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At least 19 recordsLinked to original sources
PubMed1990

Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.

Cryoprotectant toxicity is a fundamental obstacle to the full potential of artificial cryoprotection, yet it remains in general a poorly understood phenomenon. Unfortunately, most relevant biochemical studies to date have not met the basic criteria required for demonstrating mechanisms of toxicity. A model biochemical study of cryoprotectant toxicity was that of Baxter and Lathe, which demonstrated that alteration of a specific enzyme (fructose diphosphatase, or FDPase) was the cause of impaired glycolysis after treatment with and removal of dimethyl sulfoxide (D). FDPase alteration by D was reported to be preventable by the simultaneous presence of amides. This protection could be due to a "counteracting solute" effect similar to that employed by nature, but we find no meaningful correlation between the general protein stabilizing or destabilizing tendency of the cryoprotectant medium and its toxicity. Baxter and Lathe postulated that the effect of D arises from hydrogen bonding between D and the epsilon amino groups of surface lysine residues on FDPase, and it was found that molecules which resembled this group could block the alteration induced by D, presumably by competing with lysine residues for association with D. However, we find that the interaction between D and lysine in the presence of water is actually thermochemically repulsive, and that the presence of formamide does not affect the interaction between D and lysine, implying no useful complex formation between formamide and D. We were also unable to demonstrate that the blocking compounds consistently reduce toxicity when added to D rather than substituting for D, contrary to predictions based on complex formation between blocking compounds and D. In summary, it seems that present concepts of cryoprotectant toxicity are in need of serious revision.

Amides↗
PubMed1991

Rapid freezing of the mouse blastocyst: effects of cryoprotectants and of time and temperature of exposure to cryoprotectant before direct plunging into liquid nitrogen.

This study investigates the effects of time and temperature of exposure to a high concentration (4.5 M) of dimethyl sulfoxide (DMSO), glycerol, 1,2-propanediol (PROH), or a mixture of DMSO and glycerol (DG) in a solution containing 0.25 M sucrose, on the survival and development of rapidly frozen mouse blastocysts. Embryos had significantly (P less than 0.01) higher rates of survival and development when exposed to cryoprotectant at 0 degree C compared with room temperature. The time of exposure to cryoprotectant at either 0 degree C or room temperature before being plunged into liquid nitrogen significantly (P less than 0.01) affected the survival and development of frozen-thawed embryos. Survival and development of blastocysts in vitro and in vivo was significantly (P less than 0.05) higher when exposed at 0 degree C for 10 min to DG, DMSO and glycerol than to PROH. It is concluded that, unlike early-cleavage stage embryos, blastocysts need to be equilibrated at a low temperature (0 degree C) with high concentrations of cryoprotectant before rapid freezing. Exposure of blastocysts to 4.5 M cryoprotectant and 0.25 M sucrose at room temperature either was toxic or else markedly reduced their viability after freezing and thawing, depending on the duration of the initial exposure.

Animals↗
PubMed1991

Insights into the cryoprotective mechanism of dimethyl sulfoxide for phospholipid bilayers.

Dimethyl sulfoxide (Me2SO) is a widely used cryoprotectant for biological structures such as membranes. Despite hundreds of studies on the effects of this molecule, surprisingly little is known about its cryoprotective mechanism. This study investigates the ability of various Me2SO analogs to serve as cryoprotectants for liposomes. The data show that an increase in hydrophobicity progressively reduces the cryoprotective effect of sulfoxides. Additional experiments using phospholipid vesicles of varying composition demonstrate the Me2SO is markedly less effective on liposomes carrying a net negative charge. In fact, cryoprotection by Me2SO was virtually eliminated in vesicles composed of 30% phosphatidylserine (a negatively charged lipid). Based on these results, we suggest that the polar sulfoxide moiety of Me2SO interacts electrostatically with phospholipid membranes and that this interaction is critical for Me2SO's cryoprotective effect for membranes.

Binding Sites↗
PubMed1985

Effect of cryoprotectants on the viability and function of unfrozen human polymorphonuclear cells.

High concentrations of membrane permeable cryoprotectants are necessary to protect human polymorphonuclear leukocytes from osmotic stress injury during freezing, but there are reports that some cryoprotectants are chemically toxic. Cells were exposed to various concentrations of glycerol, dimethyl sulfoxide, or ethylene glycol for 5 min to 2 hr at 37, 22 or 0 degree C, adding or removing the cryoprotectant either slowly or rapidly. Assays included cell number recovery, membrane integrity, phagocytosis, microbicidal ability, and chemotaxis. We conclude that (1) 1 and 2 M concentrations generally are not toxic if they are added and removed slowly at 22 degrees C; (2) addition and removal of glycerol at 0 degree C was injurious even at 1 M; (3) slow addition and removal allowed better recovery than rapid addition or removal; (4) salt concentration in cryoprotectant solutions should be adjusted to isotonic on the basis of moles per liter of solution, rather than moles per kilogram of water; (5) the toxicity reported by other investigators can be largely explained by osmotic stress or dilution shock rather than chemical toxicity; and (6) ethylene glycol is the easiest cryoprotectant to add to and remove from these cells.

Blood Preservation↗
PubMed1991

New cryoprotectant for cryorefractive surgery.

Cryorefractive surgeries, keratomileusis, keratophakia, and epikeratophakia cause destruction of keratocytes, which may result in postoperative corneal haze. We examined the effects of two cryoprotectants on keratocyte survival following freeze injury. We compared the ability of CPTES and the standard cryoprotectant KM-26 to prevent keratocyte death by altering the length of time corneal tissue was exposed to the cryoprotectant. When corneal stroma was immersed in CPTES for five minutes prior to freezing, 66.5% of the keratocytes survived; when tissue was immersed in KM-26 for the same length of time, 27.5% survived (P less than .01). Immersion for one to 30 minutes in CPTES prior to freezing produced keratocyte viabilities that were 40% to 80% of those of fresh, unfrozen tissue; immersion in KM-26 produced keratocyte viabilities of 20% to 60%. We compared the ability of these cryoprotectants to reduce corneal haze following freeze injury using our rabbit model of lamellar keratoplasty. The postoperative data were comparable to those in the cell culture experiments. Based on our findings in rabbit corneas, a cryoprotective medium such as CPTES may promote cell survival and thereby speed recovery from cryorefractive procedures in humans.

Animals↗
PubMed1992

Influence of the cryoprotective agents glycerol and hydroxyethyl starch on red blood cell ATP and 2,3-diphosphoglyceric acid levels.

Because hydroxyethyl starch (HAES) is used for volume replacement therapy and as a cryoprotectant for frozen red blood cells (RBCs), this compound, in contrast to glycerol, does not require labor-intensive removal from thawed cells prior to transfusion. We here report the effect of both glycerol and HAES on the RBC organic phosphates ATP and 2,3-diphosphoglyceric acid (2,3-DPG). The CPD-A1-stabilized RBCs of 20 healthy donors (3 females, 17 males) were separately frozen in either 40% glycerol or 6% HAES, of molecular weight 200,000. ATP and 2,3-DPG concentrations were determined in CPD-A1 RBCs before addition of cryoprotectant and in cryopreserved thawed RBCs after 24 h storage at -80 degrees C (glycerol) and -196 degrees C (HAES). It appears that HAES, but not glycerol, significantly reduces ATP concentrations whereas both lead to a reduction of 2,3-DPG concentrations; this reduction was more pronounced with glycerol than with HAES. Experiments with the blood of 6 donors demonstrated that HAES affects autohemolysis by 16%, in contrast to glycerol, after which cryoprotectant autohemolysis was affected by 3.1% only. RBC recoveries were comparable using glycerol or HAES as cryoprotectants. A distinct pattern of reduction of 2,3-DPG levels by glycerol and less by HAES, and of ATP levels by HAES but not by glycerol, emerges. Our findings may be of importance if HAES is to be introduced as a convenient cryoprotectant.

2,3-Diphosphoglycerate↗
PubMed1992

[Freeze preservation of swine corneas with combinations of intra- and extracellular cryoprotective agents].

Clinically employed methods of corneal cryopreservation usually use the intracellular cryoprotectant dimethyl sulfoxide (DMSO). However, it has been demonstrated that extracellular cryoprotectants such as chondroitin sulfate (ChS) also display effective cryoprotection. The purpose of our study was to investigate the effect of combinations of intra- and extracellular cryoprotectants in corneal cryopreservation. Porcine corneas were cryopreserved in a cryopreservation medium consisting of MEM-medium containing 20% fetal calf serum and 2% chondroitin sulfate. The medium was varied by the addition of 2%, 4% and 8% DMSO. Sixty corneas were cryopreserved at -196 degrees C and thawed at 37 degrees C in a water bath. Morphometric evaluation was not performed directly after thawing but after a 24-h storage period in organ culture. Cryopreservation in medium without DMSO revealed the best results concerning endothelial cell density (2581 cells/mm2). Addition of 2% or 4% DMSO revealed no significant changes in endothelial cell density. Addition of 8% DMSO, however, resulted in a significant decrease (1312 +/- 319 cells/mm2) combined with a significantly higher amount of necrotic areas in the central corneal surface. We conclude that combining intra- and extracellular cryoprotectants does not enhance endothelial cell density after corneal cryopreservation. Higher concentrations of DMSO added to the cryopreservation medium appear to have a negative impact on endothelial cell viability.

Animals↗
PubMed1986

Cryoprotection of phosphofructokinase with organic solutes: characterization of enhanced protection in the presence of divalent cations.

Phosphofructokinase (PFK) purified from rabbit skeletal muscle is fully inactivated after being frozen in liquid nitrogen for 30 s and thawed. The addition of 500 mM trehalose, sucrose, or proline to the enzyme solution prior to freezing results in a recovery of over 70% of the original activity after thawing. Slightly less stabilization is imparted by maltose and 4-hydroxyproline whereas glucose, glycine, inositol, and glycerol at concentrations up to 500 mM are relatively ineffective at protecting PFK. With 50 mM trimethylamine-N-oxide, almost 50% of the prefreeze activity is recovered, and this same level of cryoprotection is noted at concentrations up to 500 mM. The addition of ionic zinc to enzyme-organic solute mixtures prior to freezing greatly enhances the cryoprotection imparted by all of the solutes tested. This effect is not simply due to the summation of the individual cryoprotective capacities of zinc and the organic solute because in many instances a great degree of cryoprotection is noted when each component is present at a concentration at which, by itself, it is totally ineffective. In the presence of a constant 50 mM organic solute concentration, freeze-thaw stabilization of PFK is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.6 mM) and organic solute concentration varied, the maximum cryoprotection, in most cases, is noted with less than 50 mM organic solute. At higher solute concentrations the degree of enhancement decreases such that with 500 mM organic solute the addition of zinc results in only a slight increase in protection. The addition of ionic copper, cadmium, nickel, and cobalt to trehalose-PFK solutions prior to freezing also increases the percentage of activity recovered after thawing. Magnesium, manganese, and calcium are ineffective in this respect.

Amino Acids↗
PubMed1975

Cryoprotective leaf proteins.

Leaves of frost-resistant plants contain a number of soluble proteins which are capable of protecting isolated biomembranes against inactivation during freezing. Such proteins have not been found in non-hardy summer material. The pattern of protective proteins was not uniform in hardy material of different origin and appeared to change with the season. Cryoprotective proteins were isolated by preparative gel electrophoresis. Molecular weights of different proteins as determined by their electrophoretic mobility in sodium dodecyl sulfate gels were between 10000 and 20000. Circular dichroism measurements failed to indicate helical structures. The amino acid composition of 2 active proteins revealed a high content of polar amino acids. The proteins were heat-stable. They were, on a molar basis, more than 1000 times as effective in protecting thylakoid membranes against freezing damage as low-molecular-weight cryoprotectants such as sucrose, glycerol or dimethylsulfoxide. Very low concentrations of the proteins increased cryoprotection provided by sucrose. Of a number of oligopeptides of known composition, only a few were cryoprotective. Their activity was very small as compared with that of the active proteins. The concentration of the cryoprotective proteins in hardy leaves appeared to be high enough for a significant contribution of the proteins to the frost tolerance of resistant plants.

Amino Acids↗
PubMed1987

Cryoprotection by dimethyl sulfoxide and dimethyl sulfone.

Preservation of cells and tissues at low temperatures requires the presence of effective cryoprotectants with low toxicity to which cells are relatively permeable. Two similar compounds, dimethyl sulfoxide (DMSO) and dimethyl sulfone (DMSO2), exhibit both features for cryoprotectants, yet DMSO is a very effective cryoprotectant while DMSO2 is ineffective. This anomaly was investigated by relating observations on the phase behavior of DMSO and DMSO2 in aqueous solutions to the recovery of human lymphocytes frozen in the presence of these compounds. The lack of cryoprotection in the presence of DMSO2 appears to be due to the precipitation of DMSO2 from the solution at subzero temperatures. The observation of reduced cell recovery after freezing with increasing concentrations of DMSO2 implies that cell damage is related to the amount of solid DMSO2 present. Precipitation of DMSO2 occurs both intra- and extracellularly, but it is argued that intracellular precipitation of DMSO2 is the damaging phenomenon. Cryoprotective compounds are normally selected based on the criteria of low toxicity and permeability to the plasma membrane. An additional condition, solubility, must be included for interpretation of experimental data and for development of effective protocols for cryopreservation.

Cell Survival↗
PubMed1992

Cryoprotection of purified rat kidney transamidinase by polyethylene glycol.

Polyethylene glycol is a water-soluble polymer which is widely used in the pharmaceutical, cosmetic, and chemical industries. In this study, it is shown that polyethylene glycol is an effective cryoprotectant of rat kidney transamidinase purified from both the mitochondria and cytosol. Much of the activity is lost when the purified enzyme is frozen and thawed in sodium-potassium phosphate buffer in the absence of cryoprotectants. Polyethylene glycols with molecular weights of 4000 to 10,000 were effective cryoprotectants. However, polyethylene glycols with a molecular weight of 1000 or lower inhibited the purified enzyme. A concentration of only 0.01% polyethylene glycol 4000, 8000, or 10,000 was required for complete cryoprotection. In addition to polyethylene glycol, 0.5 mM ethylenediaminetetraacetic acid was required in the phosphate buffer for complete cryoprotection. The stabilization of purified transamidinase by polyethylene glycol will facilitate characterization experiments designed to compare the properties of the mitochondrial and cytosolic isozymes.

Amidinotransferases↗
PubMed1991

Interaction between advancing ice fronts and erythrocytes. Mechanism of erythrocyte destruction upon freezing and influence of cryoprotective agents.

It can be shown theoretically and experimentally that in purely aqueous suspension, cells (as well as microsolutes) are excluded by advancing freezing fronts. This puts the cells under considerable osmotic stress and may be considered to be the major source of cell destruction upon freezing. It is also shown theoretically and experimentally that in aqueous suspensions, admixed with appropriate concentrations of a cryoprotectant (e.g., glycerol), cells are engulfed by advancing freezing fronts: Under such conditions, cells do not undergo any osmotic stress and remain undamaged when frozen. The influence of various common cryoprotectants is discussed, as is the reason why penetrating as well as nonpenetrating agents can be equally effective cryoprotective agents. The reason why leukocytes require lower cryoprotectant concentrations than erythrocytes is also elucidated.

Cell Communication↗
PubMed1989

Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa.

A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.

Acrosome↗
PubMed1992

Cryoprotection of red blood cells by a 2,3-butanediol containing mainly the levo and dextro isomers.

A 2,3-butanediol containing 96.7% (w/w) racemic mixture of the levo and dextro isomers and only 3.1% (w/w) of the meso isomer (called 2,3-butanediol 97% dl) has been used for the cryoprotection of red blood cells. The erythrocytes were cooled to -196 degrees C at rates between 2 and 3500 degrees C/min, followed by slow or rapid warming. Up to 20% (w/w) of this polyalcohol, only the classical peak of survival is observed, as with up to 20% (w/w) 1,2-propanediol or 1,3-butanediol. Twenty percent 2,3-butanediol 97% dl can protect red blood cells very efficiently. The maximum survival, of 90%, as with 20% glycerol, is a little lower than with 20% 1,2-propanediol and higher than with 20% 1,3-butanediol. Fifteen percent 2,3-butanediol protects fewer red blood cells than 15% glycerol or 1,2-propanediol, with a maximum survival of about 80%. The best cryoprotection by 30% 2,3-butanediol 97% dl is obtained at the slowest cooling and warming rates, where survival approaches 90%. After a minimum, an increase of survival is observed at the fastest cooling rates, which would correspond to complete vitrification. These rates are lower than with 30%, 1,2-propanediol or 1,3-butanediol, in agreement with the higher glass-forming tendency of 2,3-butanediol 97% dl solutions. In agreement with the remarkable physical properties of its aqueous solutions, the present experiments also suggest that 2,3-butanediol containing mainly the levo and dextro isomers could be a very useful cryoprotectant for organ cryopreservation. However, it would perhaps be better to use it in combination with other cryoprotectants, since it is a little more toxic than glycerol or 1,2-propanediol at high concentrations.

Blood Preservation↗
PubMed1989

Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. I. Mouse and hamster.

The achievement of successful pregnancies and births after in-vitro fertilization and embryo transfer of frozen-thawed human oocytes has stimulated further work on improving the established methodology. The present investigation was conducted on 1837 mouse oocytes, 1785 mouse pronuclear stage embryos, 1400 hamster oocytes and 1024 hamster pronuclear-stage embryos. In an effort to study the advantages and disadvantages of a newly introduced, 1,2-propanediol (1,2-PROH)-based system over the conventionally used dimethylsulphoxide (DMSO)-based methodology, a direct, prospective comparison between the two cryoprotectants was undertaken in a randomized trial. The combination of 1,2-PROH and DMSO potentiates their cryoprotective effect on mouse and hamster eggs. 1,2-PROH seems to improve cryopreservation in the animal system, though not significantly. Four different protocols were used to evaluate the effects of two changing experimental parameters. These were, firstly, the intermediary temperature attained before placing the cells into liquid nitrogen and, secondly, the modification of the method of adding and removing the cryoprotectant. The morphological survival rate, fertilization rate and developmental rate were significantly better in the low intermediary temperature compared with the high intermediary temperature system of cryopreservation. In addition, the rate of zona pellucida breakdown diminishes considerably in the former compared with the latter system. The 'rapid' sucrose method of cryoprotectant equilibrium and removal showed, in most groups, results which were lower than, or equal to, the traditionally used 'multiple-step', 'slow' method.

Animals↗
PubMed1989

Extraction from prawn shells of substances cryoprotective for Vibrio cholerae.

Substances cryoprotective for Vibrio cholerae were detected from prawn shells immersed in phosphate-buffered saline. This cryoprotective activity was heat resistant and sensitive to treatment with trypsin. For the exhibition of its full activity, the presence of Mg ion was indispensable. The cryoprotective activity of this substance was more active than that of other known cryoprotectants, like glycerol or serum.

Animals↗
PubMed1985

A new instant cryoprotectant for human sperm.

A new instant cryoprotectant, mainly containing culture medium, human serum albumin, and kallikrein, and its laboratory handling in the cryoprotection of semen are described. A comparative study distributed to three cryobiological centers using either egg yolk citrate medium or instant cryoprotectant showed that neither had a significantly better or worse mean percentage recovery rate. The advantage of the new instant cryoprotectant presented consists clinically in the absence of egg yolk and technically in its easy handling. Therefore, ICP is judged as an alternative to ECM in the cryopreservation of human sperm.

Citrates↗
PubMed1991

The ultrastructure of cryo-sections and intact vitrified cells--the effects of cryoprotectants and acceleration voltage on beam induced bubbling.

Chemically fixed pancreas was infiltrated with various cryoprotectants to obtain homogeneously vitrified samples upon cooling. The suitability of these samples for cryoultramicrotomy was tested. Contrast was hardly detectable initially in thin cryo-sections but increased upon irradiation, irrespective of the cryoprotectant (glycerol, propylene glycol, methanol) used. Contrast and beam damage were analyzed in vitrified thin films from collagen, phospholipid vesicles and various concentrations of glycerol. Glycerol increased the beam sensitivity of both collagen and phospholipid vesicles, but diminished the contrast between matrix and lipid vesicles or collagen fibers. The effects of glycerol as observed in thin films explain some of the effects of cryoprotectants in thin cryo-sections. To reduce beam damage in vitrified specimens two approaches are proposed. Firstly, when vitrified films are prepared, dilute suspensions should be used without cryoprotectant. In some cases, such as (thin) intact cells, the composition of the suspended material can only be marginally influenced. Then a second approach can be used involving the application of higher accelerating voltages (e.g. 300 kV). This has two advantages; the increase in mean free path-length of the electrons causes less beam damage on one hand and allows better resolution of thick specimens on the other hand. Micrographs from E. coli bacteria vitrified from suspension illustrate some of the potentials of "intermediate voltage" cryo-electron microscopy.

Animals↗