Effect of cooling and rewarming rates on glycerolated human erythrocytes.

Human erythrocytes suspended in a sodium-free buffered salt solution containing glycerol in 1 m concentration (1 part of packed cells to 4 parts buffered salt solution) were frozen by slow, moderately rapid, or very rapid cooling to various subzero C temperatures. The frozen specimens, after a 5-min storage period at a given temperature, were thawed at low, moderately high, or very high rates. The hemolysis in the frozen and thawed samples was measured by a colorimetric determination of the hemoglobin released from the damaged cells. At ?10 °C, the highest freezing temperature employed, nearly 100% recovery of intact erythrocytes was obtained irrespective of the cooling and rewarming conditions. The extent of the hemolysis after exposure to lower freezing temperatures depended upon the cooling and rewarming conditions. Moderately rapid and very rapid freezing to, and thawing from temperatures below ?40 °C permitted significantly higher recoveries of intact cells than the other freezing/ thawing combinations. In the temperature range ?15 to ?30 °C the combination slow cooling and slow rewarming afforded maximum protection. Very rapid freezing/ slow thawing was the most damaging combination throughout the entire freezing range. The results were interpreted in part by a conventional two-factor analysis, lower cooling rates allowing concentrated salts to determine hemolysis, higher cooling rates destroying the cells by intracellular freezing. Apparent anomalies were explained in terms of a generalized “thermal/osmotic” shock according to which the erythrocytes were subject to greater hemolysis the higher the rates of cooling and/or warming.     

Multifaceted freezing injury in human polymorphonuclear cells at high subfreezing temperatures

Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.     

A histological analysis of liver injury in freezing storage

As part of a more extensive study on the use of high subzero freezing for cryopreservation of mammalian livers we have tried to single out the effects of freezing and thawing on tissue damage. We compared the morphology of livers after freezing and thawing with what we considered an optimal high subzero cryopreservation protocol with the morphology of livers preserved under the same thermal conditions and in the same solution in a supercooled state, without freezing. The results show that while hepatocytes survive high subzero cryopreservation, detachment of endothelial cells occurs in every freezing experiment. On the other hand, the endothelial cells in livers that are not frozen are intact. This suggests that endothelial cell damage is caused by freezing and may be an important factor in high subzero freezing cryopreservation of the liver.     

Thermal shock and dilution shock as the causes of freezing injury

We suggest that during slow freezing, cellular membranes are altered by the hypertonic solutions produced. This alteration in itself does not cause membrane leakage of normally impermeant solutes but it renders the cells susceptible to solute leakage on the application of a stress, which is provided during freezing by the reduction in temperature (thermal shock) and during thawing by dilution (dilution shock).During slow freezing the effects of cooling rate changes are due to the different times available for the hypertonic solutions to affect the membrane. At a given cooling rate cryoprotective agents reduce the effect on the cells at each temperature during freezing perhaps by reducing the ionic strength. The thermal shock stress during cooling and the dilution shock during thawing thus damages the cells less. With rapid freezing, there is insufficient time for these effects to take place during cooling, which allows the cells to reach low temperatures without thermal shock damage. However, the presence of extracellular ice and the formation of intracellular ice provide hypertonic conditions that render the cells liable to dilution shock on thawing. The slower the rate of thawing of rapidly cooled cells the greater will be the damage from this dilution shock.     

Survival of tissue culture cells frozen by a two-step procedure to -196 degrees C. I. Holding temperature and time.

A two-step freezing procedure has been examined in order to separate some of the causes of damage following freezing and thawing. Different holding temperatures and times have been studied during the freezing of Chinese hamster tissue culture cells in dimethyl sulphoxide (5%, $\text{v}\text{v}$). Damage following rapid cooling to, time at, and thawing from different holding temperatures was found to increase at lower holding temperatures and at longer times. Damage on subsequent cooling from the holding temperature to ?196 °C and thawing was found to diminish at lower holding temperatures and longer times. The net result was that optimal survival from ?196 °C was obtained after 10 min at ?25 °C. Protection against the second step of cooling to ?196 °C was acquired at the holding temperature itself and was absent at ?15 °C without freezing.It seems that this technique will allow the different phases of freezing injury to be separated. These phases may include thermal shock to the holding temperature, hypertonic damage at the holding temperature and dilution shock on thawing from ?196 °C.     

Manifestations of cell damage after freezing and thawing

The nature of the primary lesions suffered by cells during freezing and thawing is unclear, although the plasma membrane is often considered the primary site for freezing injury. This study was designed to investigate the nature of damage immediately after thawing, by monitoring several functional tests of the cell and the plasma membrane. Hamster fibroblasts, human lymphocytes, and human granulocytes were subjected to a graded freeze-thaw stress in the absence of cryoprotective compound by cooling at -1 degree C/min to a temperature between -10 and -40 degrees C, and then were either warmed directly in water at 37 degrees C or cooled rapidly to -196 degrees C before rapid warming. Mitochondrial function in the cells was then assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), fluorescein diacetate (FDA), colony growth, and osmometric response in a hypertonic solution. Cells behaved as osmometers after cooling at -1 degree C/min to low temperatures at which there were no responses measured by other assays, indicating that the plasma membrane is not a primary site for injury sustained during slow cooling. These results also indicate that the FDA test does not measure membrane integrity, but reflects the permeability of the channels through which fluorescein leaves the cells. Fewer cells could respond osmotically after cooling under conditions where intracellular freezing was likely, implying that the plasma membrane is directly damaged by the conditions leading to intracellular freezing. A general model of freezing injury to nucleated mammalian cells is proposed in which disruption of the lysosomes constitutes the primary lesion in cells cooled under conditions where the cells are dehydrated at low temperatures.     

Survival of frozen mouse embryos after rapid thawing from -196 degrees C.

The effect of the rate of rewarming on the survival of 8-cell mouse embryos and blastocysts was examined. The samples were slowly cooled (0.3--0.6 degrees C/min) in 1.5 M-DMSO to temperatures between -10 and -80 degrees C before direct transfer to liquid nitrogen (-196 degrees C). Embryos survived rapid thawing (275--500 degrees C/min) only when slow cooling was terminated at relatively high subzero temperatures (-10 to -50 degrees C). The highest levels of survival in vitro of rapidly thawed 8-cell embryos were obtained after transfer to -196 degrees C from -35 and -40 degrees C (72 to 88%) and of rapidly thawed blastocysts after transfer from -25 to -50 degrees C (69 to 74%). By contrast, for embryos to survive slow thawing (8 to 20 degrees C/min) slow cooling to lower subzero temperatures (-60 degrees C and below) was required before transfer to -196 degrees C. The results indicate that embryos transferred to -196 degrees C from high subzero temperatures contain sufficient intracellular ice to damage them during slow warming but to permit survival after rapid warming. Survival of embryos after rapid dilution of DMSO at room temperature was similar to that after slow (stepwise) dilution at 0 degrees C. There was no difference between the viability of rapidly and slowly thawed embryos after transfer to pseudopregnant foster mothers. It is concluded that the behaviour of mammalian embryos subjected to the stresses of freezing and thawing is similar to that of other mammalian cells. A simpler and quicker method for the preservation of mouse embryos is described.     

Effect of varying freezing and thawing rates in experimental cryosurgery

Six different freezing/thawing programs, which varied freezing rate, duration of freezing, and thawing rates, were used to investigate the effect of these factors on cell destruction in dog skin. The range of tissue temperatures produced was from -15 to -50 degrees C. The extent of destruction was evaluated by skin biopsies 3 days after cold injury. In single, short freezing/thawing cycles, the temperature reached in the tissue was the prime factor in cell death. Longer freezing time and slow thawing were also important lethal factors which increased destruction of cells. Cooling rate, whether slow or fast, made little difference in the outcome. The experiments suggested that present-day, commonly employed cryosurgical techniques, which feature fast cooling, slow thawing, and repetition of the freeze/thaw cycle, should be modified by the use of maintenance of the tissue in the frozen state for several minutes and slow thawing. Thawing should be complete before freezing is repeated. These modifications in technique will maximize tissue destruction, an important consideration in cancer cryosurgery.     

Exposure to subfreezing temperature and a freeze-thaw cycle affect freezing tolerance of winter wheat in saturated soil

Winter wheat is sown in the autumn and harvested the following summer, necessitating the ability to survive subfreezing temperatures for several months. Autumn months in wheat-growing regions typically experience significant rainfall and several days or weeks of mild subfreezing temperatures at night, followed by above-freezing temperatures in the day. Hence, the wheat plants usually are first exposed to potentially damaging subfreezing temperatures when they have high moisture content, are growing in very wet soil, and have been exposed to freeze-thaw cycles for a period of time. These conditions are conducive to freezing stresses and plant responses that are different from those that occur under lower moisture conditions without freeze-thaw cycles. This study was conducted to investigate the impact of mild subfreezing temperature and a freeze-thaw cycle on the ability of 22 winter wheat cultivars to tolerate freezing in saturated soil. Seedlings that had been acclimated at +4°C for 5 weeks in saturated soil were frozen to potentially damaging temperatures under three treatment conditions: (1) without any subzero pre-freezing treatment; (2) with a 16-h period at ?3°C prior to freezing to potentially damaging temperatures; and (3) with a freeze-thaw cycle of ?3°C for 24 h followed by +4°C for 24 h, followed by a 16-h period at ?3°C prior to freezing to potentially damaging temperatures. In general, plants that had been exposed to the freeze-thaw cycle survived significantly more frequently than plants frozen under the other two treatments. Plants that had been exposed to 16 h at ?3° (without the freeze-thaw cycle) before freezing to potentially damaging temperatures survived significantly more frequently than plants that were frozen to potentially damaging temperatures without a subzero pre-freezing treatment. These results indicated that cold-acclimated wheat plants actively acclimate to freezing stress while exposed to mild subfreezing temperatures, and further acclimate when allowed to thaw at +4°C for 24 h. The cultivar Norstar had the lowest LT₅₀ (temperature predicted to be lethal to 50% of the plants) of the 22 cultivars when frozen with either of the subzero pre-freezing treatments, but several cultivars had lower LT₅₀ scores than Norstar when frozen without a subzero pre-freezing treatment. We conclude it may be possible to improve winterhardiness of wheat grown in saturated soil by combining the ability to effectively respond to mild subzero pre-freezing temperatures with a greater ability to withstand freezing to damaging temperatures without a subzero pre-freezing exposure.     

Freezing damage of bovine erythrocytes: Simulation using glycerol concentration changes at subzero temperatures

When a cell is frozen and thawed, it is exposed to (i) lowered temperature, (ii) increased solute concentration during freezing, and (iii) decreased solute concentration during thawing. Without actually freezing the cells, an attempt has been made to simulate physical-chemical changes to which bovine erythrocytes are exposed when frozen and thawed in glycerol solutions. Experimentally, the study consisted of suspending erythrocytes in 1, 2, or 3 glycerol at 20 °C for various times and then exposing them to each of several dilution sequences. The dilution sequences were: (i) transfer from the initial glycerol concentration at 20 °C into the same concentration at −5 °C, (ii) transfer into an increased glycerol concentration at 20 °C, (iii) transfer into an increased followed by a decreased glycerol concentration at 20 °C, (iv) transfer into an increased glycerol concentration at −5 °C, and (v) transfer into an increased followed by a decreased glycerol concentration at −5 °C. This last sequence is analogous to the exposure that cells undergo at subzero temperatures to increased solute concentration during freezing and decreased solute concentration during thawing. This dilution sequence yielded a survival pattern very similar to that obtained when bovine erythrocytes are frozen and thawed, and thus does appear to mimic freezing damage. It is concluded that a major factor in freezing damage is the extent to which a cell must shrink or swell to achieve osmotic equilibrium at subzero temperatures in partially frozen or thawed solutions.     

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

Cryopreservation of Unfertilized Mouse Oocytes: The Effect of Replacing Sodium with Choline in the Freezing Medium

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stagein vitro.Specifically, the effects of cooling to subzero temperatures, thawing rate, LN₂plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or −7.0°C in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN₂from −10, −20, or −33°C at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, −0.3 C/min, plunging at −33°C) rapid thawing by direct submersion in 30°C water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.     

Cryopreservation of isolated blood vessels

Canine saphenous veins were immersed in fetal calf serum (FCS) containing various cryoprotective agents, slowly frozen and stored for several weeks at subzero temperatures. Pharmacological investigations of frozen/thawed tissues revealed considerable attenuation of the contractile force of frozen stored veins as compared to unfrozen veins. The best recovery after thawing of frozen stored canine veins was obtained on tissues which had been frozen slowly to -70 degrees C and stored in liquid nitrogen while being immersed in FCS containing 1.8 mol/l dimethyl sulfoxide (DMSO). Though the maximum response to noradranline of helical strips prepared from these veins was diminished to about 60% the evidence suggests that there may be a very good preservation of the main biochemical properties, such as monoamine oxidase activity, endogenous prostaglandin synthesis and neuronal uptake mechanism in veins stored under these conditions. The same method of cryopreservation was applied to store samples of human veins. Comparison of the pD2 values for various agonists and of the blocking activities of various antagonists of both 5-HT receptors and alpha-adrenoceptors yielded an excellent correlation between the parameters determined on frozen/thawed and unfrozen human veins. It is concluded that freezing isolated blood vessels may be considered an effective means of preserving and storing vascular tissues for pharmacological investigations.     

Microtubules in mesophyll cells of nonacclimated and cold-acclimated spinach : visualization and responses to freezing, low temperature, and dehydration

Responses of cortical microtubules in spinach (Spinacia oleracea L. cv Bloomsdale) mesophyll cells to freezing, thawing, supercooling, and dehydration were assessed. Microtubules were visualized using a modified procedure for indirect immunofluorescence microscopy. Leaf sections of nonacclimated and cold-acclimated spinach were slowly frozen to various temperatures, fixed while frozen, and microtubules immunolabelled. Both nonacclimated and cold-acclimated cells exhibited nearly complete microtubule depolymerization after ice formation. After 1 hour thawing at 23°C, microtubules in both nonacclimated and cold-acclimated cells repolymerized. With time, however, microtubules in nonacclimated cells again depolymerized. Since microtubules in cells of leaf tissue frozen slowly are subjected to dehydration as well as subzero temperatures, these stresses were applied separately and their effects on microtubules noted. Supercooling induced microtubule depolymerization in both nonacclimated and cold-acclimated cells, but to a smaller extent than did freezing. Exposing leaf sections to solutions of sorbitol (a cell wall-penetrating osmoticum) or polyethylene glycol 10,000 (a nonpenetrating osmoticum) at room temperature caused microtubule depolymerization. The effects of low temperature and dehydration are roughly additive in producing the observed microtubule responses during freezing. Only small differences in microtubule stability were resolved between nonacclimated and cold-acclimated cells.     

Influence of simulated acid snow stress on leaf tissue of wintering herbaceous plants

Acid snow might be an environmental stress factor for wintering plants since acid precipitates are locally concentrated in snow and the period in which ice crystals are in contact with shoots might be longer than that of acid precipitates in rain. In this study, 'equilibrium' and 'prolonged' freezing tests with sulfuric acid, which simulate situations of temperature depression and chronic freezing at a subzero temperature with acid precipitate as acid snow stress, respectively, were carried out using leaf segments of cold-acclimated winter wheat. When leaf segments were frozen in the presence of sulfuric acid solution (pH 4.0, 3.0 or 2.0) by equilibrium freezing with ice seeding, the survival rate of leaf samples treated with sulfuric acid solution of pH 2.0 decreased markedly. Leaf samples after supercooling to -4 and -8 degrees C in the presence of sulfuric acid solution (pH 2.0) without ice seeding were less damaged. When leaf samples were subjected to prolonged freezing at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0), the survival rates of leaf samples exposed to sulfuric acid decreased more than those of leaf samples treated with water. On the other hand, leaf samples were less damaged by prolonged supercooling at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0). The results suggest that an acid condition (pH 2.0) in the process of extracellular freezing and/or thawing promotes freezing injury of wheat leaves.     

The response of multilamellar liposomes to freezing and thawing

The use of liposomes as a model system for investigating the mechanism of freezing injury was investigated. Modification of the liposome phospholipid and cholesterol content allows a correlation to be made between the composition of a membrane system and its response to the stresses of freezing and thawing. The data on phase transitions are contradictory in the sense that liposomes become more sensitive to freezing injury following treatments which both increase or decrease phase transition temperature. In contrast the effect of cholesterol in sensitizing membranes to the stresses of freezing and thawing appears to be more fundamental. Direct cryomicroscope observations of liposomes during slow cooling indicate that they are osmotically active at low temperatures and upon thawing morphological alterations to the membranes occur. The response of liposomes following cooling at a range of rates to ?196 °C and the effects of cryoprotective additives are similar to those observed with many cell types. These results indicate that liposomes are a valid model for investigating the biochemistry of membrane damage induced by the stresses of freezing and thawing.     

Evaluation of freezing injury and freeze-thaw injury to membranes of Saskatoon serviceberry twigs by measuring HCN release

The influence of thawing on freeze-injured Saskatoon serviceberry ( Amelanchier alnifolia Nutt.) twigs was evaluated by refreezing freeze-thawed twigs and comparing the HCN release at -5°C fro these twigs to the HCN release at -5°C from twigs that had not been thawed. An effect of thawing depended on the physiological state of the twigs, on the degree of freezing stress, or on both. Manifestation of membrane injury does not have an absolute dependence on thawing. Post-thaw temperature influences manifestation of injury, since twigs warmed to 30°C released more HCN than twigs warmed to 1°C when refrozen to -5°C. Although thawing and post-thaw conditions can influence the magnitude of membrane injury, the critical event leading to injury occurs while plants are frozen.     

Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws

Experiments were conducted to evaluate two extenders (egg-yolk Tris and egg-yolk lactose), varying concentrations of two cryopreservatives (glycerol and dimethyl sulfoxide), and rates for cooling to 5 degrees C, cooling from 5 to -100 degrees C, and warming for canine spermatozoa packaged in 0.5-ml French straws. At optimal concentrations of glycerol, egg-yolk Tris extender was superior to egg-yolk lactose in preserving spermatozoal motility. Addition of dimethyl sulfoxide, alone or in combination with glycerol in either extender, was not beneficial to spermatozoal survival after thawing. Canine spermatozoa withstood a range of cooling and equilibration times with no detrimental effect on spermatozoal motility prior to freezing. However, there were differences in spermatozoal motility immediately after thawing; these differences were variable, resulting in a cooling time by equilibration time interaction. Spermatozoal motility after thawing was best preserved by freezing in egg-yolk Tris extender containing 2-4% glycerol, using a moderate rate of cooling from 5 to -100 degrees C (-5 degrees C/min from 5 to -15 degrees C, then -20 degrees C/min from -15 to -100 degrees C). Three of 12 bitches inseminated intravaginally with semen frozen using this protocol became pregnant.     

Cryoinjury in human granulocytes and cytoplasts.

Although most isolated cells can be successfully cryopreserved, human granulocytes have little functional recovery after cryopreservation, even under optimized conditions. Cytoplasts, which are vesicles created from human granulocytes by depletion of organelles including granules and the nucleus, can carry out some of the complex functions of the parent granulocyte such as phagocytosis of bacteria, even after cryopreservation. Human granulocytes and cytoplasts were used in this comparative study of low-temperature responses to assess the relative importance of the plasma membrane and the granules in cryoinjury to human granulocytes. Boyle-van't Hoff plots of cell volume as a function of the reciprocal of osmolality showed that granulocytes and cytoplasts have similar osmometric behavior and equivalent osmotically inactive fractions. The hydraulic conductivities were also similar, indicating that the osmotic properties of the plasma membrane and cytoplasm were retained during preparation of the cytoplasts. Assessment of membrane integrity using fluorescein diacetate after graded freezing stresses showed that the low-temperature responses of cytoplasts were similar to those of human lymphocytes and hamster fibroblasts, with recoveries much higher than those of human granulocytes, particularly after post-thaw incubation at 37 degrees C. The results indicate that the plasma membrane is not the primary site of injury to granulocytes during freezing and thawing, and suggest that activation of cytoplasmic elements, such as granules, may constitute the early events in cryoinjury to human granulocytes. These studies have significance in approaches to the cryopreservation of granulocytes and other types of cells, such as platelets, with increased sensitivity to the conditions encountered during freezing and thawing.