An experimental study on the freezing of red blood cells with and without hydroxyethyl starch

Red blood cells were frozen in small capillaries down to ?196 °C at different linear cooling rates with or without the cryoadditive HES; the thawing rate was 3000 or 6500 °C/min. Hematocrit and hydroxyethyl starch concentration varied independently. The hemolysis of red blood cells was determined photometrically after 250-fold dilution and compared to totally hemolyzed samples. The typical U-shaped curves for hemolysis as a function of the cooling rate were obtained for all cell suspensions investigated. Relative optimum cooling rates were determined for the respective combinations of HES and hct. The results show that increasing hct causes an increased hemolysis; increased HES concentration C_HES reduces the optimum cooling rate B_opt; increased hct results in higher optimal cooling rates. The findings allow one to establish a linear correlation of the HES concentration and the optimum cooling rates when the dilution of the extracellular medium by the cell water efflux during freezing is taken into account. A comparison with results from larger volumes frozen (25 ml) shows that the established relationship between hematocrit, HES concentration, and optimal cooling rate remains valid.     

Freezing injury to erythrocytes. I. Freezing patterns and post-thaw hemolysis.

The extent of hemolysis of human red blood cells suspended in different concentrations of glycerol and frozen at various cooling rates was investigated on the basis of morphological observation in the frozen state. Hemolysis of the cells in the absence of glycerol showed a V-shaped curve in terms of cooling rates. There was 70% hemolysis at an optimal cooling rate of approximately 10³ °C/min and 100% hemolysis at all other rates tested. Morphologically, a lower than optimal cooling rate resulted in cellular shrinkage, while a higher than optimal rate resulted in the formation of intracellular ice.The cryoprotective effect of glycerol was dependent upon its concentration and on the cooling rate. Samples frozen at 10³ and 10⁴ °C/min showed freezing patterns which differed from cell to cell. The size of intraand extracellular ice particles became smaller, and there was less shrinkage or deformation of cells as the rate of cooling and concentration of glycerol were increased.There was some correlation between the morphology of frozen cells and the extent of post-thaw hemolysis, but the minimum size of intracellular ice crystals which might cause hemolysis could not be estimated. As a cryotechnique for electron microscopy, the addition of 30% glycerol and ultrarapid freezing at 10⁵ °C/min are minimum requirements for the inhibition of ice formation and the prevention of the corresponding artifacts in erythrocytes.     

The mechanism of cryohemolysis: by direct observation with the cryomicroscope and the electron microscope.

Blood films (3–8 μm thick) supported between two glass coverslips were frozen to ?20 °C. In the extracellular areas, ice cavities of the order of 0.2 μm separated by bands of dense plasma were evident when examined with the electron microscope; intracellular ice was not observed with the light microscope. Electron microscopy also showed the presence of intracellular ice particles of the order of 0.2–0.7 μm, these appeared as fine reticulations when observed with the light microscope. Upon gradual rewarming the following changes were observed: recrystallization in the extracellular matrix (?18 to ?8 °C), intracellular recrystallization (?13 to ?10 °C), transfer of water from erythrocytes to extracellular areas (?9 to ?7 °C), and melting and hemolysis (?6 to ?2 °C).Freezing of blood at ?3 °C and subsequent thawing did not cause hemolysis of the red cells. In blood frozen at ?3 °C and cooled to ?20 °C or frozen by abrupt exposure to 20 °C the erythrocytes hemolyzed in 7/16–11/16 of a second, whereas in blood frozen at ?3 °C and cooled to ?10 °C the cells hemolyzed in 5–15 sec even though the mode if lysis (i.e., uniform seepage of hemoglobin from the surface of the cell) was similar in all cases. This indicates that the presence of intracellular ice does not seem to play a major role in the injury to the erythrocytes. The mechanism of cryoinjury demonstrated by hemolysis has been discussed.     

Studies with dextran 40 in cryopreservation of blood

Whole blood from healthy donors was washed twice with phosphate-buffered saline (PBS) and then resuspended in sufficient PBS to give a final concentration of 2 × 10⁹/cells/ml. Aliquots were combined with equal volumes of the required diluents to give final dextran 40 concentrations of 0, 5, 10, 15, and 20% in PBS. Fifty-lambda samples in 50-lambda Micropets (Clay Adams) were frozen in alcohol baths at temperatures ranging from ?10 to ?80 °C. The specimens were frozen either for 1 min or 16 min, rapidly thawed, and resuspended in PBS or PBS plus dextran. Percentage of hemolysis was determined colorimetrically. Results indicate that concentraitons as low as 5% dextran exert a cryoprotective effect. Increased dextran concentration increases cryoprotection at high subzero bath temperatures (?10 ° and ?20 °C). Dextran concentrations beyond 12% have a damaging effect at low subzero bath temperatures (below ?30 °C). Based on this a two-factor hypothesis for cryopreservation is proposed. Apparent partial recovery of red blood cells without dextran or with 5% dextran during subzero storage was demonstrated.     

Freeze-thaw survival of the free-living nematode Caenorhabditis briggsae.

Approximately 75% or more of the L2 and L3 juvenile stages of the free-living nematode Caenorhabditis briggsae survived freezing and thawing without loss of fertility. Optimum survival depended upon a combination of conditions: (1) pretreatment with 5% DMSO at 0 °C for 10 min, (2) 0.2 °C per minute cooling rate from 0 to ?100 °C prior to immersion into liquid nitrogen, and (3) a 27.6 °C per minute warming rate from ?196 °C to ?10 °C. Storage at ?196 °C for more than 100 days was without effect on viability or fertility. Some of the L4 (about 50%) and adult (about 3%) stages survive the routine freeze-thaw treatment. However, there was no recovery of either embryonic stages or embryonated eggs from ?196 °C under these standard conditions. Either very fast cooling (about 545 °C/min) or fast warming (about 858 °C/min) rates diminished survival of the L2 and L3 stages drastically.Scanning electron microscopy revealed that freeze-thaw survivors with aberrant swimming behavior had cuticular defects. In juvenile forms, the altered swimming motion was lost after a molt whereas as abnormal adults grew, sinusoidal movement resumed. In the L4 and adult forms the cuticular abnormalities lowered viability and fertility. It is concluded that survival of nematodes from a freeze-thaw cycle is contingent upon establishing specific cryobiological conditions by varying aspects of the procedure that gave high recoveries of L2 and L3 stages.     

Membrane leakage of solutes after thermal shock or freezing

Washed human erythrocytes were cooled at different rates from +37 °C to 0 °C in hypertonic solutions of either NaCl (1.2 m) or of a mixture of sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). Thermal shock hemolysis was measured and the surviving cells were examined for their mass and cell water content and also for net movements of sodium, potassium, and ¹⁴C-sucrose. The results were compared with those obtained from cells in sucrose (40% $\text{w}\text{v}$) initially, cooled at different rates to ?196 °C and rapidly thawed.The cells cooled to 0 °C in NaCl (1.2 m) showed maximal hemolysis at the fastest cooling rate studied (39 °C/min). In addition in the surviving cells this cooling rate induced the greatest uptake of ¹⁴C-sucrose and increase in cell water and cell mass and also entry of sodium and loss of cell potassium. A different dependence on cooling rate was seen with the cells cooled from +37 °C to 0 °C in sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). In this solution, survival decreased both at slow and fast cooling rates correlating with the greatest uptake of cell sucrose and increase in cell water. There was extensive loss of cell potassium and uptake of sodium at all cooling rates, the cation concentrations across the cell membrane approaching unity.The cells frozen to ?196 °C at different cooling rates in sucrose (40% $\text{w}\text{v}$) initially, also showed sucrose and water entry on thawing together with a loss of cell potassium and an uptake of cell sodium. More sucrose entered the cells cooled slowly (1.8 ° C/min) than those cooled rapidly (318 ° C/min).These results show that cooling to 0 °C in hypertonic solutions (thermal shock) and freezing to ?196 °C both induce membrane leaks to sucrose as well as to sodium and potassium. These leaks are not induced by the hypertonic solutions themselves but are due to the effects of the added stress of the temperature reduction on the membranes modified by the hypertonic solutions. The effects of cooling rate are explicable in terms of the different times of exposure to the hypertonic solutions. These results indicate that the damage observed after thermal shock or slow freezing is of a similar nature.     

Cryoprotection of murine lymphocyte subpopulations using a microprocessor-controlled cooling system

Single-cell suspensions of splenic lymphocytes from 5- to 6-month-old C57BL/6 mice were cryopreserved using cooling rates ranging from ?0.25 to ?10.0 °C/min with the microprocessor-controlled cooling system developed in our laboratory. The cells (30 × 10⁶ cells/ml) were suspended in RPMI 1640 containing 10% FCS and 10% DMSO, and a total volume of 1.75 ml was frozen. Fluorescein-diacetate staining identified viable cells in unfrozen controls and frozen-thawed suspensions. Functional capacity was assessed in vitro by the incorporation of [³H]thymidine by dividing cells activated with graded concentrations of the T-lymphocyte mitogens, PHA-P and Con A, and the B-lymphocyte mitogen, LPS. High percentages of viable cells were recovered after cooling at rates ranging from ?0.5 to ?5.0 °C/min, as compared with those of unfrozen control suspensions. Incorporation of [³H]thymidine by T and B cells reached similar levels after cooling at rates ranging from ?0.25 to ?5.0 °C/min, and the percentage incorporation of [³H]thymidine as compared with that of unfrozen cells was 80–100%, except for T lymphocytes activated with PHA-P after cooling at ?5.0 °C/min. The relative response of cell suspensions to T- and B-cell mitogens, the relative mitogenic index, was unchanged from that of unfrozen controls in suspensions cooled at all rates including two (?0.25 and ?10.0 °C/min), which permitted recovery of only 55% of unfrozen cells. The importance of the constant cooling rates and rapid compensation for heat released at the phase change using the microprocessor-controlled system and of precise determinations of cellular viability and function are discussed and related to the apparent protection conferred on subpopulations of murine lymphocytes using cooling rates ranging from ? 0.25 to ?10.0 °C/min.     

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.     

Effect of dimethylsulphoxide on the functional characteristics of isolated perfused rat liver

Exposure of rat liver, perfused with 7% BSA in Krebs-Ringer bicarbonate buffer, to 1.4 m Me₂SO at 35 °C had no effect on the release of potassium from the livers, but the rate of urea synthesis fell from 0.6 to 0.1 μmol/min. Bile production also decreased and the total amount collected during perfusion was only half that produced by controls. After perfusion for 4 hr at 35 °C control livers and those exposed to Me₂SO started to release GOT into the perfusate but livers exposed to the cryoprotective compound released the enzyme at a faster rate.Exposure of livers to Me₂SO at 5 °C resulted in potassium being released at a slower rate (0.98 μmol/min) than from cooled controls (1.19 μmol/min) and urea synthesis was decreased from 0.8 to 0.2 μmol/min. Bile production also declined but, because bile flow normally ceases during hypothermia, the effect on this aspect of liver function was probably less than was found at 35 °C. Release of GOT from livers exposed to Me₂SO at 5 °C was quite different from that observed at 35 °C; the enzyme appeared in the perfusate after about 8 hr and it was present in much lower concentration than was found with appropriately cooled controls which started to release the enzyme after 6 hr.Thus, exposure of rat liver to Me₂SO at 5 °C appears to be slightly less damaging than exposure at 35 °C and it may even have a beneficial effect on some aspects of liver function in vitro.     

Optimizing conditions for cryopreservation of an insect cell line

A cell line (UM-BGE-2) derived from embryos of the cockroach Blattella germanica was frozen to ?196 °C under a variety of conditions and cell viability was assayed after warming. It was found that cell viability was affected by the cooling rate, the warming rate, the controlled cooling endpoint temperature, and the type and concentration of cryoprotectant. The best survival for cells suspended in Grace's tissue culture medium containing 1 M Me₂SO was obtained when cells were cooled at 1 °C/ min to at least ?90 °C before being placed in liquid nitrogen and warmed at more than 900 °C/min. Cultures initiated from these frozen cells produce typical growth curves and appear normal after several passages.     

Survival of the parasitic protozoan, Babesia bigemina, in blood cooled at widely different rates to -196 degrees C

Survival of the parasitic protozoan, Babesia bigemina, in blood cooled at widely different rates to ? 196°C. International Journal for Parasitology4: 169–172. The infectivity of Babesia bigemina in blood containing 2 m DMSO was tested in 99 cattle after the blood had been cooled to ? 196°C at eight rates ranging from 0·73–3070°C/min. Blood cooled at each rate was infective; 95 of the recipients became infected, the exceptions being four of the seven cattle inoculated with blood cooled at 3070° C/min. The infectivity of blood cooled at 39, 82 and 212°C/min was higher than that of blood cooled at slower or faster rates. Least depression of infectivity occurred at 82°C/min.     

The effect of cooling and warming rate on cortical cell function of glycerolized rabbit kidneys

Experiments previously reported (I. A. Jacobsen, D. E. Pegg, H. Starklint, J. Chemnitz, C. J. Hunt, P. Barfort, and M. P. Diaper, Cryobiology19, 668, 1982) suggested that rabbit kidneys permeated with 2 M glycerol are least damaged during freezing and thawing if they are cooled very slowly (1 °C/ hr). Using similar techniques of glycerolization, cooling, storage at ?80 °C, rewarming, and deglycerolization, active cell function in cortical tissue slices prepared from such kidneys has now been studied. Oxygen uptake, tissue $\text{K}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ ratio after incubation, and slice/medium PAH ratio after incubation were measured. Kidneys cooled at 3.1 °C/min and warmed at 4.2 °C/min gave poor results in the previous studies and the lowest levels of cell function in the present experiments. Kidneys cooled at 1 °C/hr exhibited degrees of slice function that were dependent on warming rate: warming at 1 °C/min was better than warming at either 1 °C/hr or c.20 °C/min. These results refine the previously drawn conclusions, (loc cit) and indicate optimal cooling and warming rates for rabbit kidneys containing 2 M glycerol, in the region of 1 °C/hr cooling and 1 °C/min warming. These rates are much lower than have hitherto been used by others for any system. Some implications of these findings are discussed.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Permeability of the 17-day fetal rat pancreas to glycerol and dimethylsulfoxide

Experimentally induced diabetes in rats can be reversed by the transplantation of several fresh or frozen-thawed fetal pancreases. An important question to both the mechanistic and practical aspects of cryobiology is the role played by the permeation of protective additives during freezing, thawing, and subsequent dilution. Answers require knowledge of the kinetics of permeation of the specific additive into the cell or tissue. In this paper, we report isotopic measurements of the rate of permeation of 2 M glycerol and 1 and 2 M dimethylsulfoxide (Me₂SO) into 17-day fetal pancreases at 0 and 22 °C. In Me₂SO, equilibrium was achieved in about 10–15 min at 0 °C and in less than 10 min at 22 °C. In glycerol, equilibrium was attained in about 60 min at 22 °C; but at 0 °C permeation was only 65% complete after 180 min. In general, Me₂SO permeated 10–30 times more rapidly than glycerol at 0 °C, and glycerol permeated about 10 times more rapidly at 22 than at 0 °C.The kinetics of permeation were more characteristic of a two-compartment than a single-compartment system. In all probability, the two compartments are the intercellular space and the intracellular space. The permeability data suggest that each compartment occupies about half the total volume.     

Influence of cell concentration,temperature, and press performance on flow characteristics and disintegration in the freeze-pressing of Saccharomyces cerevisiae with the X-press

The pressure required for initiation of flow when freeze-pressing with the X-press is related to the phase boundaries of water, particularly those between ice I and liquid even at temperatures around ?25°C and lower. Widening the orifice of the pressure chamber to diameters larger than 2.5 mm leads to lower pressures and less extensive cell disintegration. Pressing Saccharomyces cerevisiae slowly with the aid of a manual hydraulic jack at ?25°C produces a disintegration of 60–75% irrespective of cell concentration. Pressing at ?35°C shows no clear differences. Pressing more rapidly with the aid of a motor-driven hydraulic press produces a similar extent of disruption of diluted cell suspensions (5.4 mg/g) as slow pressing. However, freeze-pressing a paste of baker's yeast (270 mg/g) increases the degree of disintegration. Under these conditions the disintegration is further enhanced by a lower temperature, ?35°C, and by a high velocity of flow through the orifice, such that more than 95% of the S. cerevisiae is disrupted by one pressing at less than 2 × 10⁸ Pa. Mechanisms for flow through the X-press are suggested and discussed in relation to the phase diagram of water.     

Effect of polyphenols extracted from tamarind (Tamarindus indica L.) seed coat on pathophysiological changes and red blood cell glutathione peroxidase activity in heat-stressed broilers

The purpose of this study was to determine the effect of polyphenols extracted from the tamarind seed coat (PETSC) on glutathione peroxidase (GPx) activity, red blood cell parameters and bilirubin in heat-stressed broilers. One hundred forty-seven broilers, 18-days old were divided into two groups. In group 1, broilers were maintained at an environmental temperature of 26?±?2 °C throughout the experimental period. In group 2, the broilers were maintained at 38?±?2 °C (cyclic temperature: 26?±?2 °C; ?38?±?2 °C; and ?26?±?2 °C, and broilers were maintained at 38?±?2 °C for 6 h/ day) and received PETSC at a concentration of 0, 100, 200, 300, 400 or 500 mg/kg in their diet ad libitum. Parameters were investigated on days 1, 7, 14 and 21 of the experimental period. Results showed that GPx activity of heat-stressed broilers that received 100 mg/kg of PETSC in their diet was lower (P?<?0.05) than that in broilers fed the other concentrations. The mean total red blood cell count and hemoglobin concentration of heat-stressed broilers that received 100 mg/kg PETSC was higher (P?<?0.05) than those in broilers in group 1 and those fed the other concentrations. The mean bilirubin level in the excreta of heat-stressed broilers that received 100 mg/kg of PETSC was lower (P?<?0.05) than that in broilers that received 0, 300, 400 and 500 mg/kg of PETSC. This showed that PETSC could reduce GPx activity and bilirubin in feces, and increase red blood cell parameters in heat-stressed broilers.     

The effect of freeze rate,duration of phase transition,and warming rate on survival of frozen canine kidneys

The effect of cooling rate, warming rate, and duration of phase transition upon survival of frozen canine kidneys was investigated. In the present study, 11 kidneys out of 14 rapidly cooled (2–4 °C/min) to ?22 °C and thawed (70–110 °C/min) were viable following contralateral nephrectomy. The serum creatinine and BUN levels rose to a maximum of 8.4 and 30 mg%, respectively, on the eighth day post-contralateral nephrectomy. Average survival time was 10 days; however, two of the dogs in this group were allowed to survive, one for 3 months and one for over 2 years. Eight kidneys out of 16 slowly cooled (0.25–1.0 °C/min) and either rapidly or slowly warmed (20–30 °C/min) had function to produce small amounts of urine; however, they did not survive more than 5 days after contralateral nephrectomy.Cooling rates of 0.1 and 10 °C/min were too harmful to the kidney to have renal function after reimplantation.The minimum renal cell damage as assessed by LDH and GOT in the post-freeze perfusate was found in the 2–4 °C/min cooling rate following rapid warming (70 °–110 °C/min).Correlation of the duration of phase transition time to renal cell damage was linear for LDH and GOT (r = 0.93). This result suggests that the duration of phase transition time also is an important factor during the freezing process, affecting postthaw survival of canine kidneys.     

Visualization of freezing damage. II. Structural alterations during warming

There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from ?20 ° to ?50 °C and then rapidly cooled to ?196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above ?40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as ?45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as ?20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.     

A novel endogenous inhibitor from the hepatopancreas of the Kamchatka crab Paralithodes camtschaticus

A novel endogenous inhibitor from the hepatopancreas of the Kamchatka crab Paralithodes camtschatica has been isolated by affinity chromatography on gramicidin C-diasorb followed by gel filtration on Sephadex G-100. The inhibitor is a protein with M of 66 kDa. It has an optimum of activity at 15?C20°C, is stable in the range of 4?C40°C, and is completely inactivated upon heating to 50°C and above. For the manifestation of the inhibitory effect of the protein, the presence of NaCl in buffer at a concentration of 0.9?C20% is necessary. The inhibitor slows down the spreading of cells in vitro. The effectiveness of the inhibition of cell spreading depends on the cell type and probably the degree of malignization: the effect is most clearly pronounced in fibroblasts (normal and transformed), is less marked in epithelial cells, and is not pronounced in fibrosarcoma cells.     

Interaction of rate of latent heat removal during freezing and rates of subsequent cooling and warming on survival of the blood protozoan, Babesia rodhaini

Babesia rodhaini parasites in murine blood containing 1.5 m DMSO were frozen at two rates, as judged by the duration of the “freezing plateau”, then cooled to ?196 °C and rewarmed at two rates to detect interactions between the duration of the plateau and rates of subsequent cooling and rewarming. Infectivity tests showed that fast and slow freezing (plateau times of about 1 sec and 30 sec, respectively) had similar effects on parasite survival when cooling was at 130 °C/min and warming was at 800 °C/min. However, when either the cooling rate was increased to 3500 °C/min or the warming rate was decreased to 2.3 °C/min, fast freezing decreased parasite survival more than did slow freezing. It is suggested that fast freezing accentuated the damaging effects of fast cooling and slow warming by increasing intracellular ice formation.