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PubMed1997

Development of a protocol for the quantitative evaluation of contrast in NMR images of cryoprotective solvents in intact tissues.

In a recent study (Fyfe et al., Magn. Res. Chem. 32, 1994), chemical shift selective nuclear magnetic resonance (NMR) imaging was used to monitor the location and concentration of water and Me2SO in test samples. This study verifies that the technique can be applied to quantification of Me2SO in rat kidney and liver at temperatures commonly used for introducing cryoprotectants into tissues. A brief practical guide is provided for cryobiologists interested in using NMR imaging for such studies.

Animals↗
PubMed1997

Measurement of cryoprotective solvent penetration into intact organ tissues using high-field NMR microimaging.

Existing methods are not able to monitor accurately the penetration of cryoprotective solvents (CPS) into intact tissues. In this study, NMR imaging is shown to be a noninvasive nondestructive way to measure penetration rates and effective diffusion coefficients of Me2SO into samples of rat kidney and rat liver tissues. This new method is unique in that the measurements obtained are not averaged over the entire tissue volume but may be made at any site in the tissue. Measurements of penetration rates yield values which are similar to literature values, and the effective diffusion coefficients fall within the expected range. The images also suggest an explanation of why CPSs fail to completely protect organs from freezing damage.

Animals↗
PubMed1998

Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants.

A total of 498 porcine embryos at various stages of development collected from superovulated gilts was used to investigate cryopreservation. First, blastocysts (BL), expanded blastocysts (ExB), and hatched blastocysts (HB) were used to determine the effect of exposure to concentrated solutions of ethylene glycol as cryoprotective additives (CPAs) on embryo survival. Then, survival of other embryos after vitrification by rapid cooling was determined. Based on their development after 48 h in culture, embryos were not injured by being exposed to 2.0 M ethylene glycol (EG) for 15 min or to 2.0 M EG for 5 min and then to a solution of 8.0 M EG in 7% polyvinylpyrrolidone (PVP) for 1 min. The CPAs were removed from the embryos by diluting them with 1.7 M galactose. To vitrify the embryos, they were exposed to 2.0 M EG for 5 min and then were pipetted directly into short columns of 8.0 M EG-PVP contained within (1.25-ml plastic straws and separated from long columns of 1.7 M galactose by an air bubble. The straws were plunged directly into LN2. After the straws were warmed rapidly in a 25 degrees C water bath, the embryos were immediately mixed with galactose within the straws by shaking them vigorously to mix the contents. In sequential experiments, three methods were used to dilute the CPA solutions. Method 1: Embryos in the EG-PVP-galactose mixture were expelled from the straws and rinsed and cultured in modified CZB medium (mCZB). Method II: Embryos in the mixture were placed briefly into 1.5 M EG and then rinsed and cultured in mCZB. Method III: Embryos in the mixture were rinsed in 1.0 M EG and then in 0.5 M EG and finally rinsed with mCZB and cultured. After 48 h in culture, the respective percentages of survival of embryos vitrified as BL, ExB, or HB were: Method I, 21, 32, and 13%; Method II, 9, 40, and 24%; Method III, 35, 85, and 71%. Of 20 additional ExB vitrified embryos diluted by Method III and transferred into a recipient, four developed into live piglets; two other recipients failed to litter although one had been pregnant for 65 days. These results demonstrate that porcine embryos can be successfully cryopreserved by rapid cooling in EG-PVP and by careful dilution of the CPA after warming.

Animals↗
PubMed1998

Hydraulic conductivity (Lp) and its activation energy (Ea), cryoprotectant agent permeability (Ps) and its Ea, and reflection coefficients (sigma) for golden hamster individual pancreatic islet cell membranes.

Long-term cryopreservation of islets of Langerhans would be advantageous to a clinical islet transplantation program. Fundamental cryobiology utilizes knowledge of basic biophysical characteristics to increase the understanding of the preservation process and possibly increase survival rate. In this study several of these previously unreported characteristics have been determined for individual islet cells isolated from Golden hamster islets. Using an electronic particle counting device and a temperature control apparatus, dynamic volumetric response of individual islet cells to anisosmotic challenges of 1.5 M dimethyl sulfoxide (DMSO) and 1.5 M ethylene glycol (EG) were recorded at four temperatures (8, 22, 28, and 37 degreesC). The resulting curves were fitted using Kedem and Katchalsky equations which describe water flux and cryoprotectant agent (CPA) flux based on hydraulic conductivity (Lp), CPA permeability (Ps), and reflection coefficient (final sigma) for the membrane. For Golden hamster islet cells, Lp, Ps, and final sigma for DMSO at 22 degreesC were found to be 0.23 +/- 0.06 microm/min/atm, 0.79 +/- 0.32 x 10(-3) cm/min, and 0.55 +/- 0.37 (n = 11) (mean +/- SD), respectively. For EG at 22 degreesC, Lp equaled 0.23 +/- 0.06 microm/min/atm, Ps equaled 0.63 +/- 0.20 x 10(-3) cm/min, and final sigma was 0.75 +/- 0.17 (n = 9). Arrhenius plots (ln Lp or ln Ps versus 1/temperature (K)) were created by adding the data from the other three temperatures and the resulting linear regression yielded correlation coefficients (r) of 0.99 for all four plots (Lp and Ps for both CPAs). Activation energies (Ea) of Lp and Ps were calculated from the slopes of the regressions. The values for DMSO were found to be 12.43 and 18.34 kcal/mol for Lp and Ps (four temperatures, total n = 52), respectively. For EG, Ea of Lp was 11.69 kcal/mol and Ea of Ps was 20.35 kcal/mol (four temperatures, total n = 58).

Animals↗
PubMed1999

Membrane integrity, mitochondrial activity, ATP content, and motility of the European catfish (Silurus glanis) testicular spermatozoa after freezing with different cryoprotectants.

The extent of cellular damage was investigated after freeze-thawing of the European catfish testicular sperm with various cryoprotectants. The best protection was given by dimethylacetamide (10 and 15%) in a sucrose solution. Under these conditions, the percentage of cells with an intact membrane was high (90%), and the protection of the activity of the mitochondria was medium (47%). It was shown that the addition of dimethylacetamide largely increased the ATP content of the spermatozoa. It is suggested that this phenomenon is a decisive factor for the freezing resistance of European catfish testicular spermatozoa in the presence of dimethylacetamide (60% motility after thawing versus 90% before freezing).

Acetamides↗
PubMed2000

Rapid MR imaging of cryoprotectant permeation in an engineered dermal replacement.

Magnetic resonance (MR) imaging is a powerful technique for monitoring the permeation of cryoprotective agents (CPAs) inside tissues. However, the techniques published until now suffer from inherently long imaging times, limiting the application of these techniques to slow diffusion processes and large CPA concentrations. In this study, we present a rapid MR imaging technique based on a CHESS-FLASH scheme combined with Keyhole image acquisition. This technique can image the fast permeation of Me(2)SO solutions into freeze-dried artificial dermal replacements for concentrations down to 10% v/v. Special attention is given to evaluating the technique for quantitative analysis.

Cryopreservation↗
PubMed1986

The effects of time of equilibration with cryoprotectants at 0 degree C prior to freezing on the survival of mouse embryos frozen by the two-step method.

Mouse embryos were frozen by the two-step method after equilibration for 0.1-60 min with cryoprotectants at 0 degree C. No survival or a very low survival was obtained after equilibration for only 0.1 min. The morulae showed the highest survival rates when equilibration time was 5-30 min with 2 M DMSO, 20-30 min with 2 M glycerol, 5-10 min with 2 M ethylene glycol and 20-30 min with 2 M propylene glycol, respectively.

Animals↗
PubMed2006

Influence of freezing and low molecular weight cryoprotectants on microsomal membrane structure: a study by multiparametric fluorescent probe.

The influence of low molecular weight cryoprotectants (CPs) such as glycerol (GL), 1,2-propanediol (PD) and dimethylsulfoxide (DMSO) on the structure of rat liver microsomal membranes on the stages of equilibration and upon freezing up to -196 degrees C was studied using a multiparametric fluorescent probe of flavonol nature. It was estimated that the studied CPs have individual concentration ranges defining low amplitude of their action on biomembranes. An exceeding of these ranges strongly increases the violation of membrane native structure already at the stage of incubation with CPs, strengthening it during the freezing procedure. According to the perturbation effect on microsomal membranes the studied CPs can be arranged in a sequence: DMSO>PD>GL.

Animals↗
PubMed1988

Cryoprotection of antibody by organic solutes and organic solute/divalent cation mixtures.

Antibodies are globular glycoproteins that protect animals from microbial and toxic insult. These proteins have proven to have substantial commercial and research value but are variably susceptible to freeze-thaw damage, thus limiting their usefulness. Several carbohydrates and divalent cations were examined alone and in combination to determine whether they could protect antibody from freeze-thaw damage. The amino acid proline was also tested. Two antibodies, derived from different sources and specific for different antigens, were tested by a direct enzyme-linked immunosorbent assay (ELISA). Confirmation of antibody freeze-lability was obtained by rocket electrophoresis and radial immunodiffusion tests. Neither carbohydrate nor divalent cation alone fully protected antibody activity from freeze-thaw damage. However, several combinations protected antibody activity completely when compared to their effect on antibody maintained at room temperature. In the case of affinity-purified antibody, full protection of antibody activity relative to an untreated control was obtained. In several instances, cryoprotection of antibody by solute-divalent cation combinations was synergistic and not an additive effect of each component. Alkaline phosphatase, an enzyme typically linked to antibody for an ELISA, was not freeze-thaw labile. These results indicate that antibody function can be fully protected from damage due to freeze-thaw treatment by organic solutes in combination with divalent cations.

Alkaline Phosphatase↗
PubMed1991

The effects of glycerol on the phase behaviour of hydrated distearoylphosphatidylethanolamine and its possible relation to the mode of action of cryoprotectants.

A phase diagram for 1,2-distearoylphosphatidylethanolamine (DSPE) dispersed in glycerol/water mixtures was constructed using data obtained from differential scanning calorimetry and time-resolved X-ray diffraction measurements. The phase sequence seen on heating the lipid remains the same for samples containing up to 70 wt% glycerol. Depending on the hydration conditions, the samples are either in a metastable lamellar gel (L beta) or one or other of two possible sub-gel phases (Lc and Lc') at low temperatures. These phases convert first to a lamellar liquid crystalline (L alpha) and then to an inverted hexagonal (HII) phase on heating. On cooling, the samples revert first to the L alpha and then to the L beta phase. Although the phase sequence is preserved, marked changes are seen in the transition temperatures between the different phases. The temperature of the transition between the L alpha and the HII phases decreases strongly with increasing glycerol concentration while that of the Lc and Lc' phases to L alpha, and to a lesser extent that of the L beta to L alpha transition, increases. Substantial changes in phase behaviour are seen if the glycerol concentration is increased above 70 wt%. Under these conditions, the Lc and Lc' phases transform directly into the HII phase on heating (a similar direct transition from the L beta to the HII phase is seen above 80 wt% glycerol). An exothermic transition from the L beta phase to the Lc' phase is observed and there is also an increasing tendency for the samples to revert to the Lc or Lc' phases on storage. These changes in relative stability of the different phases are discussed in terms of a possible membrane Hofmeister effect and their relevance to the mode of action of cryoprotectants is explored.

Calorimetry, Differential Scanning↗
PubMed1984

Factors influencing renal cryopreservation. I. Effects of three vehicle solutions and the permeation kinetics of three cryoprotectants assessed with rabbit cortical slices.

A renal cortical slice model was used to assess the effects on viability of three vehicle solutions-Krebs-Henseleit (K-H), solution A, and RPS-2--at 25 degrees C. After 120 min incubation no differences in [K+]/[Na+] ratios were found. Tracer techniques were used to study the osmotic effects and permeation kinetics at 25 degrees C of three cryoprotectants (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) and the effect of the vehicle solution (K-H or RPS-2) on Me2SO kinetics. It was found that Me2SO was most permeable and ethylene glycol least, and that ethylene glycol had unusual effects which suggest that it may not act as a simple solute. Differences were found when Me2SO was introduced in K-H and RPS-2 that are believed to be related to the binding properties of Me2SO to cell constituents.

Animals↗
PubMed1984

Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in nonfrozen rabbit cortical slices.

The [K+]/[Na+] ratio of rabbit renal cortical slices was used to examine, at 25 degrees C, the effects on viability of three cryoprotectant agents (CPA) (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) in combination with three vehicle solutions (Krebs-Henseleit (K-H), solution A, and RPS-2). Viability assessment by [K+]/[Na+] for all test solutions was made after incubating the slices in modified Cross-Taggart solution (C-T). With K-H and solution A, all concentrations of ethylene glycol and glycerol resulted in lowered ratios, whereas with Me2SO, concentrations greater than 1.4 M are required to reduce [K+]/[Na+]. With RPS-2 no decrease in the ratios was found until concentrations greater than 2.8 M were reached for all three CPAs. Binding of Me2SO to albumin, studied using [14C]Me2SO, was inhibited by RPS-2 when compared to K-H. Introduction and removal of Me2SO at 10 degrees C allowed an improvement in viability, at higher Me2SO concentrations, as compared to 25 degrees C.

Animals↗
PubMed1984

Comparison of the cryoprotection of red blood cells by 1,2-propanediol and glycerol.

Red blood cells are cooled in buffered solutions containing 10, 15, 20, 30, or 35% (w/w) 1,2-propanediol or glycerol. Cell survival is measured after cooling to -196 degrees C at rates between 1 and 3500 degrees C/min, followed by rewarming rapidly, except in a few cases. At low cooling rates, where the injuries are due to solution effects, for the same (w/w) concentrations of 15 or 20% (w/w), 1,2-propanediol protects erythrocytes better than glycerol. Differences are still observed when the two cryoprotectants are compared on a mole-fraction basis. At high cooling rates the survival passes through a minimum and then increases again. For the same concentrations, the minimum occurs at much lower cooling rates with 1,2-propanediol than with glycerol, in agreement with the better glass-forming tendency of 1,2-propanediol solutions. These cooling rates almost coincide with those at which the quantity of ice crystallized begins to decrease in the corresponding solutions. Thus, survival seems to be closely related to the glass-forming tendency at the survival minimum, and at higher cooling rates. After the fastest cooling rates, the warming rates necessary to avoid damage on warming are much smaller than those necessary to avoid devitrification. Therefore, in the present experiments the survivals are not related to the stability of the wholly amorphous state. However, injury follows the presumed transition from cubic to hexagonal ice, in erythrocytes as well as in other kinds of cells.

Blood Preservation↗
PubMed1985

The influence of temperature on the function of renal cortical slices frozen in various cryoprotectants.

Renal cortical slices were frozen to various subzero temperatures after treatment with 2.1 M of one of three cryoprotectants, dimethyl sulfoxide (Me2SO), ethylene glycol, or glycerol. The effects on tissue [K+]/[Na+] of cooling to these temperatures were tested (using identical procedure times, cooling rates, and warming rates) by holding the slices at each experimental temperature for appropriate periods of time prior to rewarming. The effects of the holding time were assessed by comparison with slices which were cooled and rewarmed with no intermediate holding time. Slices treated with ethylene glycol or glycerol were found to exhibit a continuous decrease in [K+]/[Na+] with lowered temperatures, in contrast to those treated with Me2SO. Slices treated with Me2SO actually experienced a continuous increase in [K+]/[Na+] with lowered temperature (-12 to -33 degrees C). Me2SO does exhibit toxic effects at subzero temperatures. Adverse effects of holding time on viability are seen for Me2SO-treated slices at higher subzero temperatures. These effects were alleviated as the temperature is reduced, suggesting that temperature has a greater effect on survival of renal cortical tissue than Me2SO concentration. However, the toxicity observed at higher subzero temperatures is expected to be of importance, particularly for slowly cooled tissues which are exposed to these temperatures for relatively long periods of time.

Animals↗
PubMed1986

Effects of cryoprotectants on enzyme structure.

The interaction between organic cosolvents and proteins is considered, especially from the point of view of effects on protein stability. It is concluded that each protein-cosolvent system constitutes a unique situation, making generalized predictions of expected effects difficult. Two classes of cosolvents are distinguished, based on the nature of their interactions with the protein surface. The thermodynamic instability to the system introduced by the presence of the cosolvent can be accommodated (i) by preferential exclusion of the cosolvent from the vicinity of the protein, (ii) by major structural changes of the protein, or (iii) by aggregation. Polyols tend to undergo preferential exclusion due to unfavorable interactions with nonpolar surface groups, whereas monohydric alcohols and other more hydrophobic cosolvents may undergo preferential exclusion due to adverse interactions with charged groups on the protein surface. Typical cosolvent effects on the structural and catalytic properties of enzymes are illustrated with data for ribonuclease and beta-lactamase with alcohol cosolvents. The relative hydrophobicity of the cosolvent is the major determinant of the effect of a cryosolvent on the enzyme stability and properties. Thus the position of the unfolding transition in cryosolvent will be decreased more by a more nonpolar cosolvent. Different cosolvents can have significantly different effects on the catalytic and structural properties of the same enzyme. Conversely the same cosolvent can have significantly different effects on similar proteins. The number and distribution of the nonpolar and charged groups on the protein's surface probably are the major determinants of the protein contribution to the solvent-protein interaction. The large temperature dependence of the rates of protein unfolding and refolding can be beneficially utilized in cryoprotectant studies of living cells.

Alcohols↗
PubMed1986

Comparison with the theory of the kinetics and extent of ice crystallization and of the glass-forming tendency in aqueous cryoprotective solutions.

The glass-forming tendency and stability of the wholly amorphous state of various cryoprotective solutions has been studied in recent years (5-10, 20). A lot of experimental data including heats of ice crystallization at various cooling rates and devitrification temperatures have been given. In this article these data have been compared with analytical expressions using a semiempirical model. The theoretical variation of the total quantity of ice crystallized with the cooling rate fits very well with the experimental data, adjusting only one parameter. Using the same model, theoretical differential scanning calorimeter (DSC) crystallization peaks have been obtained for cooling or rewarming. The general shape, height, and width of the theoretical peaks are very similar to those of the experimental peaks. The differences are comparable to the random variations of the experimental peaks from one experiment to another. The analytical expressions obtained here could be used to study the relationship between the kinetics of ice crystallization and cell damage when ice crystallizes incompletely inside or outside the cells. These expressions have been applied to ice crystallization for applications in cryobiology. But they could also probably be used in other fields of research such as crystallization from silicates or other mineral or organic glasses.

Cryoprotective Agents↗
PubMed1988

Cryoprotection of red blood cells by 1,3-butanediol and 2,3-butanediol.

1,3-Butanediol and 2,3-butanediol have been used in buffered solutions with 20, 30, or 35% (w/w) alcohol to cool erythrocytes to -196 degrees C at different cooling rates between 1 to 3500 degrees C/min, followed by slow or rapid rewarming. 1,3-butanediol shows the same shapes of red blood cell survival curves as 1,2-propanediol. Having nearly the same physical properties, they have comparable effects on cell survival. The classical maximum of survival for intermediate cooling rates and an increase for the highest cooling rates are observed. This increase seems to be correlated with the glass-forming tendency of the solution. After the fastest cooling rates, a warming rate of 5000 degrees C/min is sufficient to avoid cell damage, but a warming rate of 100-200 degrees C/min is not. Yet both of these rates would be insufficient to avoid the intracellular ice crystallization on warming. The damage on warming after fast cooling seems once again to be correlated with the transition from cubic to hexagonal ice. For all our results, 1,3-butanediol is like a "second" 1,2-propanediol and could be useful as a cryoprotectant for preservation by total vitrification. 2,3-Butanediol always gives extremely low survival rates, though it presents good physical properties. The crystallization of its hydrate seems to be lethal on cooling or on rewarming.

Blood Preservation↗
PubMed1988

Introduction and removal of cryoprotective agents with rabbit kidneys: assessment by transplantation.

Rabbit kidneys were perfused with up to 4 M glycerol or propane-1,2-diol (propylene glycol, PG) in three vehicle solutions: one normokalemic and made hypertonic with mannitol (HP5), one hyperkalemic but without mannitol (HP6), and one hyperkalemic and with mannitol (HP7). Subsequent function was assessed by autotransplantation. Up to 3 M glycerol in HP5 was well tolerated but not in HP6 or HP7. Conversely, up to 3 M PG in HP7 was compatible with excellent post-transplant function, but the same concentration in HP5 was severely damaging. PG (4 M) in either solution was severely injurious and no kidneys survived perfusion with this concentration. Vascular resistance was well controlled by the vehicle solutions with mannitol, but it was generally higher during perfusion with the hyperkalemic HP7 compared with the normokalemic HP5. No kidneys perfused with 3 M solutions of either of the cryoprotective agents and cooled briefly to -6 degrees C without freezing had any post-transplant function, and neither did kidneys perfused with 3 M PG or 4 M glycerol tolerate slow cooling to -80 degrees C and warming. The need to optimize perfusate composition for the CPA being used is clear, and the dramatic increase in toxicity of PG when the concentration exceeds 3 M supports the suggestion that mixtures of PG and glycerol should be considered. The observation of damage at high subzero temperatures, before freezing has occurred, requires further detailed study.

Animals↗