Effects of freezing and thawing on glycerol mutants of Escherichia coli

The effects of freezing and thawing on the viability of three glycerol mutants of Escherichia coli were determined when glycerol was absent or present in either the intracellular, extracellular, or both intra- and extracellular milieux.The recovery of nonglycerolated cells was related to the combination of freezing and thawing rates. Cell survival was significantly increased when subjected to the same rates of freezing and thawing.The ability of glycerol to protect against irreversible freeze-thawing injury was related to its cellular localization. Survival was markedly enhanced by extracellular glycerol and further increased by the presence of intracellular glycerol. However, intracellular glycerol alone failed to increase cell recovery. The rate of recovery, in respect to extracellular glycerol, was dependent upon both the rate of freezing and the combination of freezing and thawing rates.     

The cryopreservation of Chlamydomonas.

A cryophilic strain of the unicellular green alga Chlamydomonas, C. nivalis was found to be more resistant to the stresses both of freezing and thawing and of shrinkage and rehydration than was a mesophilic strain C. reinhardii. C. nivalis was found to have a higher degree of unsaturation of phospholipid fatty acids. Following freezing and thawing of C. reinhardii there was a direct correlation between reduction in cell viability and loss of membrane selective permeability. Activation of intracellular phospholipases occurred at an early stage of freezing injury. Attempts to cold harden C. reinhardii were unsuccessful. For C. reinhardii methanol was the only effective cryoprotectant for freezing to and thawing from ?196 °C and the effects of cooling rate upon cellular survival are presented.     

31P NMR study of changes in the intracellular pH of Escherichia coli after freezing-thawing

Influence of two types of freezing, at -196 and -50 degrees C with following thawing of Escherichia coli cells at 37 degrees C on the value of intracellular pH has been studied by means of 31P NMR spectroscopy. All the cycles of freeze-thawing have been shown to result in acidification of intracellular medium on 1.0 unit pH apart from the freezing type. The extracellular medium (pHex) was acidified too, but the degree of pHex changes after freeze-thawing depended on the freezing depth.     

The effect of cooling and warming rates on the survival of cryopreserved L-cells

A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to 10.0 °C/min) and warming rate (0.3 to 200 °C/min) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow cooling and rapid warming (~1 and 200 °C/ min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3 °C/min. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent.     

Ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

One phase of a study on cryosurvival and cryoprotection of mammalian cells, in terms of ultrastructural alteration of rough endoplasmic reticulum (RER) within rat pancreatic acinar cells, is presented. Small (2–3 mm) squares of tissue, 0.7–0.9 mm in thickness, were compared as unfrozen controls, with (w) and without (wo) glycerol pretreatment (15% $\text{v}\text{v}$ in mammalian Ringer's solution) at 0 °C and 22 °C (to regulate glycerol permeability); as well as parallel frozen-thawed samples, after combinations of slow (3.8 °C/min) freezing (SF) and rapid (38 °C/sec) freezing (RF) with either slow (1.5 °C/min) thawing (ST) or rapid (8 °C/sec) thawing (RT). Regimens compared were SFRT, SFST, RFRT, and RFST, all w and wo glycerol pretreatment at 0 °C and 22 °C. Tissue from each treatment was prepared for electron microscopic observations. The results on rates of freezing and thawing and relative cryoprotection of intracellular and extracellular glycerol under conditions described are intended to serve as a correlative basis for subsequent parallel studies on function (protein synthesis) and ultrastructure of the frozen state. They now indicate the following: (1) Cryoinjury of RER, which occurred during all treatments compared, was manifested in irregularity, dilatation, vesiculation, and altered matrix density of cisternae, and ribosomal derangement or disjunction. Least injury was shown by some disorientation and dilatation with increasing degrees of damage involving accentuation of these and other alterations. Such ultrastructural alterations to RER are not unique to cryoinjury, since they have been induced by treatments and agents other than freeze-thawing in experimental pathology. (2) Cryoinjury is unique, however, in that it can be regulated to demonstrate a spectrum of degrees of injury to cells and their organelles, immediately after cryoexposure. Controlled cryoinjury is suggested as a research tool for studies on injury, in general, on an ultrastructural-functional level. (3) Glycerol is injurious or toxic during pretreatment. Toxicity, which resembles cryoinjury, is greater during 22 ° C (intracellular) than 0 °C (extracellular) glycerol pretreatment, especially with respect to dilatation of cisternae. (4) Extra-cellular glycerol is cryoprotective during both slow and rapid freezing followed by either slow or rapid thawing, while little or no cryoprotection is afforded when glycerol is located simultaneously in the intracellular and extracellular location. (5) Rate of freezing is more important than rate of thawing as a factor in cryosurvival. Rapid freezing is more injurious than slow freezing, in the absence of glycerol or in the presence of extracellular glycerol, with slight or no differences seen as a function of thawing rate. Neither rate of freezing nor rate of thawing is of serious consequence when glycerol is intracellular. (6) Rate of thawing has importance after slow freezing, when slow thawing is more injurious than rapid, but not after rapid freezing, either in the presence or absence of extracellular glyeerol.     

Meristem clusters in cortical layer of thallus cells of the cultured red alga Palmaria palmata

For the first time somatic spotlike formations of 1.5 mm in diameter and height up to 0.5 mm were revealed in thalli of the red alga Palmaria palmata reared in an aerated stirred culture. It was determined that some cortical cells of the thallus are able to divide in the periclinal and anticlinal directions forming spots. Deep freezing and subsequent thawing of thalli showed that the cortical cells of spots were meristematic cells. In certain conditions they enabled the formation of prolifications, which germinated plantlets. These clusters of meristem tissue in the cortical layer of cells of the thallus of P. palmata, which were formed in the culture as in nature, function as growth cells facilitating growth of thalli in thick and natural “planting material” formed upon thalli fragmenting after their freezing in the winter season.     

Protein denaturation during freezing and thawing in phosphate buffer systems: monomeric and tetrameric beta-galactosidase

During freezing in sodium and potassium phosphate (NaP and KP) buffer solutions, changes in pH may impact the stability of proteins. Since the degradation pathways for the model proteins, monomeric and tetrameric beta-galactosidase (beta-gal), chosen for this study are governed by conformational changes (i.e., physical instability) as opposed to chemical transformations, we explored how the stresses of freezing and thawing alter the protein's native structure and if preservation of the native conformation during freeze-thawing is a requisite for optimal recovery of activity. During freezing in NaP buffer, a significant pH decrease from 7.0 to as low as 3.8 was observed due to the selective precipitation of the disodium phosphate; however, the pH during freezing in KP buffer only increased by at most 0.3 pH units. pH-induced inactivation was evident as seen by the lower recovery of activity when freeze-thawing in NaP buffer as compared to KP buffer for both sources of beta-gal. In addition, we investigated the effects of cooling rate and warming rate on the recovery of activity for monomeric and tetrameric beta-gal. Optimal recovery of activity for the NaP samples was obtained when the processing protocol involved a fast cool/fast warm combination, which minimizes exposure to acidic conditions and concentrated solutes. Alterations in the native secondary structure of monomeric beta-gal as measured by infrared spectroscopy were more significant when freezing and thawing in NaP buffer as opposed to KP buffer. Conformational and activity analyses indicate that pH changes during freezing in NaP buffer contribute to denaturation of beta-gal. These results suggest that proteins formulated in NaP buffer should be frozen and thawed rapidly to minimize exposure to low pH and high buffer salts.     

Freezing injury in spinach leaf tissue: Effects on water-soluble proteins, protein-sulfhydryl and water-soluble non-protein-sulfhydryl groups

Freezing of spinach leaf discs ( Spinacia aleracea L. cv. Estivato) resulted in an irreversible and parallel loss of protein-sulfhydryl (SH) and water-soluble protein. This decrease was inversely related to the increase in freezing injury as determined by the loss of electrolytes from the tissue after thawing. Loss of proteins and protein-SH occurred during freezing of the tissue and was not enhanced by thawing. The parallel decreases in content of soluble proteins and SH groups make it impossible to determine whether oxidation of protein-SH groups is the primary step in decline of protein content. During freezing the content of non-protein-SH compounds, mainly glutathione (GSH), was decreased to a lesser extent than that of protein-SH. Contrary to protein-SH, the levels of non-protein-SH declined substantially after thawing. The data indicate that GSH is not directly involved in protection of soluble proteins against freezing-induced denaturation.     

Proceedings: Freezing and freeze-drying of Spirulina platensis

A species of blue-green alga, Spirulina platensis, is extremely susceptible to freezing and drying. However, young cells grown autotrophically with a high intensity of light were resistant to freeze-thawing if the rate of temperature change in the operation was in the range of 20–50 °C/min. Several amino acids, gum arabic, and gelatin were effective in protecting cells from injury caused by freezing.In the case of drying only gum arabic and gelatin could protect cells from injury. The gum arabic-plug method of freeze-drying was shown to be the most suitable method for the maintenance of viability in this alga.The freeze-thawed or freeze-dried cells grew to form abnormally long cells after a period of long lag in the first stage of transfer.     

Ultrastructure-function correlative studies for cardiac cryopreservation. V. Absence of a correlation between electrolyte toxicity and cryoinjury in the slowly frozen,cryoprotected rat heart

Hearts were frozen to ?17 °C in the initial presence of 2.1 m DMSO. Attempts were made to prevent or minimize the consequences of an osmotic shock based on Lovelock's classical hypothesis of freezing injury. Substitution of mannitol or potassium for NaCl before freezing did not improve the results, nor did perfusion of thawed hearts with hyperosmotic perfusate. It was found that freezing and thawing resulted in a significant attenuation of coronary flow and that, as a result of this, DMSO was apparently retained within the heart after thawing. DMSO was also difficult to remove at 30 °C in the absence of prior freezing and caused a significant drop in coronary flow upon institution of DMSO washout with balanced salt solution. The blanching of freezing and thawing was also seen, in milder form, in nonfrozen hearts. For both frozen-thawed and nonfrozen hearts, the blanching was associated with DMSO washout with balanced salt solution. Flow was improved by perfusion with hyperosmotic perfusate in both nonfrozen and in frozen-thawed hearts, but the improvement was largely temporary. Evidence from earlier studies indicates that electrolyte concentrations during freezing cannot be correlated with cardiac cryoinjury, in support of the present findings. It is suggested instead that cryoprotectant toxicity may be the chief agent of injury under the conditions studied.     

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

Human red blood cells were frozen at temperatures down to ?9 °C in solutions containing sucrose, and the hemolysis on thawing was measured. This was compared with the hemolysis caused by exposing the cells to high concentrations of sucrose and then resuspending them in more dilute solutions at 4 °C. The effects of the hypertonic solutions of sucrose on potassium, sodium, and sucrose movements were also investigated. It was found that sucrose does not prevent damage to the cells by very hypertonic solutions (whether during freezing and thawing or at 4 °C) but it does reduce hemolysis of cells previously exposed to these solutions if present in the resuspension (or thawing) solution. Evidence is presented that the damaging effects of the hypertonic solutions of sucrose occurring during freezing are associated with changes in cell membrane permeability but that posthypertonic hemolysis is not primarily associated with a “loading” of the cells with extracellular solutes in the hypertonic phase. It is concluded that sucrose may reduce hemolysis of red blood cells by slow freezing and thawing by reducing colloid osmotic swelling of cells with abnormally permeable membranes.     

The significance of culture for successful cryopreservation of isolated pancreatic islets of Langerhans

It was the aim of the present study to investigate the significance of culture before and after freeze-thawing of isolated mouse pancreatic islets. To evaluate the impact of culture before freezing (5 degrees C/min; 2 M dimethyl sulfoxide), islets were frozen either directly after isolation or after 2, 4, or 7 days of culture in medium RPMI 1640. The culture period after thawing was 7 days. Islets immediately frozen exhibited virtually no (pro)insulin biosynthesis and also a severe inhibition of glucose-stimulated insulin release. The precultured (2-7 days), frozen islets synthesized and released insulin at rates comparable to those of nonfrozen, cultured islets. Studies of the effects of culture after freeze-thawing were performed after a 3-day culture period prior to freezing. The (pro)insulin biosynthetic rates did not differ between islets cultured for 0-7 days after thawing. There was an apparent increase of glucose-stimulated insulin release when the islets were cultured for more than 2 days after thawing. It may be that the decreased viability of islets frozen immediately after isolation was due to minor cell damage induced by the collagenase incubation. During culture the islets may recover and become more resistant to freeze-damage. The beneficial effect of culture after thawing may reflect the loss of damaged cells, which otherwise would influence the results of the viability tests.     

Artifactual staining of proteins on polyacrylamide gels by nitrobluetetrazolium chloride and phenazine methosulfate

Different purified proteins were shown to give purple formazan bands corresponding to the protein stain following electrophoresis on polyacrylamide gels, in the presence of nitrobluetetrazolium (NBT) and phenazine methosulfate (PMS). Both PMS and NBT are needed for formazan production which has a favorable pH at 8.5. Sulfhydryl blockers in the incubation medium inhibited this color development to different extents. While proteins with free SH groups like bovine serum albumin, ovalbumin, and urease showed this pyridine nucleotide independent artifact, nonthiol proteins, viz., bovine pancreatic ribonuclease A, and riboflavin-binding protein from chicken egg white failed to do so. The nonenzymatic formazan formation observed with different proteins could also be shown in an in vitro assay system. It is clear that the “nothing dehydrogenase” phenomenon observed in several cases may be due to the thiol group-mediated artifactual staining of proteins.     

Proceedings: Repair of injury in Salmonella anatum cells after freezing and thawing in milk

Fast freezing and slow thawing of Salmonella anatum cells in nonfat milk solids resulted in about 20% death and 50% injury of the cells surviving the treatment. Death was defined as the inability to form colonies on a nonselective plating medium [xylose-lysine-peptone agar (XLP)] after freezing and thawing. Injury was defined as the inability to form colonies on a selective plating medium (XLP with 0.2% sodium desoxycholate added). The injured cells repaired rapidly and within 2 hr at 25 °C, in the presence of 0.1% milk solids; all the injured cells regained the ability to form colonies on the selective medium. The treated cells showed a 1-hr extended lag phase of growth as compared to the unfrozen cells. Milk solids concentration in the freezing and repair menstrua influenced injury, repair of injury, and death. The repair process was affected by the pH and temperature of environment in which the injured cells were incubated. Maximum repair occurred at pH values between 6.0 and 7.4 and temperatures from 25 to 42 °C. The data suggested repair did not require the synthesis of protein, ribonucleic acid, or cell-wall mucopeptide but did require energy synthesis.     

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.     

Reduction of Cold Injury by Superoxide Dismutase and Catalase

The pathophysiology of cold injury was examined by cooling a hind leg of an anesthetized New Zealand white rabbit. A flow probe and a thermocouple were placed in the leg to be cooled to monitor the blood flow and tissue temperature. After baseline measurements, the leg was cooled with a freezing mixture up to 0°C. which was followed by rewarming. The other leg served as control. In the experimental group, liposome-bound superoxide dismutase and catalase were infused through the femoral vein 15 minutes prior to putting the freezing mixture on the leg. Salicylic acid was injected through the femoral vein at the end of some experiments to assay hydroxy radical (OH). Our results demonstrated reduction of local blood flow in cold-exposed leg, indicating development of ischemia. Creatine kinase and lactage dehydrogenase were increased during rewarming in conjunction with hydroxyl radical formation, phospholipid breakdown, and lipid peroxidation. Treatment with superoxide dismutase and catalase reduced OH formation, prevented phospholipid degradation, and decreased creatine kinase. lactate dehydrogenase. and malonaldehyde formation. These results indicate that rewarming of cooled tissue is associated with “rewarming injury” similar to “reperfusion injury”, and that oxygen-derived free radicals play a signidcant role in the pathophysiology of such injury.     

Specific cooperative induction by KLH or invertebrate hemolymphs of mouse polyclonal T-cell-mediated cytolysis

We previously showed that cells from mice primed in vivo with xenogeneic vertebrate serum can generate cytolytic T cells in vitro after boosting with the same serum. We investigated whether this would occur using any antigenic stimulus. We found in fact that (1) a number of conventional antigens were relatively inefficient at inducing cytolysis, (2) in contrast, KLH (a partially purified preparation of keyhole limpet hemocyanin) was efficient at inducing cytolysis, (3) induction in this case was KLH specific while cytolysis once induced had a polyclonal specificity for syngeneic and allogeneic tumor target cells, (4) mixtures of irradiated KLH-primed cells and normal spleen cells led to the generation of cytolytic cells, which was consistent with the existence of a first population of KLH-specific “promoter” cells triggering a second population of cells to polyclonally differentiate into cytolytic cells, and (5) not only vertebrate xenosera and KLH (a component of an invertebrate hemolymph) but also invertebrate hemolymphs themselves could specifically induce T-cell-mediated cytolysis. The question is raised in the Discussion section as to the nature and possible homology of those components present in both vertebrate sera and invertebrate hemolymphs, which are especially efficient at stimulating promoter cells. Also, from these and other results it is clear to us that indirectly KLH may do much more to the T-cell immune system than just stimulate KLH-specific cells.     

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.     

Water Loss during Contracture of Muscle

The relationship of contracture and exudation of water in frozenthawed frog muscle was studied. With maximum shortening, there was a water loss of 35 per cent of the weight of muscle. By restricting the contraction, it was demonstrated that the amount of water loss was proportional to the degree of shortening, there being no significant loss with isometric contraction. Muscle already shortened by tetanic stimulation also exuded water on subsequent freezing and thawing. The force of contraction could be reduced by depleting the muscle of calcium and it was shown that the amount of water exuded was also proportional to the tensile ability of the muscle. In a smooth muscle (anterior byssus retractor of Mytilus) which did not contract vigorously only a little water exuded. Contracture produced by caffeine was similarly associated with a loss of water. Microscopic studies revealed a disruption of the sarcomeres of the frozen-thawed muscle which contracted; glycerol-extracted and calcium-depleted muscles, which did not contract on freeze-thawing, did not show such disruption. Freezing and thawing of actomyosin caused a reversible syneresis of the protein. It is concluded that the exudation of the water is not merely due to the freezing and thawing but is also dependent on the contractile events.     

Ultrastructural studies of muscle cells and vascular endothelium immediately after freeze-thaw injury

Little is known about the ultrastructural changes which occur in vascular endothelium immediately after an in vivo freezethaw insult, although most investigators will agree that tissue viability relates directly to the degree of vascular damage. In this study an electron microscopic examination of an in vivo model for frostbite injury was initiated. The horseradish peroxidase technique was utilized to follow early alterations in capillary flow and the independent effects of hypoxia, cooling to 2 °C, supercooling, and a single freeze-thaw insult were assessed. No precipitous changes in muscle cell mitochondria or capillary endothelium were detected as a result of brief hypoxia, cooling at 2 °C, or supercooling to ?13 °C. Reducing the temperature by 1 °C/min until freezing occurred, continuing to cool for 10 min postheat of fusion, and rapidly rewarming resulted in consistent mitochondrial damage in muscle cells and marked degeneration of associated capillaries. Peroxidase injected iv prior to thawing was rarely localized in the capillaries of previously frozen muscle. Since peroxidase was found in the capillaries of unfrozen legs of the same animals, it is inferred that little or no flow occurred in most capillaries postthaw. Ultrastructural integrity of capillaries immediately after thawing may be a good index for predicting tissue loss.“In conducting the research described in this report, the investigators adhered to the ‘Guide for Laboratory Animal Facilities and Care,’ as promulgated by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council.”