the ultrastructure of heart cells in suspension and in tissue culture after freezing ,at
AND
of Applied
AND
and at the nonoptimal cooling rate preceding and following the optimal rate in the series 1, 5, 10, 30, and 50”C/min ( 1). Immediately after thawing and washing out the DMSO, the cells were fixed in Karnovsky’s fixative (8) for a period of at least 24 hr and subsequently rinsed in 0.1 M phosphate buffer, pH 7.4, at 4°C for 1 hr. After postfixation in a 1% osmium tetroxide solution in phosphate buffer, pH 7.5 ( 19), the material was dehydrated by increasing concentrations ‘of ethanol and embedded in Epon. Ultrathin sections were cut from two cell blocks on a Reichert ultramicrotome and stained with a saturated uranyl acetate solution in aqua destillata for 20 min sand with lead hydroxide according to Millonig (20) for 10 min. After evaporation with carbon the sections were examined with a Philips EM 300 electron microscope. In the same way, electron microscopical preparations were made of cells treated only with DMSO as well as of untreated control cells. We especially studied the integrity of the cell membrane, the mitochondria and the nucleus in untreated and unfrozen, treated and unfrozen, and treated and frozen muscle and nonmuscle cells. The ultrastructure of unfrozen and frozen-thawed myoblasts after 8 days of culturing was ,also investigated. After fixation in Karnovsky’s fluid the cultured cells were separated from the surface of the dish by
METHODS
Hearts of neonatal rats ( Wistar), l-3 days old, were trypsinized according to a method described previously ( 1). The neonatal rat heart ‘cells were brought into suspension in culture medium and frozen with 2.5, 5, 7.5, or 10% DMSO. Addition of DMSO in culture medium to the cell suspensions befgore freezing and dilution with medium B after thawing were done slowly at room temperature. The cells were cooled at the ‘optimal cooling rate for each DMSO concentration, 5”C/min for 2.5, 5, and 10% DMSO and 30”C/min for 7.5% DMSO, Received July 1, 1975. 1 These investigations were supported by the Foundation for Medical Research FUNGO, The Netherlands. 305 Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
ALINK
306
ET AL.
tt-FIG. 1. 1 Electron micrograph of an untreated, unfrozen neonatal rat heart muscle cell showwirn well spread spreaa chrocnroing intact cell membrane N )1 with memtxane ( C), mitochondria ( M ), and nucleus ((Iu matin. X 10,000.
scratching ,and then prepared for electron microscopy according to the method described above. RESULTS
The electron microscopical preparation of untreated, unfrozen muscle cells (Figs. 1 and la) showed mostly round cells with an intact cell membrane, mitochondria, rough endoplasmic reticulum, and a nucleus with well-spread chromatin and glycogen granules. Most of the myofibrils were disorganized; often parts of Z-lines were visible. Nonmuscle celIs (Fig. 2) were characterized by the absence of myofibrils and glycogen granules. The shape of these cells was more irregular. In sections of untreated, unfrozen cells, a few cells of both types always were partly or completely damaged, probably because of the isolation procedure.
Muscle cells treated with DMSO only or frozen in the presence of DMSO appeared to have different stages of cellular damage (Table 1) ; in nonmuscle cells no damage was observed. Muscle cells treated with 2.5% DMSO showed no ultrastructural difference compared to untreated cells. After treatment with 5% DMSO, a space occurred between the cytoplasmic matrix and the cell membrane (Fig. 3). With 7.5% DMSO most of the cells showed membrane damage similar to that with 5% DMSO, and in addition many cells showed swollen mitochondria. In nearly all cells treated with 10% DMSO the cell membrane was interrupted, or it disappeared completely. The majority of the mitochondria was swollen or destroyed, and in many cells the perinuclear space was dilated and the nuclear chromatin was clumped (Fig. 4).
ULTRASTRUCTURE
OF FROZEN-THAWED
HEART
CELLS
FIG. la. Detail of untreated, unfrozen myoblast, showing disorganized parts of :Z-lines (Z), and the cristae pattern in mitochondria (M). X35,000. TABLE
myofibrils
(My),
1
and Nucleus and the Survival in Culture of Myoblasts The Integrity of Cell Membrane, Mitochondria, DMSO Treatment and after Freezing with DMSO at Optimal and Nonoptimal Cooling Rates Cooling rate (T/min) 0 *iMrSO
2.5 5 7.5 10
n. n. n. n.
c.~ c. c. c.
Integrity= Cell membrane
Mitochondria
NUCIIXIS
after
SurvivaP (%I
++ + f -
++ ++ + f
++ ++ ++ +
100 97 51 32
2.5 2.5 2.5
1 5 10
f f f
f f f
+ + +
1 23 17
5 5 5
1 5 10
+ + +
f ++ +
+ ++ ++
33 72 40
7.5 7.5 7.5
1 30 50
f + f
f + f
i-f -I-+ f
24 59 29
1 5 10
f f f
f f f
+ + +
34 40 23
10 10 10
a Cells with ,a normal structure: b Survival, based on contracting c n. c.,]no-cooling.
++, 75-100%; +, 50-7570; f, 2550%; -, O-25%. area, on the second day of culturing; previous experiments
(1).
308
ALINK
ET AL.
FIG. 2. Electron micrograph of untreated, unfrozen fibroblast showing the irregular shape of the cell membrane (C). The rough endoplasmic reticulum (ER) is well developed and myofibrils are absent. X 15,000.
FIG. 3. Myoblast after treatment with 5% DMSO at room temperature. There is a space (S) between the cytoplasmic matrix and the cell membrane. Further, the morphological appearance does not differ from an untreated myoblast. ~15,000. _ .
ULTRASTRUCTURE
OF FROZEN-THAWED
HEART
CELLS
FIG. 4. Myoblast after treatment with 10% DMSO at room temperature. The cell membrane is severely damaged as is shown by the numerous interruptions (arrows), The mitochondria (M) are swollen and the nuclear chromatin (Ch) is clumped. X15,000.
Muscle cells frozen with 2.5% DMSO had mostly undulating *and interrupted cell membranes, swollen and frequently destroyed mitochondria. Many cells had clumped nuclear chromatin (Fig. 5). No difference in morphological damage was observed between cells cooled at optimal or at nonoptimal cooling rates. Freezing at the optimal ‘cooling rate with 5% DMSO had an effect on the cell integrity other than freezing at the nonoptimal cooling rate. At the optimal cooling rate of S”C/ min, only a part of the cells showed a space between cytoplasm and cell membrane (Fig. 6). In addition, at lO”C/min many cells appeared to have swollen mitochondria. At l.“C/min the number of cells with swollen rnitochondria increased, .and cells with clumped nuclear chromatin appeared (Fig. 7). Muscle cells frozen with 7.5% DMSO showed the smallest number of damaged cells when frozen at the optimal cooling rate of 30”C/min. The nuclei of most cells showed a normal appearance at
rates of 1 and 30”C/min. At 5O”C/min most cells had undulating and interrupted cell membranes, swollen mitochondria, and damaged nuclei. After freezing with 10% DMSO at optimal or nonoptimal cooling rates, most of the cells showed undulated and interrupted cell membranes, swollen and occasionally destroyed mitochondria; about 50% of the cells showed clumping of the nuclear chromatin. Comparing the morphological damage caused by DMSO with the damage caused by freezing, it appears that in both cases the nucleus of the cells suffers less damage than the cell membrane and the mitochondria. After DMSO treatment only, the cell membranes are already damaged at lower DMSO concentrations than the mitochondria. After freezing and thawing, the cell membranes and mitochondria are usually damaged at the same DMSO concentrations. Ultrastructure of myoblasts after prolonged culturing. After 8 days of culturing
310
ALINK
ET AL.
FIG. 5. Severely damaged muscle cell after freezing at lO”C/min with 2.5% DMSO. The cell membrane ( C ) is undulating and shows numerous interruptions ( arrows). The mitochondria (M) are swollen and have destroyed cristae which have partly or completely disappeared (Me). In the nucleus the chromatin ( Ch) is clumped. X15,000.
FIG. 6. Myoblast after freezing at B”C/min with 5% DMSO. There is a space (S) between the cytoplasm and the cell membrane. Further, the morphological appearance does not differ from an untreated unfrozen myoblast. X15,000.
ULTRASTRUCTURE
OF FROZEN-THAWED
HEART
CELLS
FIG. 7. Damaged muscle cell after freezing at l”C/min with 5% DMSO. The cell membrane (C) is undulating and shows numerous interruptions (arrows) as is seen in Fig. 5. The mitochondria (M) are swollen and the nuclear chromatin (Ch) is clumped. X15,000.
FIG. 8. Electron micrograph of frozen-thawed rat heart muscle cells after 8 days of culturing. The cells show well organized myofibrils (My) and Z-lines (Z); intercalated disks (I) are established. The cell membranes (C) and the mitochondria (M) have a normal appearance. X23,000.
ALINK
312
ET AL.
FIG. 8a. Detail of frozen-thawed muscle cell after 8 days of culturing, showing nucleus (N) and myofibri1 (My) with Z-line. ~37,000.
no difference between the ultrastructure of control cells and ‘cells treated with DMSO or frozen with DMSO was seen, This occurred at all DMSO concentrations and at all cooling rates which were studied. The myoblasts showed very well organized myofibrils and Z-lines. The intercalated disks were well established. The cell membrane, the mitochondria, and the nucleus of frozen cells had a normal #appearance (Fig. 8, 8a). DISCUSSION
The ultrastructure of neonatal rat heart cells in suspension and after a few days of culturing has been described by several investigators (11, 12, 13). Some structural alterations after freezing and thawing were reported in embryonic chicken heart cells ( 32). Ultrastructure-survival correlation studies were performed in the rat heart (2527), smooth muscle (5), HeLa cells (31)) foeta1 human lung cells (4), hamster lung cells (2), and yeast cells (3). Extensive studies on the ultrastructural damage after freezing and thawing were done on
mouse hepatic cells (28-30) and on the mitochondria of renal cortex cells of mice (23, 24). In our freezing studies on the ultrastructural damage, DMSO was used as a cryoprotectant (1). The toxic properties of DMSO are well known (9, 10, 16, 22, 25). Because it also decreases the viability of heart cells ( 1, 6), we studied the effect of DMSO on the ultrastructure. In our experiments, 2.5% DMSO added at room temperature to cells in suspension was the highest concentration of DMSO which caused no morphological damage. With 5% DMSO a space developed between cell membrane and cytoplasm, and with 7.5% DMSO swelling of mitochondria occurred. With 10% DMSO, clumping of chromatin in the nucleus was observed and the cell membrane and the mitochondria were often ruptured. The increase of cellular damage correlated well with the previously observed ( 1) decrease in survival of the cells, measured as contracting area on the second day of culturing.
ULTRASTRUCTURE
OF FROZEN-THAWED
Ultrastructural damage at relatively low con’centrations of DMSO was described by Malinin (15) in monkey kidney cells. He reported damage to cell membranes, mitochondria, and nuclei after treatment of cultured cells with 7.5% DMSO at room temperature. It is ‘also reported that morphological dalmage after treatment with higher concentrations of DMSO correlates with function. Iln guinea pig smooth muscle (5) and in the rat heart (25), increasing DMSO concentrations caused an increase in structural damage to the mitochondria and the nucleus, which correlated with a decrease in the contractility. In these organs the cell membranes were intact which is in contrast with our results and with those of Malinin ( 15). Weiss and Armstrong (31) found intact cell Imembranes, nuclei, and swollen mitochondria after treatment of HeLa cells for 75 min with 15% glycerol. These cells showed normal growth in culture. After freezing at optimal, suboptimal, or supraoptimal cooling rates with different DMSO concentrations, we found that in the presence of 5 or 7.5% DMSO a high survival of the cells occurred at the optimal cooling rates. The ultrastructure of those cells was better than that of the cells frozen at nonoptimal cooling rates, which caused a low or moderate survival. After freezing with 2.5 or 10% DMSO at optimal and nonoptimal cooling rates we found moderate or low survival. However, these differences in survival were not reflected in the ultrastructure of the cells. In a study by Trump et al. (29) on mouse hepatic cells, at lower cooling rates alterations were largely confmed to the cell membrane; in tissue which was frozen rapidly, thle alterations involved numerous intercellular membrane systems as well. Better integrity of cell membranes and mitochondria was observed after sl’ow freezing with 1’5% DMSO (30) than after slow freezing without a cryoprotectant. Beadle and Harris ( 4 ) reported no difference in ultrastructural damage in human foetal lung
HEART
CELLS
313
cells after freezing at a low and a high cooling rate if the recovery was low. If freezing resulted in a high recovery, the structural damage was less. Bank and Mazur (2) reported that, if the survival of hamster lung cells after freezing under different conditions was 5 or 20%, no differences in morphologioal alterations could be observed between both groups. On the other hand when survival was ,about equal (20-25s ), structural characteristics ranged from highly abnormal to relatively normal, depending on the freeze-thaw procedure. Only at optimal freezing conditions (80% survival), was the ultrastructure of the cells quite normal. In another study Bank and Mazur ( 3), using a freeze-cleaving, freezeetching technique, showed in yeast cells that, even at optimal cooling rates (survival, 60% ), the ultrastructure of the cells can differ greatly from the well-organized structure in the frozen state at ultrarapid cooling rates. Weiss and Armstrong (31) described the ultrastructure of HeLa cells after freezing with and without 15% glycerol. Although there was survival of cells after freezing with glycerol but not without glycerol, the ultrastructural damage was equal in both groups. They described the cell membranes as being intact, although there were empty spaces just inside these membranes which were folded. In our experiments such cell membranes were defined as being damaged. Mitochondria and nuclei were damaged in the HeLa cells, as we observed in heart muscle cells. A good correlation between ultrastructure and function was described by Farrant et al. (5) in guinea pig smooth muscle after stepwise cooling with and without DMSO to -79°C and rewarming. Such a correl,ation was not found by Shlafer and Karow (27) who studied the cell integrity of the adult rat heart after slow cooling to -17°C at different DMSO concentrations. At 1 and 20% DMSO, mitochondria and nuclei were ruptured and no contractions of the heart were observed. After cooling with 5, 10,
314
ALINK
and 15% DMSO, the mitochondrk were swollen to different degrees and the nuclei often had clumped chromatin. Some hearts showed contractions, others did not. There was no difference in ultrastructural damage between survivors ,and nonsurvivors. The ultrastructural investigations discussed above show a great diversity in the choice of cell type and describe various aspects of the freezing process and their influence on the ultrastructure of the cells. The ultrastructure was often ‘compared to survival. One of the aspects of such studies is the fact that cryoprotectants such as DMSO and glycerol may be toxic. Cooling rate is another important aspect. The damage during slow cooling is caused by high electrolyte concentrations (14) or by OSmotic stress (18) and during rapid cooling by intercellular ice formation. At intermediate cooling rates, the least amount of damage may be expected (17). In our experiments we studied the ultrastructure of neonatal rat heart cells after freezing at optimal, suboptimal, and supraoptimal cooling rates with the DMSO concentrations previously used in freeze-survival experiments ( 1). Comparing our results with the published data discussed above, we conclude that DMSO leads to different degrees of ultrastructural damage depending on DMSO concentration, cell type, and other experimental conditions. Swelling of membrane-Iimited structures, especially mitochondria, is one of the first phenomena to occur. Subsequently, clumping of the nuclear chromatin as well as rupturing of the cell, mitochondrial, and nucIear membranes occur. After DMSO treatment there is a good correlation between ultrastructural damage and survival. After freezing with DMSO and thawing, the order of damaged structures is about the same as after DMSO treatment only. If the ultrastructural damage after freezing is compared with the survival in culture, the situation differs from DMSO treatment only. Generally it may be concluded that a good survival cor-
ET AL.
relates with a good ultrastructure and that such a correlation is not found if the survival is low or moderate. When the survival of cells is higher, the correlation with the ultrastructure seems to be better. If the survival is low or moderate, phenomena other than those which can be observed on the ultrastructural level may contribute to the cell damage after freezing
The ultrastructure of neonatal rat heart cells in suspension and in tissue culture after freezing ,at optimal, suboptimal, and supraoptimal cooling rates with 2.5, 5, 7.5, and 10% DMSO was investigated. The effect of DMSO treatment only on the structure of the cells was also studied. A comparison was made with the surviva1 in culture. Without freezing, increasing DMSO concentrations caused an increase of morphoIogioaI damage, correIating with a decrease of the survival in culture. With 2.5% DMSO there was no difference with untreated cells. At higher DMSO concentrations, the ultrastructural damage increased from spaces between cell membrane and cytoplasm at 5% DMSO to interrupted cell membranes, swollen or destroyed mitochondria, and nucIei with clumped chromatin at 10% DMSO. After freezing at optima1 or nonoptimal cooling rates with 5 or 7.5% DMSO, the ultrastructure correlated well with the survival. After freezing with 2.5 or 10% DMSO at optimal or nonoptimal cooling rates, differences in survival were found, which were not reflected in the ultrastructure of the cell. After 8 days of culturing, cells which were frozen at all the different cool-
ULTRASTRUCTURE
OF FROZEN-THAWED
ing rates and DMSO concentrations peared to have a normal structure.
ap-
ACKNOWLEDGMENTS We thank Mr. C. C. Verheul and Mr. R. Sprokholt for their skillful technical assistance. We are grateful to Mr. P. Meelker and Mr. L. R. Hafkamp for making the photographs and to Mrs. H. Grooters for typing the manuscript. REFERENCES 1. Alink, G. M., Verheul, C. C., and Offerijns, F. G. J. The effect of cooling rate and of dimethyl sulfoxide concentration on low temperature preservation of neonatal rat heart cells. Cryobiology 13, 295-304 ( 1976). 2. Bank, II., and Mazur, P. Relations between ultrastructure and viability of frozen-thawed Chinese hamster tissue-culture celIs. Exp. Cell Iles. 71, 441-454 (1972). 3. Bank, II., and Mazur, P. Visualization of freezing damage. J. Cell Biol. 57, 729-742 (1973).
4. Beadle, D. J., and Harris, L. W. Relationship between freezing rate, ultrastructure and recovery in a human diploid cell line. J. Cell Sci. 15, 419427 ( 1974). 5. Farrant, J., Walter, C. A., and Armstrong, J. A. .Preservation of structure and function of an organized tissue after freezing and thawiug. PTOC. Roy. Sot. B 168, 293-310 (1967); 6. Hak, A. M., Offerijns, F. G. J., and Verheul, C. C. Toxic effects of DMSO on cultured beating heart cells at temperatures above zero. lCryobiology 10, 244-250 ( 1973). 7. Janiszewski, E., and Wollenberger, A. Gefrierkomnservierung von Herzzellen und Herzfragmenten. Acta Biol. Med. Gem. 29, 135147 (1972). 8. Karnovsky, M. J. A. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137-138 A (1965). 9. Karow, A. M., and Webb, W. R. Toxicity of variou:; solute moderates used in hypothermia. Cryobiology 1, 270-273 ( 1965). 10. Karow, A. M., Carrier, O., and Holland, W. C. Toxicity of high dimethylsulfoxide concentrations in rat heart freezing. Cryobiology 3, 464468 ( 1967). 11. Kasten, F. H. Phase contrast observations and electron microscopy of cultured newborn rat he.art cells. J. CeU Biol. 27, 122123A (1965). 12. Kasten, I?. H. Electron microscope studies of the combined effects of trypsinization and
13.
14. 15.
16.
17.
IS.
HEART
CELLS
315
centrifugation on rat heart cells, with observations of early cultures. J. Cell Biol. 28, 131A (1966). Kessler, A. Myosingehalt und Aktivitiit der Kreatin-Phosphokinase in Rattenherz-Zellkulturen. Acta biol. med. germ. 29, 119134 (1972). Lovelock, J. E. The haemolysis of human red blood cells by freezing and thawing. Biochim. Biophys. Actu 10, 414-426 ( 1953). Malinin, G. I. Cytotoxic effect of dimethylsulfoxide on the ultrastructure of cultured rhesus kidney cells. CryobioZogy 10, 22-32 (1973). Mason, M. M. Toxicology of DMSO in animals. In “Dimethyl Sulfoxide” (S. W. Jacob, E. E. Rosenbaum, and D. C. Wood, Eds.), Vol. 1, pp. 113-131, Marcel Dekker, New York, 1971. Mazur, I’., Leibo, S. P., and Chu, E. H. Y. A two-factor hypothesis of freezing injury; evidence from Chinese hamster tissue culture cells. Exp. Cell Res. 71, 345-355 ( 1972). Meryman, H. T. Modified model for the mechanism of freezing injury in erythrocytes. Nature
(London)
218, 333-336
(1968).
19. Millonig, G. Advantages of a phosphate buffer for 0~01 solutions in fixation. J. Appl. PhysioE. 32, 1637 ( 1961). 20. Millonig, G. A modified procedure for lead staining of thin sections. J. Biophys. Biochm. CytoZ. 11, 736-739 ( 1961). 21. Offerijns, F. G. J., Freud, G. E., and Krijnen, H. W. The reanimation of myocardial cells preserved in the frozen state. Nature (London) 222, 1174 (1969). 22. Offerijns, F. G. J., and Krijnen, H. W. The preservation of the rat heart in the frozen state. Cryobiology 9, 289-295 (1972). 23. Sherman, J. K. Correlation in ultrastructural cryoinjury of mitochondria with aspects of their respiratory function. Erp. Cell Res. 66, 378-384 ( 1971). 24. Sherman, J. K. Comparison of in vitro and in situ ultrastructural cryoinjury and cryoprotection of mitochondria. Cryobiology 9, 112-122 ( 1972). 25. Shlafer, M., and Karow, A. M. Ultrastructurefunction correlative studies for cardiac preservation. I. Hearts perfused with various concentrations of dimethylsulfoxide (DMSO). Cryobiology 8, 280-289 ( 1971). 26. Shlafer, M., and Karow, A. M. Ultrastructurefunction correlative studies for cardiac preservation. II. Hearts frozen to various temperatures without a cryoprotectant. Cyobiology 8, 350-360 ( 1971).
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27. Shlafer, M., and K,arow, A. M. Ultrastructure-function correlative studies for cardiac preservation. III. Hearts frozen to -10°C and -17°C with and without dimethylsulfoxide (DMSO). Cryobiology 9, 38-50 (1972). 28. Stowell, R. E., Young, D. E., Arnold, E. A., and Trump, B. F. Structural, chemical, physical and functional alterations in mammalian nucleus, following different conditions of freezing, storage and thawing. Fed. Proc. 24, Suppl. 15, 115-141 ( 1965). 29. Trump, B. E., Goldblatt, P. J., Griffin, C. C., Waravdekar, V. S., and Stowell, R. E. Effects of freezing and thawing on the ultrastructure of mouse hepatic parenchymal cells. Lab. Inuest. 13, 967-1002 ( 1964).
ET AL. 30. Trump, B. F., and Stowell, thawing on tution and tures. Fed. (1965).
Young, D. E., Arnold, E. R., R. E. Effects of freezing and the structure, chemical constifunction of cytoplasmic strucProc. 24, SuppE. 15, 144-168
31. Weiss, L., and Armstrong, J. A. Structural changes in mammalian cells associated with cooling to -79°C. I. Biophys. Biochem. Cytot. 7, 673-677 ( 1969). 32. Wollenberger, A. Survival of potentially beating single heart cells in the frozen state. In “Factors Influencing Myocardial Contractility” (R. D. Tanz, T. Kavaler, and J, Roberts, Eds.), pp. 317-327. Academic Press, New York/London, 1967.
13, 305316
(1976)
The Effect of Cooling Rate and of Dimethyl Sulfoxide Concentration on the Ultrastructure of Neonatal Rat Heart Cells after Freezing and Thawing1 G. M. ALINK, D#epartment Netherlands
J. AGTERBERG,
A. W. HELDER,
F. G. J. OFFERIJNS
Physiology and Cryobiology, Central Laboratory of the Red Cross Blood Transfusion Service, Amsterdam, The Netherlands
Neonatal rat heart cells can be preserved in the frozen state (1, 7, 21). Their survival after freezing and thawing ,depends on cooling rate, on ,dimethyl sulfoxide (DMSO) concentration ( 1 ), and probably also on other f,actors. In order to obtain a better understanding of differences in survival we investigated the ultrastructure of heart cells in suspension and in tissue culture after freezing ,at
AND
of Applied
AND
and at the nonoptimal cooling rate preceding and following the optimal rate in the series 1, 5, 10, 30, and 50”C/min ( 1). Immediately after thawing and washing out the DMSO, the cells were fixed in Karnovsky’s fixative (8) for a period of at least 24 hr and subsequently rinsed in 0.1 M phosphate buffer, pH 7.4, at 4°C for 1 hr. After postfixation in a 1% osmium tetroxide solution in phosphate buffer, pH 7.5 ( 19), the material was dehydrated by increasing concentrations ‘of ethanol and embedded in Epon. Ultrathin sections were cut from two cell blocks on a Reichert ultramicrotome and stained with a saturated uranyl acetate solution in aqua destillata for 20 min sand with lead hydroxide according to Millonig (20) for 10 min. After evaporation with carbon the sections were examined with a Philips EM 300 electron microscope. In the same way, electron microscopical preparations were made of cells treated only with DMSO as well as of untreated control cells. We especially studied the integrity of the cell membrane, the mitochondria and the nucleus in untreated and unfrozen, treated and unfrozen, and treated and frozen muscle and nonmuscle cells. The ultrastructure of unfrozen and frozen-thawed myoblasts after 8 days of culturing was ,also investigated. After fixation in Karnovsky’s fluid the cultured cells were separated from the surface of the dish by
METHODS
Hearts of neonatal rats ( Wistar), l-3 days old, were trypsinized according to a method described previously ( 1). The neonatal rat heart ‘cells were brought into suspension in culture medium and frozen with 2.5, 5, 7.5, or 10% DMSO. Addition of DMSO in culture medium to the cell suspensions befgore freezing and dilution with medium B after thawing were done slowly at room temperature. The cells were cooled at the ‘optimal cooling rate for each DMSO concentration, 5”C/min for 2.5, 5, and 10% DMSO and 30”C/min for 7.5% DMSO, Received July 1, 1975. 1 These investigations were supported by the Foundation for Medical Research FUNGO, The Netherlands. 305 Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
ALINK
306
ET AL.
tt-FIG. 1. 1 Electron micrograph of an untreated, unfrozen neonatal rat heart muscle cell showwirn well spread spreaa chrocnroing intact cell membrane N )1 with memtxane ( C), mitochondria ( M ), and nucleus ((Iu matin. X 10,000.
scratching ,and then prepared for electron microscopy according to the method described above. RESULTS
The electron microscopical preparation of untreated, unfrozen muscle cells (Figs. 1 and la) showed mostly round cells with an intact cell membrane, mitochondria, rough endoplasmic reticulum, and a nucleus with well-spread chromatin and glycogen granules. Most of the myofibrils were disorganized; often parts of Z-lines were visible. Nonmuscle celIs (Fig. 2) were characterized by the absence of myofibrils and glycogen granules. The shape of these cells was more irregular. In sections of untreated, unfrozen cells, a few cells of both types always were partly or completely damaged, probably because of the isolation procedure.
Muscle cells treated with DMSO only or frozen in the presence of DMSO appeared to have different stages of cellular damage (Table 1) ; in nonmuscle cells no damage was observed. Muscle cells treated with 2.5% DMSO showed no ultrastructural difference compared to untreated cells. After treatment with 5% DMSO, a space occurred between the cytoplasmic matrix and the cell membrane (Fig. 3). With 7.5% DMSO most of the cells showed membrane damage similar to that with 5% DMSO, and in addition many cells showed swollen mitochondria. In nearly all cells treated with 10% DMSO the cell membrane was interrupted, or it disappeared completely. The majority of the mitochondria was swollen or destroyed, and in many cells the perinuclear space was dilated and the nuclear chromatin was clumped (Fig. 4).
ULTRASTRUCTURE
OF FROZEN-THAWED
HEART
CELLS
FIG. la. Detail of untreated, unfrozen myoblast, showing disorganized parts of :Z-lines (Z), and the cristae pattern in mitochondria (M). X35,000. TABLE
myofibrils
(My),
1
and Nucleus and the Survival in Culture of Myoblasts The Integrity of Cell Membrane, Mitochondria, DMSO Treatment and after Freezing with DMSO at Optimal and Nonoptimal Cooling Rates Cooling rate (T/min) 0 *iMrSO
2.5 5 7.5 10
n. n. n. n.
c.~ c. c. c.
Integrity= Cell membrane
Mitochondria
NUCIIXIS
after
SurvivaP (%I
++ + f -
++ ++ + f
++ ++ ++ +
100 97 51 32
2.5 2.5 2.5
1 5 10
f f f
f f f
+ + +
1 23 17
5 5 5
1 5 10
+ + +
f ++ +
+ ++ ++
33 72 40
7.5 7.5 7.5
1 30 50
f + f
f + f
i-f -I-+ f
24 59 29
1 5 10
f f f
f f f
+ + +
34 40 23
10 10 10
a Cells with ,a normal structure: b Survival, based on contracting c n. c.,]no-cooling.
++, 75-100%; +, 50-7570; f, 2550%; -, O-25%. area, on the second day of culturing; previous experiments
(1).
308
ALINK
ET AL.
FIG. 2. Electron micrograph of untreated, unfrozen fibroblast showing the irregular shape of the cell membrane (C). The rough endoplasmic reticulum (ER) is well developed and myofibrils are absent. X 15,000.
FIG. 3. Myoblast after treatment with 5% DMSO at room temperature. There is a space (S) between the cytoplasmic matrix and the cell membrane. Further, the morphological appearance does not differ from an untreated myoblast. ~15,000. _ .
ULTRASTRUCTURE
OF FROZEN-THAWED
HEART
CELLS
FIG. 4. Myoblast after treatment with 10% DMSO at room temperature. The cell membrane is severely damaged as is shown by the numerous interruptions (arrows), The mitochondria (M) are swollen and the nuclear chromatin (Ch) is clumped. X15,000.
Muscle cells frozen with 2.5% DMSO had mostly undulating *and interrupted cell membranes, swollen and frequently destroyed mitochondria. Many cells had clumped nuclear chromatin (Fig. 5). No difference in morphological damage was observed between cells cooled at optimal or at nonoptimal cooling rates. Freezing at the optimal ‘cooling rate with 5% DMSO had an effect on the cell integrity other than freezing at the nonoptimal cooling rate. At the optimal cooling rate of S”C/ min, only a part of the cells showed a space between cytoplasm and cell membrane (Fig. 6). In addition, at lO”C/min many cells appeared to have swollen mitochondria. At l.“C/min the number of cells with swollen rnitochondria increased, .and cells with clumped nuclear chromatin appeared (Fig. 7). Muscle cells frozen with 7.5% DMSO showed the smallest number of damaged cells when frozen at the optimal cooling rate of 30”C/min. The nuclei of most cells showed a normal appearance at
rates of 1 and 30”C/min. At 5O”C/min most cells had undulating and interrupted cell membranes, swollen mitochondria, and damaged nuclei. After freezing with 10% DMSO at optimal or nonoptimal cooling rates, most of the cells showed undulated and interrupted cell membranes, swollen and occasionally destroyed mitochondria; about 50% of the cells showed clumping of the nuclear chromatin. Comparing the morphological damage caused by DMSO with the damage caused by freezing, it appears that in both cases the nucleus of the cells suffers less damage than the cell membrane and the mitochondria. After DMSO treatment only, the cell membranes are already damaged at lower DMSO concentrations than the mitochondria. After freezing and thawing, the cell membranes and mitochondria are usually damaged at the same DMSO concentrations. Ultrastructure of myoblasts after prolonged culturing. After 8 days of culturing
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FIG. 5. Severely damaged muscle cell after freezing at lO”C/min with 2.5% DMSO. The cell membrane ( C ) is undulating and shows numerous interruptions ( arrows). The mitochondria (M) are swollen and have destroyed cristae which have partly or completely disappeared (Me). In the nucleus the chromatin ( Ch) is clumped. X15,000.
FIG. 6. Myoblast after freezing at B”C/min with 5% DMSO. There is a space (S) between the cytoplasm and the cell membrane. Further, the morphological appearance does not differ from an untreated unfrozen myoblast. X15,000.
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FIG. 7. Damaged muscle cell after freezing at l”C/min with 5% DMSO. The cell membrane (C) is undulating and shows numerous interruptions (arrows) as is seen in Fig. 5. The mitochondria (M) are swollen and the nuclear chromatin (Ch) is clumped. X15,000.
FIG. 8. Electron micrograph of frozen-thawed rat heart muscle cells after 8 days of culturing. The cells show well organized myofibrils (My) and Z-lines (Z); intercalated disks (I) are established. The cell membranes (C) and the mitochondria (M) have a normal appearance. X23,000.
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FIG. 8a. Detail of frozen-thawed muscle cell after 8 days of culturing, showing nucleus (N) and myofibri1 (My) with Z-line. ~37,000.
no difference between the ultrastructure of control cells and ‘cells treated with DMSO or frozen with DMSO was seen, This occurred at all DMSO concentrations and at all cooling rates which were studied. The myoblasts showed very well organized myofibrils and Z-lines. The intercalated disks were well established. The cell membrane, the mitochondria, and the nucleus of frozen cells had a normal #appearance (Fig. 8, 8a). DISCUSSION
The ultrastructure of neonatal rat heart cells in suspension and after a few days of culturing has been described by several investigators (11, 12, 13). Some structural alterations after freezing and thawing were reported in embryonic chicken heart cells ( 32). Ultrastructure-survival correlation studies were performed in the rat heart (2527), smooth muscle (5), HeLa cells (31)) foeta1 human lung cells (4), hamster lung cells (2), and yeast cells (3). Extensive studies on the ultrastructural damage after freezing and thawing were done on
mouse hepatic cells (28-30) and on the mitochondria of renal cortex cells of mice (23, 24). In our freezing studies on the ultrastructural damage, DMSO was used as a cryoprotectant (1). The toxic properties of DMSO are well known (9, 10, 16, 22, 25). Because it also decreases the viability of heart cells ( 1, 6), we studied the effect of DMSO on the ultrastructure. In our experiments, 2.5% DMSO added at room temperature to cells in suspension was the highest concentration of DMSO which caused no morphological damage. With 5% DMSO a space developed between cell membrane and cytoplasm, and with 7.5% DMSO swelling of mitochondria occurred. With 10% DMSO, clumping of chromatin in the nucleus was observed and the cell membrane and the mitochondria were often ruptured. The increase of cellular damage correlated well with the previously observed ( 1) decrease in survival of the cells, measured as contracting area on the second day of culturing.
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Ultrastructural damage at relatively low con’centrations of DMSO was described by Malinin (15) in monkey kidney cells. He reported damage to cell membranes, mitochondria, and nuclei after treatment of cultured cells with 7.5% DMSO at room temperature. It is ‘also reported that morphological dalmage after treatment with higher concentrations of DMSO correlates with function. Iln guinea pig smooth muscle (5) and in the rat heart (25), increasing DMSO concentrations caused an increase in structural damage to the mitochondria and the nucleus, which correlated with a decrease in the contractility. In these organs the cell membranes were intact which is in contrast with our results and with those of Malinin ( 15). Weiss and Armstrong (31) found intact cell Imembranes, nuclei, and swollen mitochondria after treatment of HeLa cells for 75 min with 15% glycerol. These cells showed normal growth in culture. After freezing at optimal, suboptimal, or supraoptimal cooling rates with different DMSO concentrations, we found that in the presence of 5 or 7.5% DMSO a high survival of the cells occurred at the optimal cooling rates. The ultrastructure of those cells was better than that of the cells frozen at nonoptimal cooling rates, which caused a low or moderate survival. After freezing with 2.5 or 10% DMSO at optimal and nonoptimal cooling rates we found moderate or low survival. However, these differences in survival were not reflected in the ultrastructure of the cells. In a study by Trump et al. (29) on mouse hepatic cells, at lower cooling rates alterations were largely confmed to the cell membrane; in tissue which was frozen rapidly, thle alterations involved numerous intercellular membrane systems as well. Better integrity of cell membranes and mitochondria was observed after sl’ow freezing with 1’5% DMSO (30) than after slow freezing without a cryoprotectant. Beadle and Harris ( 4 ) reported no difference in ultrastructural damage in human foetal lung
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cells after freezing at a low and a high cooling rate if the recovery was low. If freezing resulted in a high recovery, the structural damage was less. Bank and Mazur (2) reported that, if the survival of hamster lung cells after freezing under different conditions was 5 or 20%, no differences in morphologioal alterations could be observed between both groups. On the other hand when survival was ,about equal (20-25s ), structural characteristics ranged from highly abnormal to relatively normal, depending on the freeze-thaw procedure. Only at optimal freezing conditions (80% survival), was the ultrastructure of the cells quite normal. In another study Bank and Mazur ( 3), using a freeze-cleaving, freezeetching technique, showed in yeast cells that, even at optimal cooling rates (survival, 60% ), the ultrastructure of the cells can differ greatly from the well-organized structure in the frozen state at ultrarapid cooling rates. Weiss and Armstrong (31) described the ultrastructure of HeLa cells after freezing with and without 15% glycerol. Although there was survival of cells after freezing with glycerol but not without glycerol, the ultrastructural damage was equal in both groups. They described the cell membranes as being intact, although there were empty spaces just inside these membranes which were folded. In our experiments such cell membranes were defined as being damaged. Mitochondria and nuclei were damaged in the HeLa cells, as we observed in heart muscle cells. A good correlation between ultrastructure and function was described by Farrant et al. (5) in guinea pig smooth muscle after stepwise cooling with and without DMSO to -79°C and rewarming. Such a correl,ation was not found by Shlafer and Karow (27) who studied the cell integrity of the adult rat heart after slow cooling to -17°C at different DMSO concentrations. At 1 and 20% DMSO, mitochondria and nuclei were ruptured and no contractions of the heart were observed. After cooling with 5, 10,
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and 15% DMSO, the mitochondrk were swollen to different degrees and the nuclei often had clumped chromatin. Some hearts showed contractions, others did not. There was no difference in ultrastructural damage between survivors ,and nonsurvivors. The ultrastructural investigations discussed above show a great diversity in the choice of cell type and describe various aspects of the freezing process and their influence on the ultrastructure of the cells. The ultrastructure was often ‘compared to survival. One of the aspects of such studies is the fact that cryoprotectants such as DMSO and glycerol may be toxic. Cooling rate is another important aspect. The damage during slow cooling is caused by high electrolyte concentrations (14) or by OSmotic stress (18) and during rapid cooling by intercellular ice formation. At intermediate cooling rates, the least amount of damage may be expected (17). In our experiments we studied the ultrastructure of neonatal rat heart cells after freezing at optimal, suboptimal, and supraoptimal cooling rates with the DMSO concentrations previously used in freeze-survival experiments ( 1). Comparing our results with the published data discussed above, we conclude that DMSO leads to different degrees of ultrastructural damage depending on DMSO concentration, cell type, and other experimental conditions. Swelling of membrane-Iimited structures, especially mitochondria, is one of the first phenomena to occur. Subsequently, clumping of the nuclear chromatin as well as rupturing of the cell, mitochondrial, and nucIear membranes occur. After DMSO treatment there is a good correlation between ultrastructural damage and survival. After freezing with DMSO and thawing, the order of damaged structures is about the same as after DMSO treatment only. If the ultrastructural damage after freezing is compared with the survival in culture, the situation differs from DMSO treatment only. Generally it may be concluded that a good survival cor-
ET AL.
relates with a good ultrastructure and that such a correlation is not found if the survival is low or moderate. When the survival of cells is higher, the correlation with the ultrastructure seems to be better. If the survival is low or moderate, phenomena other than those which can be observed on the ultrastructural level may contribute to the cell damage after freezing
The ultrastructure of neonatal rat heart cells in suspension and in tissue culture after freezing ,at optimal, suboptimal, and supraoptimal cooling rates with 2.5, 5, 7.5, and 10% DMSO was investigated. The effect of DMSO treatment only on the structure of the cells was also studied. A comparison was made with the surviva1 in culture. Without freezing, increasing DMSO concentrations caused an increase of morphoIogioaI damage, correIating with a decrease of the survival in culture. With 2.5% DMSO there was no difference with untreated cells. At higher DMSO concentrations, the ultrastructural damage increased from spaces between cell membrane and cytoplasm at 5% DMSO to interrupted cell membranes, swollen or destroyed mitochondria, and nucIei with clumped chromatin at 10% DMSO. After freezing at optima1 or nonoptimal cooling rates with 5 or 7.5% DMSO, the ultrastructure correlated well with the survival. After freezing with 2.5 or 10% DMSO at optimal or nonoptimal cooling rates, differences in survival were found, which were not reflected in the ultrastructure of the cell. After 8 days of culturing, cells which were frozen at all the different cool-
ULTRASTRUCTURE
OF FROZEN-THAWED
ing rates and DMSO concentrations peared to have a normal structure.
ap-
ACKNOWLEDGMENTS We thank Mr. C. C. Verheul and Mr. R. Sprokholt for their skillful technical assistance. We are grateful to Mr. P. Meelker and Mr. L. R. Hafkamp for making the photographs and to Mrs. H. Grooters for typing the manuscript. REFERENCES 1. Alink, G. M., Verheul, C. C., and Offerijns, F. G. J. The effect of cooling rate and of dimethyl sulfoxide concentration on low temperature preservation of neonatal rat heart cells. Cryobiology 13, 295-304 ( 1976). 2. Bank, II., and Mazur, P. Relations between ultrastructure and viability of frozen-thawed Chinese hamster tissue-culture celIs. Exp. Cell Iles. 71, 441-454 (1972). 3. Bank, II., and Mazur, P. Visualization of freezing damage. J. Cell Biol. 57, 729-742 (1973).
4. Beadle, D. J., and Harris, L. W. Relationship between freezing rate, ultrastructure and recovery in a human diploid cell line. J. Cell Sci. 15, 419427 ( 1974). 5. Farrant, J., Walter, C. A., and Armstrong, J. A. .Preservation of structure and function of an organized tissue after freezing and thawiug. PTOC. Roy. Sot. B 168, 293-310 (1967); 6. Hak, A. M., Offerijns, F. G. J., and Verheul, C. C. Toxic effects of DMSO on cultured beating heart cells at temperatures above zero. lCryobiology 10, 244-250 ( 1973). 7. Janiszewski, E., and Wollenberger, A. Gefrierkomnservierung von Herzzellen und Herzfragmenten. Acta Biol. Med. Gem. 29, 135147 (1972). 8. Karnovsky, M. J. A. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137-138 A (1965). 9. Karow, A. M., and Webb, W. R. Toxicity of variou:; solute moderates used in hypothermia. Cryobiology 1, 270-273 ( 1965). 10. Karow, A. M., Carrier, O., and Holland, W. C. Toxicity of high dimethylsulfoxide concentrations in rat heart freezing. Cryobiology 3, 464468 ( 1967). 11. Kasten, F. H. Phase contrast observations and electron microscopy of cultured newborn rat he.art cells. J. CeU Biol. 27, 122123A (1965). 12. Kasten, I?. H. Electron microscope studies of the combined effects of trypsinization and
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centrifugation on rat heart cells, with observations of early cultures. J. Cell Biol. 28, 131A (1966). Kessler, A. Myosingehalt und Aktivitiit der Kreatin-Phosphokinase in Rattenherz-Zellkulturen. Acta biol. med. germ. 29, 119134 (1972). Lovelock, J. E. The haemolysis of human red blood cells by freezing and thawing. Biochim. Biophys. Actu 10, 414-426 ( 1953). Malinin, G. I. Cytotoxic effect of dimethylsulfoxide on the ultrastructure of cultured rhesus kidney cells. CryobioZogy 10, 22-32 (1973). Mason, M. M. Toxicology of DMSO in animals. In “Dimethyl Sulfoxide” (S. W. Jacob, E. E. Rosenbaum, and D. C. Wood, Eds.), Vol. 1, pp. 113-131, Marcel Dekker, New York, 1971. Mazur, I’., Leibo, S. P., and Chu, E. H. Y. A two-factor hypothesis of freezing injury; evidence from Chinese hamster tissue culture cells. Exp. Cell Res. 71, 345-355 ( 1972). Meryman, H. T. Modified model for the mechanism of freezing injury in erythrocytes. Nature
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19. Millonig, G. Advantages of a phosphate buffer for 0~01 solutions in fixation. J. Appl. PhysioE. 32, 1637 ( 1961). 20. Millonig, G. A modified procedure for lead staining of thin sections. J. Biophys. Biochm. CytoZ. 11, 736-739 ( 1961). 21. Offerijns, F. G. J., Freud, G. E., and Krijnen, H. W. The reanimation of myocardial cells preserved in the frozen state. Nature (London) 222, 1174 (1969). 22. Offerijns, F. G. J., and Krijnen, H. W. The preservation of the rat heart in the frozen state. Cryobiology 9, 289-295 (1972). 23. Sherman, J. K. Correlation in ultrastructural cryoinjury of mitochondria with aspects of their respiratory function. Erp. Cell Res. 66, 378-384 ( 1971). 24. Sherman, J. K. Comparison of in vitro and in situ ultrastructural cryoinjury and cryoprotection of mitochondria. Cryobiology 9, 112-122 ( 1972). 25. Shlafer, M., and Karow, A. M. Ultrastructurefunction correlative studies for cardiac preservation. I. Hearts perfused with various concentrations of dimethylsulfoxide (DMSO). Cryobiology 8, 280-289 ( 1971). 26. Shlafer, M., and Karow, A. M. Ultrastructurefunction correlative studies for cardiac preservation. II. Hearts frozen to various temperatures without a cryoprotectant. Cyobiology 8, 350-360 ( 1971).
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27. Shlafer, M., and K,arow, A. M. Ultrastructure-function correlative studies for cardiac preservation. III. Hearts frozen to -10°C and -17°C with and without dimethylsulfoxide (DMSO). Cryobiology 9, 38-50 (1972). 28. Stowell, R. E., Young, D. E., Arnold, E. A., and Trump, B. F. Structural, chemical, physical and functional alterations in mammalian nucleus, following different conditions of freezing, storage and thawing. Fed. Proc. 24, Suppl. 15, 115-141 ( 1965). 29. Trump, B. E., Goldblatt, P. J., Griffin, C. C., Waravdekar, V. S., and Stowell, R. E. Effects of freezing and thawing on the ultrastructure of mouse hepatic parenchymal cells. Lab. Inuest. 13, 967-1002 ( 1964).
ET AL. 30. Trump, B. F., and Stowell, thawing on tution and tures. Fed. (1965).
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31. Weiss, L., and Armstrong, J. A. Structural changes in mammalian cells associated with cooling to -79°C. I. Biophys. Biochem. Cytot. 7, 673-677 ( 1969). 32. Wollenberger, A. Survival of potentially beating single heart cells in the frozen state. In “Factors Influencing Myocardial Contractility” (R. D. Tanz, T. Kavaler, and J, Roberts, Eds.), pp. 317-327. Academic Press, New York/London, 1967.