Interaction between advancing ice fronts and erythrocytes

It can be shown theoretically and experimentally that in purely aqueous suspension, cells (as well as microsolutes) areexcluded 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 areengulfed 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.     

Theoretical and experimental studies on freezing point depression and vapor pressure deficit as methods to measure osmotic pressure of aqueous polyethylene glycol and bovine serum albumin solutions

For survival in adverse environments where there is drought, high salt concentration or low temperature, some plants seem to be able to synthesize biochemical compounds, including proteins, in response to changes in water activity or osmotic pressure. Measurement of the water activity or osmotic pressure of simple aqueous solutions has been based on freezing point depression or vapor pressure deficit. Measurement of the osmotic pressure of plants under water stress has been mainly based on vapor pressure deficit. However, differences have been noted for osmotic pressure values of aqueous polyethylene glycol (PEG) solutions measured by freezing point depression and vapor pressure deficit. For this paper, the physicochemical basis of freezing point depression and vapor pressure deficit were first examined theoretically and then, the osmotic pressure of aqueous ethylene glycol and of PEG solutions were measured by both freezing point depression and vapor pressure deficit in comparison with other aqueous solutions such as NaCl, KCl, CaCl(2), glucose, sucrose, raffinose, and bovine serum albumin (BSA) solutions. The results showed that: (1) freezing point depression and vapor pressure deficit share theoretically the same physicochemical basis; (2) theoretically, they are proportional to the molal concentration of the aqueous solutions to be measured; (3) in practice, the osmotic pressure levels of aqueous NaCl, KCl, CaCl(2), glucose, sucrose, and raffinose solutions increase in proportion to their molal concentrations and there is little inconsistency between those measured by freezing point depression and vapor pressure deficit; (4) the osmotic pressure levels of aqueous ethylene glycol and PEG solutions measured by freezing point depression differed from the values measured by vapor pressure deficit; (5) the osmotic pressure of aqueous BSA solution measured by freezing point depression differed slightly from that measured by vapor pressure deficit.     

Osmotic consequences of cryoprotectant permeability and its relation to the survival of frozen-thawed embryos

The freezing of a living cell involves a complex physicochemical process of heat and water transport between the cell and its surrounding medium. Embryos survive cryopreservation only in the presence of a cryoprotectant in concentrations between 1 and 2M. During the addition and dilution of a permeating cryoprotectant, the cell undergoes osmotic changes in cell size. As a consequence, if the addition or particularly the dilution are carried out inappropriately, the viability of cells can be affected. Equations which model the influx and efflux of cryoprotectants in cells can be used to calculate the optimum and most practical addition and removal method. However, the equations require the permeability coefficient of the cryoprotectant, a quantity that has only experimentally determined for a few of the developmental stages of two species.     

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.     

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.     

Evaluation of alternative cryoprotectants for preserving stallion spermatozoa

Although use of cryopreserved stallion spermatozoa is currently accepted by many breed registries, utilization of this technique remains limited due to poor fertility for some stallions. One reason for these results is osmotic stress that spermatozoa experiences when the cryoprotectant (glycerol) is added to the cells prior to freezing and removal from the cells after thawing. In an effort to minimize osmotic damage, alternative cryoprotectants, having lower molecular weights and greater membrane permeability than glycerol, were evaluated to determine their effectiveness for cryopreserving stallion spermatozoa. In the first experiment, equal molar concentrations of several amides were compared to determine if they could preserve the motility of sperm as well as glycerol. At 0.55 M concentration, addition of glycerol to a skim milk-egg yolk (SMEY) diluent resulted in higher percentages of motile sperm (61%) than methyl formamide (40%) or dimethyl formamide (38%, P<0.05), while formamide, acetamide, and methyl acetamide resulted in recovery of less than 20% motile cells (P<0.05). When methyl formamide or dimethyl formamide were increased to 0.6 or 0.9 M they resulted in percentages of motile cells (48-54%) similar to that achieved with glycerol (52%). Similarly, 0.9 M ethylene glycol also resulted in similar percentages of motile cells (43%). Replacing the glucose and fructose in the SMEY diluent with either raffinose or trehalose did not result in higher percentages of motile sperm (65 and 66%, respectively) than the control SMEY (63%). Similarly, addition of methyl cellulose also did not increase the percentages of motile spermatozoa in the samples, after cryopreservation (P>0.05). In conclusion, both methyl formamide and dimethyl formamide protected stallion spermatozoa from cryodamage as effectively as glycerol. Since these compounds permeate the plasma membrane more effectively than glycerol, they should cause less osmotic damage to stallion spermatozoa than glycerol. Therefore, these compounds may prove very effective in the cryopreservation of stallion spermatozoa, and may be particularly useful for spermatozoa from stallions that produce spermatozoa that have poor post-thaw characteristics when glycerol is used as the cryoprotectant.     

Investigating cryoinjury using simulations and experiments. 1: TF-1 cells during two-step freezing (rapid cooling interrupted with a hold time)

There is significant interest in designing a cryopreservation protocol for hematopoietic stem cells (HSC) which does not rely on dimethyl sulfoxide (Me₂SO) as a cryoprotectant. Computer simulations that describe cellular osmotic responses during cooling and warming can be used to optimize the viability of cryopreserved HSC; however, a better understanding of cellular osmotic parameters is required for these simulations. As a model for HSC, the erythroleukemic human cell line TF-1 was used in this study. Simulations, based on the osmotic properties of TF-1 cells and on the solution properties of the intra- and extracellular compartments, were used to interpret cryoinjury associated with a two-step cryopreservation protocol. Calculated intracellular supercooling was used as an indicator of cryoinjury related to intracellular ice formation. Simulations were applied to the two-step cooling protocol (rapid cooling interrupted with a hold time) for TF-1 cells in the absence of Me₂SO or other cryoprotectants and optimized by minimizing the indicator of cryoinjury. A comparison of simulations and experimental measurements of membrane integrity supports the concept that, for two-step cooling, increasing intracellular supercooling is the primary contributor to potential freezing injury due to the increase in the likelihood of intracellular ice formation. By calculating intracellular supercooling for each step separately and comparing these calculations with cell recovery data, it was demonstrated that it is not optimal simply to limit overall supercooling during two-step freezing procedures. More aptly, appropriate limitations of supercooling differ from the first step to the second step. This study also demonstrates why high cell recovery after cryopreservation could be achieved in the absence of traditional cryoprotectants.     

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.     

Cryobanking the genetic diversity in the critically endangered Iberian lynx (Lynx pardinus) from skin biopsies. Investigating the cryopreservation and culture ability of highly valuable explants and cells

Cryobanking skin samples permit preserving a maximum of genetic representation from the population biodiversity. This is a relevant aspect for threatened species, potentially menaced by an epizooty and from which it is difficult to obtain gametes. As a first step for properly cryobanking skin samples of a given species, the optimal conditions of culture and freezing have to be studied by covering a broad range of possibilities. This paper presents, for the first time, a systematic study of such conditions for the Iberian lynx (Lynx pardinus). To that end, we have analyzed twenty different culture conditions and fifteen different freezing solutions for skin explants, as well as three freezing solutions for isolated cells derived from them. The culture conditions included both two different culture strategies and several combinations of nutritional supplements and mitotic agents. For the freezing solutions, we have considered different concentrations of the permeating cryoprotectant dimethyl sulfoxide (Me₂SO) either alone (5%, 7.5%, 10%, 12.5% and 15% v/v for explants, 10% for isolated cells) or along with the non-permeating cryoprotectant sucrose (0.1 or 0.2 M). Our results have been analyzed through several quantitative parameters and show that only thawed explants cryopreserved in Me₂SO (10%) either alone or with sucrose (0.2 M) presented similar properties to those in optimal fresh cultures. In addition, for these freezing conditions, isolated thawed cells also presented high survival rates (90%) and percentages of cellular functionality (85%). These results, focussed on the most endangered felid in the world, could be also useful for other threatened/endangered species.     

Optimal concentrations of cryoprotective agents for semen from stallions that are classified 'good' or 'poor' for freezing

Cryopreserved stallion sperm displays a high degree of male-to-male variability with respect to cell viability after thawing. Animals that have semen with low viability after cryopreservation are classified as 'poor' freezers, and when post-thaw viability is high they are designated as 'good' freezers. Cryoprotective agents that are used for cryopreserving stallion sperm include glycerol, ethylene glycol, methyl formamide, and dimethylformamide, and are typically used in concentrations ranging from 1% to 4%. The aim of this study was to evaluate the osmotic stresses that stallion sperm is exposed to during cryopreservation, and to determine if sperm from 'good' and 'poor' freezers show differences in osmotic tolerance limits and in the suitability of cryoprotective agents. Concentrations of 2-3% of the above mentioned cryoprotectants with freezing extender osmolalities ranging from 580 to 895 mOsm kg(-1) showed the highest motility rates after freeze-thaw, both for 'good' and 'poor' freezers, for all cryoprotectants tested with slightly higher values for glycerol. Freeze-thawed semen from 'poor' freezers was found to have a lower percentage of progressively motile sperm compared to that of 'good' freezers. Assessment of plasma and acrosomal membrane integrity after return to isosmotic conditions revealed that cryopreserved sperm from 'poor' freezers showed lower osmotic tolerance limits as compared to sperm from 'good' freezers. Semen from 'poor' freezers that was frozen using freezing extenders supplemented with more then 2% cryoprotectant showed decreased viability and increased acrosome reaction upon return to isoosmotic conditions, whereas 'good' freezers could withstand cryoprotectant concentrations up to 3% before a decline in viability was observed.     

Protection of liposomes during dehydration or freezing.

When liposomes are subjected to dehydration or freeze-thawing, vesicle fusion and/or leakage of vesicle contents can occur. The disaccharide, trehalose and the cryoprotectant, glycerol, are known to protect vesicle integrity during dehydration and freezing respectively. Here we examine their protective abilities as a function of vesicle size and lipid composition. It is shown that fatty acyl composition, cholesterol content and, with the exception of phosphatidylglycerol, acidic lipid content do not significantly alter the retention of aqueous contents by vesicles dehydrated and rehydrated in the presence of trehalose. The susceptibility to leakage induced by both dehydration and freezing is, however, critically dependent upon vesicle size with the smallest systems (70-100 nm diameter) being most stable. The mechanism whereby trehalose protects against vesicle fusion and leakage is also discussed.     

In vitro maturation and fertilization of goat oocytes frozen at the germinal vesicle stage

The ability of frozen immature goat oocytes to undergo in vitro maturation (IVM) and fertilization (IVF) was investigated. Fully grown germinal vesicle stage (GV-stage) goat oocytes were submitted to different variables of cryopreservation: 1) exposure to propanediol before maturation but without freezing to detect the level of damage attributable to propanediol alone, 2) removal of cumulus cells to mimic damage attributable to osmotic stress during cryoprotectant exposure or freezing procedure, and 3) rapid freezing with propanediol. Maturation and fertilization rates were 82.1, 71, 65.3 and 23.7% and 71.2, 40, 58.4 and 23.1% for control, exposed, denuded and frozen oocytes, respectively. These results indicate that freezing sticto sensu (i.e., cooling and warming phases) have detrimental effects on IVM of GV-stage oocytes, whereas the reduced IVF rates of post-thaw matured oocytes are imputable to a cryoprotectant effect.     

Cryopreservation of umbilical cord blood: 2. Tolerance of CD34(+) cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing

Cryopreservation protocols for umbilical cord blood have been based on methods established for bone marrow (BM) and peripheral blood stem cells (PBSC). The a priori assumption that these methods are optimal for progenitor cells from UCB has not been investigated systematically. Optimal cryopreservation protocols utilising penetrating cryoprotectants require that a number of major factors are controlled: osmotic damage during the addition and removal of the cryoprotectant; chemical toxicity of the cryoprotectant to the target cell and the interrelationship between cryoprotectant concentration and cooling rate. We have established addition and elution protocols that prevent osmotic damage and have used these to investigate the effect of multimolar concentrations of Me(2)SO on membrane integrity and functional recovery. We have investigated the effect of freezing and thawing over a range of cooling rates and cryoprotectant concentrations. CD34(+) cells tolerate up to 60 min exposure to 25% w/w (3.2M) Me(2)SO at +2 degrees C with no significant loss in clonogenic capacity. Exposure at +20 degrees C for a similar period of time induced significant damage. CD34(+) cells showed an optimal cooling range between 1 degrees C and 2.5 degrees C/min. At or above 1 degrees C/min, increasing the Me(2)SO concentration above 10% w/w provided little extra protection. At the lowest cooling rate tested (0.1 degrees C/min), increasing the Me(2)SO concentration had a statistically significant beneficial effect on functional recovery of progenitor cells. Our findings support the conclusion that optimal recovery of CD34(+) cells requires serial addition of Me(2)SO, slow cooling at rates between 1 degrees C and 2.5 degrees C/min and serial elution of the cryoprotectant after thawing. A concentration of 10% w/w Me(2)SO is optimal. At this concentration, equilibration temperature is unlikely to be of practical importance with regard to chemical toxicity.     

Frozen/thawed platelets: importance of osmotic tolerance and platelet subpopulations.

Me2SO cryopreserved platelets circulate in vivo, reduce bleeding time, and have hemostatic properties but their functional recovery is only half that of the fresh material. Poor osmotic response is often reported as the cause of the freezing injury. Osmotic excursions on 1- and 5-day-old platelets have been studied. Platelets stored for 5 days have a lesser capability to regulate their volume particularly after an initial swelling. This is attributed to the reduction of discoid cell number, 80% vs 62% for 1-day-old and 5-day-old platelets, respectively. After freezing, hypotonic stress response is reduced from 86 to 39% for 1-day-old and 73 to 31% for 5-day-old platelets. This reduction in function is supported by a similar reduction of discoid cells from 80 to 40% for 1-day-old and 62 to 32% for 5-day-old platelets. The integrity of the cytoskeleton is critical for the osmotic response. Freezing recovery is significantly lowered in the presence of propylene glycol, which alters actin. This contrasts with the recovery of platelets treated with anti-aggregating agents. Platelets show a greater viability after freezing and thawing when PGI2 is added. It is postulated that freshly collected platelets, which are heterogeneous, contain populations of cells that are more sensitive to freezing than others. More tolerant cells remain discoid after freezing and are also less susceptible to storage lesions. Therefore, the maintenance of the integrity of the membrane and the cytoskeleton should be considered for the development of preservation methodologies.     

Urea is not a universal cryoprotectant among hibernating anurans: Evidence from the freeze-tolerant boreal chorus frog (Pseudacris maculata)

Freeze-tolerant organisms accumulate a diversity of low molecular weight compounds to combat negative effects of ice formation. Previous studies of anuran freeze tolerance have implicated urea as a cryoprotectant in the wood frog (Lithobates sylvatica). However, a cryoprotective role for urea has been identified only for wood frogs, though urea accumulation is an evolutionarily conserved mechanism for coping with osmotic stress in amphibians. To identify whether multiple solutes are involved in freezing tolerance in the boreal chorus frog (Pseudacris maculata), we examined seasonal and freezing-induced variation in several potential cryoprotectants. We further tested for a cryoprotective role for urea by comparing survival and recovery from freezing in control and urea-loaded chorus frogs. Tissue levels of glucose, urea, and glycerol did not vary significantly among seasons for heart, liver, or leg muscle. Furthermore, no changes in urea or glycerol levels were detected with exposure to freezing temperatures in these tissues. Urea-loading increased tissue urea concentrations, but failed to enhance freezing survival or facilitate recovery from freezing in chorus frogs in this study, suggesting little role for urea as a natural cryoprotectant in this species. These data suggest that urea may not universally serve as a primary cryoprotectant among freeze-tolerant, terrestrially hibernating anurans.     

Mechanisms of intracellular ice formation.

The phenomenon of intracellular freezing in cells was investigated by designing experiments with cultured mouse fibroblasts on a cryomicroscope to critically assess the current hypotheses describing the genesis of intracellular ice: (a) intracellular freezing is a result of critical undercooling; (b) the cytoplasm is nucleated through aqueous pores in the plasma membrane; and (c) intracellular freezing is a result of membrane damage caused by electrical transients at the ice interface. The experimental data did not support any of these theories, but was consistent with the hypothesis that the plasma membrane is damaged at a critical gradient in osmotic pressure across the membrane, and intracellular freezing occurs as a result of this damage. An implication of this hypothesis is that mathematical models can be used to design protocols to avoid damaging gradients in osmotic pressure, allowing new approaches to the preservation of cells, tissues, and organs by rapid cooling.     

Erythrocyte swelling after rapid dilution of cryoprotectants and its prevention

These in vitro studies on canine red blood cells confirm that cell swelling occurs after rapid dilution of Me2SO and glycerol. Cells loaded with a penetrating cryoprotectant in a medium with low Na+, high K+ composition present significantly less swelling after rapid dilution of the cryoprotectant than cells exposed to an electrolyte medium characterized by high Na+, low K+ composition. The osmotic cell stress during rapid dilution of Me2SO can be completely counteracted by the simultaneous use of the nonpenetrating sorbitol during exposure and loading. However, the addition of sorbitol is of no important benefit when glycerol is used as the intracellular cryoprotectant. This is probably due to the slower elution of glycerol. Thus utilizing a perfusion solution containing sorbitol during loading and dilution of Me2SO reduces the osmotic injury and may greatly improve the survival prospects of cryopreserved organs by avoiding "out-flow" block.     

Glycine betaine as a cryoprotectant for prokaryotes

Osmoprotectants are low molecular weight, hydrophilic, nontoxic molecules that assist a cell under osmotic stress to stabilize its concentration of internal solutes. These properties are similar to compounds used as cryoprotectants for the preservation of prokaryotic cells during freezing. This study tested the ability of a common compatible solute, glycine betaine (GB), to act as a cryoprotectant. In a series of freeze-drying studies using a variety of prokaryotes, GB performed as well, or better than, two commonly used cryoprotectants, sucrose/bovine serum albumin (S/BSA) and trehalose/dextran (T/D). GB did especially well maintaining cell viability after long-term storage (simulated equivalent of 20 years) for microorganisms like Neisseria gonorrhoeae and Streptococcus pneumoniae. GB was tested for its ability to preserve members of the genus Acidothiobacillus, a difficult genus to preserve. For two strains of Acidithiobacillus ferrooxidans that were preserved using liquid drying, GB performed as well as S/BSA. Results were more mixed for two strains of Acidithiobacillus thiooxidans; one strain could be preserved with S/BSA but not GB, the other strain gave low recoveries with both cryoprotectants. GB also proved to be a useful cryoprotectant for liquid nitrogen preservation yielding equivalent results to the cryopreservative, glycerol for halophilic archaea, and neutrophilic Fe-oxidizing bacteria. These results indicate that GB is a simple and useful cryoprotectant that works for a wide range of prokaryotic organisms under different cryopreservation regimens.