CRYOBIOLOGY
13, 278-286
Freezing
( 1976)
Injury
to Erythrocytes. I. Freezing Post-Thaw Hemolysis TOKIO
The Institute
of Low
Temperature
Received
May
14, 1975.
1976 by Academic Press, Inc. Copyrieht All rights o8 reproduction in any form reserved.
and
NE1
Science, Hokkaido
In the field of cryobiology there has been a considerable number of studies on the mechanism of freezing injury to animal and plant cells. In the majority of these studies, the viability of cells frozen under various conditions was examined by one of the physiological functions of thawed specimens. To elu’cidate freezing injury, it is important to observe both the morphology of cells in the frozen state and their physiology after thawing; however, very few investigations have been performed along these lines (1, 3, 9, 16). In the present study the survival of frozen cells was examined with regard to their appearance during freezing as well as the patterns of ice present. Vmarious cryotechniques have been #applied to the study of biological materials by electron microscopy. The ultimate aim of recent developments in cryotechniques is to eliminate the artifacts which can occur in the standard preparation proced’ures of fixation, dehydration, embedding, and sectioning and to observe the specimens in their nlative state. However, even with these new techniques, the most common artifact is ice crystal formation in the frozen specimens. It is therefore important to find a way to prevent such ice f’ormation. The freeze-fracture-replica method employed in this study is extremely useful for the ultrastructural examination of biological specimens.
Patterns
University,
Sapporo,
Japan
Human red blood cells are useful for both morphological sand physiological examination of cell injury, Hemolysis, one of the established parameters of cell membrane damage, can be measured quantitatively. In freeze-fractured specimens, ice particles formed within erythrocytes are easily identified because of the lack of intracellular organelles such as nuclei and mitochondria. The present study was carried out to investigate the interrelationship of the function of frozen and frozen-thawed erythrocytes land to examine ice crystal formation within the cells as an artifact in cryotechniques for electron microscopy. MATERIALS
AND
METHODS
I. Materials. Human red blood cells, collected as ACD (citric acid-dextrose) whole blood and kept at 4°C for 1 week or #less, were washed three times and suspended in physiological saline (0.15 M NaCI). Cell suspensions containing 10, 20, and 30% glycerol (v/v) were used for examination of the cryoprotective effect. 2. Preparation procedure. Specially designed specimen holders were employed for morphological investigations; a copper plate was made, 0.3 mm in thickness and 10 x 10 mm in size, folded into a box-like shape to fit the cold stage of an evaporator. Aliquots of cell suspension (0.01 ml), prepared as described (above, were placed on the holder. For ultrarapid cooling, 0.003 ml of suspension was placed on the holder and
FREEZING
INJURY
covered with a small metal plate, 0.1 mm in thickness and 5 x 5 mm in size ( 11). 3. Temperature measurement. The rate of cooling was measured by the use of copper-const,antan thermocouples of O.lmm diameter. Because it was difficult to measure accurately very rapid temperature changes which occur in such a small amount of specimen, only approximate values of cooling rates are described. 4. Freezing. Specimens were frozen at five different cooling sates: (i) for a lO’C/ min cooling rate, the droplet specimens were frozen :in a -20°C freezer for initial ice inoculation, transferred into a -80°C deep freezer and finally immersed in liquid nitrogen; (ii ) for lo2 “C/min, they were frozen in liquid nitrogen vapour in a Dewar flask and then dipped into liquid nitrogen; (iii) for lo3 “C/min, they were directly immersed in liquid nitrogen; (iv) for lo4 ‘C/min, the specimens were abrupt,ly immersed in Freon 22 kept at ‘approximately -150°C by cooling with liquid nitrogen; (v) for lo5 “C/min, 0.003-ml sandwiched specimens were abmptly immersed in Freon 22 cooled w:ith liquid nitrogen. After all specimens had been brought to the temperature of liquid nitrogen, they were prepared for morphological observation and measurement of hemolysis. 5. Morphological observation. The specimens frozen on the holders were transferred onto t’he cold stage installed in the evaporation chtamber, JEOL JEE-FED. After high vacuum ( 10d6 Torr) and low temperature (-100°C) were attained, the specimens were fractured, etched, and replicated according to the standard technique. The replicas thus prepared were cleaned and observed in an electron microscope, JEOL 6s. 6. Measurement of hemolysis. Frozen specimens were thawed rapidly by immersion at room temperature into quantities of the same solution as previously used for each suspending medium. The amount of hemoglobin released into the supernatant
TO ERYTHROCYTES
279
was measured spectrophotometrioally after centrifugation. The extent of hemolysis was expressed as the ratio of the supernatant hemoglobin to the hemoglobin in the control which was osmotically hemolysed with distilled water. RESULTS I.
0.15
M
NaCl Cell Suspensions
1. Hemolysis. As indicated in Fig. 1, erythrocytes suspended in physiological saline and cooled at various rates were almost completely hemolysed after thawing, except that at 103 “C/min they sh’owed approximately 70% hemolysis. 2. Freezing patterns. According to the morphological observation of freeze-fractured specimens, cells frozen at 10 and lo2 “C/min tended to clump, probably as a result of their confinement in concentrated solutions during slow freezing. Individual cells were often difficult to identify, but most of them appeared to be shrunken into various shapes (Fig. 2-la). Because the cells underwent similar changes at both these rates, photographs of cells cooled at lO”C/min were excluded from the figures in this paper. At a cooling rate of lo3 “C/min, a few cells (10% or less) showed irregular outlines and there were intriacellular ice particles of approximately 0.1 to 0.2 pm in diameter, mostly clustered at the center of the cells. The majority of the cells were shrunken but had no intracellular ice
Cooling
Rate
( “Wmin)
FIG. 1. Post-thaw hemolysis of human erythrocytes with different concentrations of glycerol, frozen at various rates of cooling. ( l ), NO glycerol; ( 0 ) 10% glycerol; ( A ), 20% glycerol; ( 0 ), 30% glycerol.
;0
TOKIO
NE1
FIG. 2. Freezing patterns of human erythrocytes with different concentrations of glycerol. frozen at various rates of cooling. Electron micrographs prepared by the freeze-etching technique. X4500. (11, No glycerol; (Z), 10% glycerol; (3), 20% glycerol; (4), 30% glycerol; (a), 10’ “C/min; (b), lo3 “C/min; (c), 104 “C/min; (d), 10’ “C/min. For example, “la” indicates the specimen without glycerol frozen at lo” “C/min.
FIG. 2 co ntinued.
particles (Fig. 2-lb). At lo4 and lo5 “C/ min, all of the cells were intracellularly frozen withy irregular outlines. The size of
the intracellular ice particles depemded upon the cooling rate; the higher the rate, the smaller their size (Figs. 2-k and -1d).
TOKIO NE1
282 II. Glycerolated
Suspensions
307’0, ‘cellular deformation and intracellular ice formation decreased. 1. Hemolysis. As illustrated in Fig. 1, the (iii) Freezing at lo4 “C/min: Different extent of hemolysis decreased according to freezing patterns were observed at the pethe concentration of glycerol added to the ripheral and the central parts of the samcell suspension. Glycerol ( 10% ) showed a ple. This might be due to different rates of marked protective effect only Iat a rate of cooling, depending upon the distance from lo2 “C/min; 20% glycerol exerted a marked the sample surface. The findings that are effect at 10 and lo2 ‘C/min, a slight effect described in this paper pertain only to the at lo5 “C/min, and no eff ect at lo3 and intermediate portion of the sample. All of lo4 “C/min. In 30% glycerol, hemolysis the ceIIs in glycero1 (10% ) were shrunken was almost totally inhibited, except at a and filled with many smlall ice particles less cooling rate of lo4 “C/min. than 0.05 pm in diameter (Fig. 2-2~). In 2. Freezing patterns. The freezing patthe cells frozen in the presence of glycerol terns of specimens examined in this study (20% ), the size of intracellular ice crystals depended upon the rates of cooling and the ,and the shrinkage of cells were decreased concentration of glycerol used. as compared to cells in 10% glycerol. (i) Freezing at lo2 “C/min: Cells frozen Among these, cells without ice particles in glycerol ( 10% ) were deformed and were scattered (Fig. 2-3~). Cells in 30% shrunken without any of the intracehular glycerol showed normal shapes without any ice formation as seen in the control cells definite ice crystals. Only a few cells looked frozen in the absence of glycero1 (Fig. 2- very finely granular. Even in this sample, 2a). In the specimens with a higher con- the extracellular region was filled with centration of glycerol, cells appeared to be small ice particles (Fig. 2-4~). In general, confined in channels, presumably formed at a freezing rate of lo4 “C/min, increasby the concentration of the surrounding ing concentrations of glycerol resulted in medium and bordered by the cavities from smaller ice crystals ‘and a smoother cell which ice had sublimed. These ceIIs showed outline. slight contrast ‘and obscure outlines, possi(iv) Freezing at lo5 “C/min: Very tiny bly because of insufficient etching (Fig. 2intracellular ice particles, around 0.01 pm 3a). The cells in glycerol (30% ) appeared in diameter, and slight cell deformation to have retained their original size and were found only in the cells in 10% glycshape and showed only slight deformation erol (Fig. 2-2d). In 20% glycerol the cells (Fig. 2-4a). appeared almost to maintain their original (ii) Freezing at lo3 “C/min: Cells frosize and shape (Fig. 2-3d). Specimens in zen in glycerol (10% ) showed an appearglycerol (30% ) approached their natural ance similar to th,at of the nonglycerinated configuration; there were no particular ice cells described above, but the size of intraparticles in any part ‘of the samples (Fig. cellular ice particles was smaller, 0.1 to 0.05 2-4d). pm (Fig. 2-2b). Most of the cells in glycerol (20% ) had no ice crystals but showed III. Relation between Morphology slight shrinkage. S,ome cells contained ice and Hemolysis crystals of less than 0.1 pm (Fig. 2-3b). Cells in glycerol (30% ) looked almost norAt the slow rates of cooling such as 10 mal with very slight deformation, but ob- and lo2 “C/min, the extent of post-thaw hemolysis was reduced from 100 to 0% viously large extracellular ice ‘crystals still with increased concentrations of glycerol remained (Fig. 2-4b). Thus, ‘as the glycerol concentration was increased from 10 to from 0 to 30%, but there were no remark-
FREEZING
INJURY TO ERYTHROCYTES
able ‘changes in the freezing patterns, only a slight restoration of the cellular shape. At the rapid rates of lo4 to IO5 “C/min, the most important morphological finding was that of intracellular ice crystal formation. Such ice crystals disappeared with the increase in cooling rate and glycerol concentration. However, the minimum size of intracellular ice particles which might cause hemolysis could not be determined. For example, cells in 10% glycerol frozen at lo5 “C/min arrd cells in 20% glycerol frozen at lo4 “C/mm both showed 100% hemolysis and nearly the same freezing pattern; some of the cells contained ice particles of less than 0.01 pm in diameter while others had no ice particles. DISCUSSION
Many studies have been made to elucidate the mechanism of freezing injury to living ~ce1l.son the basis of morphology and function. However, only #a few investigations have been conducted to compare the frozen state and the post-thaw survival, i.e. specimens in recovery, of corresponding terms of cooling rates. Moor ( 9) reported the freezing patterns of yeast cells without protective additives frozen tat slow and rapid rates as follows: shrunken protoplasm at 0.01 ‘C/set, large ice partic1e.s at 1°C/sec, small ice particles at lO”C/sec, and no ice formation at 1O,OOO”C/sec. In the experiment to examine the viability of cells, post-thaw survival w,as graphically shown to be a V-shaped curve with the minimum value at O.S’C/ sec. When glycerol was added as a cryoprotective ‘agent, survival curves shifted to higher percentages parallel to that of nonglycerol control specimens. Bank and Mazur (3) ‘compared their own morphological findings on yeast with the previously reported survival curve. While the cells were shrunken due to extracellular freezing at l’ow cooling rates of 6 and l.S”C/min, intracellular ice crystals were formed tat the high cooling rates of 7500 and 15,060”C/min. The optimum rate for
283
survival was found to be lO”C/min. They concluded that their results were essentially consistent with those reported by Moor (9). Although the freezing patterns show a similar trend in terms of cooling rates, there is some discrepancy on survival ‘curves in these two sets of data. Albrecht et al. (1) tried to attain ultrarapid freezing by the use of a Milhpore filter. Several bacteria - S&I as Staphylococcus aureus, Escherichia coli, and Serratia marcescens showed shrinkage and little or no intracellular ice with a survival rate of almost 100% after rapid thawing. Shimada and Asahina ( 16) observed small intracellular ice crystals in rat ascites tumor cells frozen very rapidly. The sizes of those ice crystals were approximately 0.03, 0.05, and 0.1 pm at the cooling rates of 800, 500, and 3OO”C/ set ,as well as of the shrunken cells frozen at the lower rate of 16”C/sec. From this fact, it was suggested that the cells could survive intracellular freezing, provided that the ice crystals were too small to be injurious to the cells during freezing and thawing. The minimum size of intracellular ice crystals large enough to cause cell dlamage was assumed by Moor to be 100 A in yeast cells but assumed by Shimada to be 0.05 to 0.1 pm in ascites tumor cells. Many studies on freezing injury have been done with mammalian erythrocytes in the context of hemolysis caused by freezing and thawing. In this study, humtan red blood cells were employed for examination of morphology and physi’ology of frozen cells and similar results were obtained. An examination of the hemolysis curve with regard to cooling rates showed an optimal rate around lo3 ’ C/min. Hemolysis increased at cooling rates both higher and lower than the optimal. The freezing patterns of the cells indicated some shrinkage and deformation, probably due to extracellular freezing at the ‘lower rates and intracellular ice formation at the higher rates. The optimal rate determined for red cells was a higher value than that of yeast cells
284
TOKIO
reported by Moor (9) and by Bcank and Mazur (3). Mazur et al. (7) also reported that the rate which shows maximal survival varies with each cell type. Suoh variation may be due to differences in the permeability to water in each cell type. At the lower cooling rate, cells might be shrunken due to dehydration by extracellular freezing according to the mechanism of hemolysis that has long been explained by the salt injury theory proposed by Lovelock (5). The cryoprotective effect of glycerol on erythrocytes has been reported by several investigators (6, 10, 14, 15). This effect was explained by the buffering action of glycerol against a concentration of other solutes. Although definite conclusions cannot be drawn from the morphological observations alone, it was noticed that the cells confined in the concentrated glycerol solution appeared to recover from shrunken to normal shape as the extent
13, 278-286
Freezing
( 1976)
Injury
to Erythrocytes. I. Freezing Post-Thaw Hemolysis TOKIO
The Institute
of Low
Temperature
Received
May
14, 1975.
1976 by Academic Press, Inc. Copyrieht All rights o8 reproduction in any form reserved.
and
NE1
Science, Hokkaido
In the field of cryobiology there has been a considerable number of studies on the mechanism of freezing injury to animal and plant cells. In the majority of these studies, the viability of cells frozen under various conditions was examined by one of the physiological functions of thawed specimens. To elu’cidate freezing injury, it is important to observe both the morphology of cells in the frozen state and their physiology after thawing; however, very few investigations have been performed along these lines (1, 3, 9, 16). In the present study the survival of frozen cells was examined with regard to their appearance during freezing as well as the patterns of ice present. Vmarious cryotechniques have been #applied to the study of biological materials by electron microscopy. The ultimate aim of recent developments in cryotechniques is to eliminate the artifacts which can occur in the standard preparation proced’ures of fixation, dehydration, embedding, and sectioning and to observe the specimens in their nlative state. However, even with these new techniques, the most common artifact is ice crystal formation in the frozen specimens. It is therefore important to find a way to prevent such ice f’ormation. The freeze-fracture-replica method employed in this study is extremely useful for the ultrastructural examination of biological specimens.
Patterns
University,
Sapporo,
Japan
Human red blood cells are useful for both morphological sand physiological examination of cell injury, Hemolysis, one of the established parameters of cell membrane damage, can be measured quantitatively. In freeze-fractured specimens, ice particles formed within erythrocytes are easily identified because of the lack of intracellular organelles such as nuclei and mitochondria. The present study was carried out to investigate the interrelationship of the function of frozen and frozen-thawed erythrocytes land to examine ice crystal formation within the cells as an artifact in cryotechniques for electron microscopy. MATERIALS
AND
METHODS
I. Materials. Human red blood cells, collected as ACD (citric acid-dextrose) whole blood and kept at 4°C for 1 week or #less, were washed three times and suspended in physiological saline (0.15 M NaCI). Cell suspensions containing 10, 20, and 30% glycerol (v/v) were used for examination of the cryoprotective effect. 2. Preparation procedure. Specially designed specimen holders were employed for morphological investigations; a copper plate was made, 0.3 mm in thickness and 10 x 10 mm in size, folded into a box-like shape to fit the cold stage of an evaporator. Aliquots of cell suspension (0.01 ml), prepared as described (above, were placed on the holder. For ultrarapid cooling, 0.003 ml of suspension was placed on the holder and
FREEZING
INJURY
covered with a small metal plate, 0.1 mm in thickness and 5 x 5 mm in size ( 11). 3. Temperature measurement. The rate of cooling was measured by the use of copper-const,antan thermocouples of O.lmm diameter. Because it was difficult to measure accurately very rapid temperature changes which occur in such a small amount of specimen, only approximate values of cooling rates are described. 4. Freezing. Specimens were frozen at five different cooling sates: (i) for a lO’C/ min cooling rate, the droplet specimens were frozen :in a -20°C freezer for initial ice inoculation, transferred into a -80°C deep freezer and finally immersed in liquid nitrogen; (ii ) for lo2 “C/min, they were frozen in liquid nitrogen vapour in a Dewar flask and then dipped into liquid nitrogen; (iii) for lo3 “C/min, they were directly immersed in liquid nitrogen; (iv) for lo4 ‘C/min, the specimens were abrupt,ly immersed in Freon 22 kept at ‘approximately -150°C by cooling with liquid nitrogen; (v) for lo5 “C/min, 0.003-ml sandwiched specimens were abmptly immersed in Freon 22 cooled w:ith liquid nitrogen. After all specimens had been brought to the temperature of liquid nitrogen, they were prepared for morphological observation and measurement of hemolysis. 5. Morphological observation. The specimens frozen on the holders were transferred onto t’he cold stage installed in the evaporation chtamber, JEOL JEE-FED. After high vacuum ( 10d6 Torr) and low temperature (-100°C) were attained, the specimens were fractured, etched, and replicated according to the standard technique. The replicas thus prepared were cleaned and observed in an electron microscope, JEOL 6s. 6. Measurement of hemolysis. Frozen specimens were thawed rapidly by immersion at room temperature into quantities of the same solution as previously used for each suspending medium. The amount of hemoglobin released into the supernatant
TO ERYTHROCYTES
279
was measured spectrophotometrioally after centrifugation. The extent of hemolysis was expressed as the ratio of the supernatant hemoglobin to the hemoglobin in the control which was osmotically hemolysed with distilled water. RESULTS I.
0.15
M
NaCl Cell Suspensions
1. Hemolysis. As indicated in Fig. 1, erythrocytes suspended in physiological saline and cooled at various rates were almost completely hemolysed after thawing, except that at 103 “C/min they sh’owed approximately 70% hemolysis. 2. Freezing patterns. According to the morphological observation of freeze-fractured specimens, cells frozen at 10 and lo2 “C/min tended to clump, probably as a result of their confinement in concentrated solutions during slow freezing. Individual cells were often difficult to identify, but most of them appeared to be shrunken into various shapes (Fig. 2-la). Because the cells underwent similar changes at both these rates, photographs of cells cooled at lO”C/min were excluded from the figures in this paper. At a cooling rate of lo3 “C/min, a few cells (10% or less) showed irregular outlines and there were intriacellular ice particles of approximately 0.1 to 0.2 pm in diameter, mostly clustered at the center of the cells. The majority of the cells were shrunken but had no intracellular ice
Cooling
Rate
( “Wmin)
FIG. 1. Post-thaw hemolysis of human erythrocytes with different concentrations of glycerol, frozen at various rates of cooling. ( l ), NO glycerol; ( 0 ) 10% glycerol; ( A ), 20% glycerol; ( 0 ), 30% glycerol.
;0
TOKIO
NE1
FIG. 2. Freezing patterns of human erythrocytes with different concentrations of glycerol. frozen at various rates of cooling. Electron micrographs prepared by the freeze-etching technique. X4500. (11, No glycerol; (Z), 10% glycerol; (3), 20% glycerol; (4), 30% glycerol; (a), 10’ “C/min; (b), lo3 “C/min; (c), 104 “C/min; (d), 10’ “C/min. For example, “la” indicates the specimen without glycerol frozen at lo” “C/min.
FIG. 2 co ntinued.
particles (Fig. 2-lb). At lo4 and lo5 “C/ min, all of the cells were intracellularly frozen withy irregular outlines. The size of
the intracellular ice particles depemded upon the cooling rate; the higher the rate, the smaller their size (Figs. 2-k and -1d).
TOKIO NE1
282 II. Glycerolated
Suspensions
307’0, ‘cellular deformation and intracellular ice formation decreased. 1. Hemolysis. As illustrated in Fig. 1, the (iii) Freezing at lo4 “C/min: Different extent of hemolysis decreased according to freezing patterns were observed at the pethe concentration of glycerol added to the ripheral and the central parts of the samcell suspension. Glycerol ( 10% ) showed a ple. This might be due to different rates of marked protective effect only Iat a rate of cooling, depending upon the distance from lo2 “C/min; 20% glycerol exerted a marked the sample surface. The findings that are effect at 10 and lo2 ‘C/min, a slight effect described in this paper pertain only to the at lo5 “C/min, and no eff ect at lo3 and intermediate portion of the sample. All of lo4 “C/min. In 30% glycerol, hemolysis the ceIIs in glycero1 (10% ) were shrunken was almost totally inhibited, except at a and filled with many smlall ice particles less cooling rate of lo4 “C/min. than 0.05 pm in diameter (Fig. 2-2~). In 2. Freezing patterns. The freezing patthe cells frozen in the presence of glycerol terns of specimens examined in this study (20% ), the size of intracellular ice crystals depended upon the rates of cooling and the ,and the shrinkage of cells were decreased concentration of glycerol used. as compared to cells in 10% glycerol. (i) Freezing at lo2 “C/min: Cells frozen Among these, cells without ice particles in glycerol ( 10% ) were deformed and were scattered (Fig. 2-3~). Cells in 30% shrunken without any of the intracehular glycerol showed normal shapes without any ice formation as seen in the control cells definite ice crystals. Only a few cells looked frozen in the absence of glycero1 (Fig. 2- very finely granular. Even in this sample, 2a). In the specimens with a higher con- the extracellular region was filled with centration of glycerol, cells appeared to be small ice particles (Fig. 2-4~). In general, confined in channels, presumably formed at a freezing rate of lo4 “C/min, increasby the concentration of the surrounding ing concentrations of glycerol resulted in medium and bordered by the cavities from smaller ice crystals ‘and a smoother cell which ice had sublimed. These ceIIs showed outline. slight contrast ‘and obscure outlines, possi(iv) Freezing at lo5 “C/min: Very tiny bly because of insufficient etching (Fig. 2intracellular ice particles, around 0.01 pm 3a). The cells in glycerol (30% ) appeared in diameter, and slight cell deformation to have retained their original size and were found only in the cells in 10% glycshape and showed only slight deformation erol (Fig. 2-2d). In 20% glycerol the cells (Fig. 2-4a). appeared almost to maintain their original (ii) Freezing at lo3 “C/min: Cells frosize and shape (Fig. 2-3d). Specimens in zen in glycerol (10% ) showed an appearglycerol (30% ) approached their natural ance similar to th,at of the nonglycerinated configuration; there were no particular ice cells described above, but the size of intraparticles in any part ‘of the samples (Fig. cellular ice particles was smaller, 0.1 to 0.05 2-4d). pm (Fig. 2-2b). Most of the cells in glycerol (20% ) had no ice crystals but showed III. Relation between Morphology slight shrinkage. S,ome cells contained ice and Hemolysis crystals of less than 0.1 pm (Fig. 2-3b). Cells in glycerol (30% ) looked almost norAt the slow rates of cooling such as 10 mal with very slight deformation, but ob- and lo2 “C/min, the extent of post-thaw hemolysis was reduced from 100 to 0% viously large extracellular ice ‘crystals still with increased concentrations of glycerol remained (Fig. 2-4b). Thus, ‘as the glycerol concentration was increased from 10 to from 0 to 30%, but there were no remark-
FREEZING
INJURY TO ERYTHROCYTES
able ‘changes in the freezing patterns, only a slight restoration of the cellular shape. At the rapid rates of lo4 to IO5 “C/min, the most important morphological finding was that of intracellular ice crystal formation. Such ice crystals disappeared with the increase in cooling rate and glycerol concentration. However, the minimum size of intracellular ice particles which might cause hemolysis could not be determined. For example, cells in 10% glycerol frozen at lo5 “C/min arrd cells in 20% glycerol frozen at lo4 “C/mm both showed 100% hemolysis and nearly the same freezing pattern; some of the cells contained ice particles of less than 0.01 pm in diameter while others had no ice particles. DISCUSSION
Many studies have been made to elucidate the mechanism of freezing injury to living ~ce1l.son the basis of morphology and function. However, only #a few investigations have been conducted to compare the frozen state and the post-thaw survival, i.e. specimens in recovery, of corresponding terms of cooling rates. Moor ( 9) reported the freezing patterns of yeast cells without protective additives frozen tat slow and rapid rates as follows: shrunken protoplasm at 0.01 ‘C/set, large ice partic1e.s at 1°C/sec, small ice particles at lO”C/sec, and no ice formation at 1O,OOO”C/sec. In the experiment to examine the viability of cells, post-thaw survival w,as graphically shown to be a V-shaped curve with the minimum value at O.S’C/ sec. When glycerol was added as a cryoprotective ‘agent, survival curves shifted to higher percentages parallel to that of nonglycerol control specimens. Bank and Mazur (3) ‘compared their own morphological findings on yeast with the previously reported survival curve. While the cells were shrunken due to extracellular freezing at l’ow cooling rates of 6 and l.S”C/min, intracellular ice crystals were formed tat the high cooling rates of 7500 and 15,060”C/min. The optimum rate for
283
survival was found to be lO”C/min. They concluded that their results were essentially consistent with those reported by Moor (9). Although the freezing patterns show a similar trend in terms of cooling rates, there is some discrepancy on survival ‘curves in these two sets of data. Albrecht et al. (1) tried to attain ultrarapid freezing by the use of a Milhpore filter. Several bacteria - S&I as Staphylococcus aureus, Escherichia coli, and Serratia marcescens showed shrinkage and little or no intracellular ice with a survival rate of almost 100% after rapid thawing. Shimada and Asahina ( 16) observed small intracellular ice crystals in rat ascites tumor cells frozen very rapidly. The sizes of those ice crystals were approximately 0.03, 0.05, and 0.1 pm at the cooling rates of 800, 500, and 3OO”C/ set ,as well as of the shrunken cells frozen at the lower rate of 16”C/sec. From this fact, it was suggested that the cells could survive intracellular freezing, provided that the ice crystals were too small to be injurious to the cells during freezing and thawing. The minimum size of intracellular ice crystals large enough to cause cell dlamage was assumed by Moor to be 100 A in yeast cells but assumed by Shimada to be 0.05 to 0.1 pm in ascites tumor cells. Many studies on freezing injury have been done with mammalian erythrocytes in the context of hemolysis caused by freezing and thawing. In this study, humtan red blood cells were employed for examination of morphology and physi’ology of frozen cells and similar results were obtained. An examination of the hemolysis curve with regard to cooling rates showed an optimal rate around lo3 ’ C/min. Hemolysis increased at cooling rates both higher and lower than the optimal. The freezing patterns of the cells indicated some shrinkage and deformation, probably due to extracellular freezing at the ‘lower rates and intracellular ice formation at the higher rates. The optimal rate determined for red cells was a higher value than that of yeast cells
284
TOKIO
reported by Moor (9) and by Bcank and Mazur (3). Mazur et al. (7) also reported that the rate which shows maximal survival varies with each cell type. Suoh variation may be due to differences in the permeability to water in each cell type. At the lower cooling rate, cells might be shrunken due to dehydration by extracellular freezing according to the mechanism of hemolysis that has long been explained by the salt injury theory proposed by Lovelock (5). The cryoprotective effect of glycerol on erythrocytes has been reported by several investigators (6, 10, 14, 15). This effect was explained by the buffering action of glycerol against a concentration of other solutes. Although definite conclusions cannot be drawn from the morphological observations alone, it was noticed that the cells confined in the concentrated glycerol solution appeared to recover from shrunken to normal shape as the extent
