Absence Of Cryoprotective Agents Research Articles

Ultraflexible lipid vesicles allow topical absorption of cyclosporin A

Psoriasis and atopic dermatitis are widespread pathologies with a need to improve their treatment. Topical administration of cyclosporine A (CyA) could be used if targeted to the skin, thus avoiding systemic levels. Unfortunately, CyA molecular weight and lipophilicity prevent its diffusion through human skin. Four novel lipid vesicles have been prepared by different methodologies to overcome this problem. The vesicles were characterized in terms of particle size, size polydispersity, Z-potential, morphology, drug encapsulation, phospholipid content, and vesicle flexibility. Freeze-drying in presence and absence of cryoprotective agents was also performed, to guarantee long-term stability. The ability to deliver CyA into the skin was assessed using human epidermis in Franz diffusion cells and compared with the delivery of drug solutions with enhancers. The technical characteristics of four types of vesicle make them suitable to carry drugs. Moreover, these liposomal formulations were able to effectively deliver CyA in vitro into the skin. The present work introduces a promising approach for the topical treatment of skin pathologies with an immune component.

Modification of cellular membranes conveys cryoprotection to cells during rapid, non-equilibrium cryopreservation.

Rapid cooling and re-warming has been shown promising to cryopreserve living cells, which cannot be preserved by conventional slow freezing methods. However, success is limited by the cytotoxicity of highly concentrated cryoprotective agents. Recent results have shown that cryoprotective agents do not need to suppress intracellular ice crystals completely to allow for survival after cryopreservation. Cryoprotective agents like DMSO or ethylene glycol can also lead to a tolerance of cells towards intracellular ice. It is however unclear by which mechanism this tolerance is achieved. These substances are also known to modulate properties of cellular membranes. It is shown here that cryoprotective DMSO and ethylene glycol have a clear influence on the mobility of lipids in the plasma membrane of HeLa cells. To isolate changes of the properties of plasma membranes from effects on ice formation, the membrane properties were modulated in absence of cryoprotective agents. This was achieved by changing their sterol content. In cells with elevated sterol content, an immobile lipid fraction was present, similar to cells treated with DMSO and ethylene glycol. These cells showed also significantly increased plasma membrane integrity after rapid freezing and thawing in the absence of classical cryoprotective agents. However, their intracellular lysosomes, which cannot be enriched with sterols, still got ruptured. These results clearly indicate that a modulation of membrane properties can convey cryoprotection. Upon slow cooling, elevated sterol content had actually an adverse effect on the plasma membranes, which shows that this effect is specific for rapid, non-equilibrium cooling processes. Unraveling this alternative mode of action of cryoprotection should help in the directed design of novel cryoprotective agents, which might be less cytotoxic than classical, empirically-found cryoprotective agents.

Video analysis of osmotic cell response during cryopreservation

Cellular response during the freeze–thaw process strongly affects the cryopreservation outcome including cell morphology and cell viability. Cryomicroscopy was used to individually analyze the osmotic response of human pulmonary microvascular endothelial cells (HPMECs) during slow cooling (1°C/min) to −60°C and fast rewarming to 4°C (100°C/min). The ice nucleation temperature was controlled (Tn=−8°C). Different concentrations of different cryoprotectant agents, dimethyl sulfoxide, ethylene glycol, proline, ectoin, and trehalose resulted in various cell volume changes. The described methods for image processing and computer vision allows for a fully automatic and individual analysis of the osmotically driven cell response under a temporal resolution of 2frames per second. As a result, we show that in the presence of dimethyl sulfoxide or ethylene glycol cells shrink during cooling to a high degree, especially at intermediate molar concentrations in the range between 0 and 2M, while during rewarming cells swell to isotonic volumes gradually. Comparative cell vitality tests, membrane integrity, and viability tests after 24h recultivation, under these conditions show a high cell survival. In the absence of cryoprotective agents or with proline, ectoin or trehalose, osmotic shrinkage did not meet our expectations: a freeze-induced swelling was detected during cooling and an extreme swelling was observed after rewarming, which was accompanied by lower comparative cell viability. A linear correlation between the cellular membrane integrity after cryopreservation and the maximal relative cell volume was derived (R2=96). The results clearly show that it is crucial to analyze cells within a sample individually due to their individual different osmotic response.

Chondrocyte Viability in Fresh and Frozen Large Human Osteochondral Allografts: Effect of Cryoprotective Agents

Chondrocyte survival is a major goal for the effective storage and clinical performance of human osteochondral allografts. The majority of animal and human cryopreservation studies conducted so far have been performed in small osteochondral cylinders. Using human tibial plateaus as a model for large osteochondral pieces, this work sought to evaluate the cryoprotective efficiency of glycerol and dimethylsulfoxide (DMSO), and to identify cryopreservation conditions suitable for use in tissue banks. Human tibial plateaus harvested from 7 cadaveric tissue donors were incubated in the presence or absence of cryoprotective agents (CPA): 10% or 15% glycerol and 10% DMSO in a Ham F-12 nutrient mixture. Chondrocyte viability was assessed immediately after thawing, using the MTT reduction assay and a fluorescence microscopic method. The tibial plateaus frozen in the absence of CPA showed a significant decrease in chondrocyte viability. The use of CPA significantly increased chondrocyte viability compared with cartilage frozen without CPA (nearly 50% versus 80% living chondrocytes with 10% glycerol versus 10% DMSO, respectively) relative to that in fresh cartilage. In this regard, 10% DMSO was slightly more effective than either 10% or 15% glycerol, eliciting the recovery of approximately 15% relative to the living chondrocyte content in fresh cartilage. In all conditions, fluorescence microscopic studies showed that surviving chondrocytes were restricted to the superficial cartilage layer. Human tibial plateaus seemed to be a good experimental model to establish cryopreservation methods applicable to large human osteochondral pieces in tissue banks.

Subzero water transport characteristics and optimal rates of freezing rhesus monkey (Macaca mulatta) ovarian tissue

The purpose of the present study was to examine the effect of two different suprazero (room temperature +25 degrees C to +4 degrees C) cooling conditions on the measured water transport response of primate (Macaca mulatta) ovarian tissue in the presence and absence of cryoprotective agents (CPAs). Freshly collected Macaca mulatta (rhesus monkey) ovarian tissue sections were cooled at either 0.5 degrees C/min or 40 degrees C/min from 25 to 4 degrees C. A shape independent differential scanning calorimeter (DSC) technique was then used to measure the volumetric shrinkage during freezing of ovarian tissue sections at a freezing rate of 5 degrees C/min in the presence and absence of three different CPAs (0.85 M glycerol, 0.85 M dimethylsulfoxide, and 0.85 M ethylene glycol). Thus, water transport during freezing of primate ovarian tissue was obtained at eight different conditions (i.e., at four different freezing media with two different suprazero cooling conditions). The water transport response of ovarian tissue cooled rapidly from 25 to 4 degrees C was significantly different (P < 0.01) than that of slow cooled tissue, in the freezing media without CPAs and with dimethylsulfoxide. However, the differences in the measured water transport response due to the imposed suprazero cooling conditions were reduced with the addition of glycerol and ethylene glycol (statistically different with P < 0.05). By fitting a model of water transport to the experimentally obtained volumetric shrinkage data the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The best-fit parameters of water transport in primate ovarian tissue sections ranged from: L(pg) = 0.7 to 0.15 microm/min-atm and E(Lp) = 22.1 to 32.1 kcal/mol (the goodness of fit parameter, R(2) > 0.96). These parameters suggest that the "optimal rates of cryopreservation" for ovarian tissue are significantly dependent upon suprazero cooling conditions and the choice of CPA.

Development of Phosphorylated Glucomannan-Coated Chitosan Nanoparticles as Nanocarriers for Protein Delivery

The aim of the present work was to develop a new nanoparticle carrier, adapted for the oral administration of proteins and their delivery to the immune system. Chitosan and phosphorylated glucomannan were chosen as major constituents of the nanoparticles. Chitosan nanoparticles were formed by ionic gelation and then coated with glucomannan. Two different protocols were adopted for the formation of the glucomannan coating: protocol I, in which chitosan nanoparticles were isolated before their coating; protocol II, in which chitosan nanoparticles were not isolated, but coated with glucomannan in the presence of free chitosan. The results showed that, under the selected formulation conditions, the sizes of the nanoparticles ranged between 170 and 300 nm and their zeta potential values were inverted from positive to negative by the glucomannan coating. The nanoparticles prepared by the two protocols could be freeze-dried, in the presence or absence of cryoprotective agents, preserving their original characteristics. The results of the stability study evidenced the positive role of the glucomannan coating in preventing the aggregation of the nanoparticles in buffered media. Finally, the association of the inmunomodulatory protein complex P1 to the chitosan-glucomannan nanoparticles was investigated. The results showed that the association was not dependent on the chitosan: sodium tripoliphosphate ratio, but it was significantly affected by the presence of sodium phosphate in the protein structure.

Effect of glycerol and cholesterol-loaded cyclodextrin on freezing-induced water loss in bovine spermatozoa

Recent experimental data show that incubating bovine sperm with cholesterol-loaded cyclodextrin (CLC) before cryopreservation increases the percentages of motile and viable cells recovered after freezing and thawing, compared with control sperm. In the present study, we report the effect of incubating bovine sperm with CLC on the subzero water transport response and the membrane permeability parameters (reference membrane permeability (L(pg)) and activation energy (E(Lp))). Water transport data during freezing of bovine sperm cell suspensions were obtained at a cooling rate of 20 degrees C/min under three different conditions: 1. in the absence of cryoprotective agents (CPAs); 2. in the presence of 0.7 M glycerol; and 3. in the presence of 1.5 mg/ml CLC and 0.7 M glycerol. With previously published values, the bovine sperm cell was modeled as a cylinder of length 39.8 microm and radius 0.4 microm, with osmotically inactive cell volume (V(b)) of 0.61 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L(pg) and E(Lp)) were determined. The predicted best-fit permeability parameters ranged from L(pg) = 0.02 to 0.036 microm/min-atm and E(Lp) = 26.4 to 42.1 kcal/mol. These subzero water transport parameters are significantly different from the suprazero membrane permeability values (obtained in the absence of extracellular ice) reported in the literature. Calculations made of the theoretical response of bovine spermatozoa at subzero temperatures suggest that the optimal cooling rate to cryopreserve bovine spermatozoa is 45-60 degrees C/min, agreeing quite closely with experimentally determined rates of freezing bovine spermatozoa.

Effects of Freezing and Cryopreservation on the Mechanical Properties of Arteries

Cryoplasty, a freezing therapy, is being used for the treatment of restenosis in peripheral arteries. In addition, cryo-preserved arteries are increasingly used in vascular grafts. While studies are being performed to establish the efficacy of such treatments, very little is known about the postcryosurgical or postcryo-preservation changes in mechanical properties of the arteries. Few studies have examined the effect of freezing in the absence of cryoprotective agents (CPAs), and the several studies done in the presence of CPAs have given mixed results. To examine this issue further, we froze pig femoral arteries in a controlled rate freezer, using an aluminum probe, both in the presence at (-80 degrees C to 1 degrees C/min) and absence (at -20 degrees C for 2 or 5 mins) of CPA and Fetal bovine serum (FBS). Following freezing, artery samples were subjected to uniaxial tensile testing. The weights of the tissue were measured before and after freezing. Our results suggest that freezing does have an effect on stress-strain properties, particularly in the low stress region corresponding to physiological conditions. The mechanisms of this change in mechanical properties may include the loss of smooth muscle cell viability, damage to extra cellular matrix (ECM), bulk redistribution of water, or changes in alignment caused by ice crystal growth. In the case of samples frozen in the absence of CPA or FBS, the results indicated a drastic reduction in weight of the tissue suggesting the importance of bulk water redistribution as one underlying mechanism. To further examine potential mechanisms, we subjected cryopreserved vessels to the same uniaxial tests. The extent of changes in mechanical properties and bulk water redistribution was greatly attenuated; reinforcing that water movement might play a role in the changes observed with freezing.

Effects of rapid cooling on articular cartilage

In order to improve the technique and protocols of cryopreservation of articular cartilage, a study was carried out to assess the effects of rapid cooling on the intact articular cartilage. Cartilage slices with a thickness ranging from 0.2 to 0.5 mm taken from bovine metacarpal-phalangeal joints were subjected to rapid cooling by immersing them in liquid nitrogen with and without treatment of the VS55 cryoprotective agent (CPA). The ultrastructure, chondrocyte viability, swelling property, and glycosaminoglycan (GAG) content were then examined before and after cryopreservation to give qualitative and quantitative evaluation on the functional state of both chondrocytes and extracellular matrix. The transmission electron microscopy study demonstrated that damage to chondrocytes without CPA was far more pronounced than those with VS55 protection while the structure of the extracellular matrix altered little in either group. The cell viability assay showed that although the exposure to VS55 led to about 36% chondrocytes losing membrane integrity, the VS55 could provide protection to chondrocytes during rapid cooling and thawing, with approximately 51% of the cells having survived rapid cooling compared to fewer than 5% in the absence of CPA. There were no significant differences in degrees of swelling or the GAG contents of cartilage slices after cryopreservation indicating rapid freezing caused little damage to the matrix. Future research activities include searching improved CPA formulation, optimising the treatment protocol and investigating the long-term effects of rapid cooling on articular cartilage.

Suprazero cooling conditions significantly influence subzero permeability parameters of mammalian ovarian tissue

To model the cryobiological responses of cells and tissues, permeability characteristics are often measured at suprazero temperatures and the measured values are used to predict the responses at subzero temperatures. The purpose of the present study was to determine whether the rate of cooling from +25 to +4 degrees C influenced the measured water transport response of ovarian tissue at subzero temperatures in the presence or absence of cryoprotective agents (CPAs). Sections of freshly collected equine ovarian tissue were first cooled either at 40 degrees C/min or at 0.5 degrees C/min from 25 to 4 degrees C, and then cooled to subzero temperatures. A shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine ovarian tissue sections. After ice was induced to form in the extracellular fluid within the specimen, the sample was frozen from the phase change temperature to -50 degrees C at 5 degrees C/min. Replicate samples were frozen in isotonic medium alone or in medium containing 0.85 M glycerol or 0.85 M dimethylsulfoxide. The water transport response of ovarian tissue samples cooled at 40 degrees C/min from 25 to 4 degrees C was significantly different (confidence level >95%) from that of tissue samples cooled at 0.5 degrees C/min, whether in the presence or absence of CPAs. We fitted a model of water transport to the experimentally-derived volumetric shrinkage data and determined the best-fit membrane permeability parameters (L(pg) and E(Lp)) of equine ovarian tissue during freezing. Subzero water transport parameters of ovarian tissue samples cooled at 0.5 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.06 to 0.73 microm/min.atm and E(Lp) = 6.1 to 20.5 kcal/mol. The corresponding parameters of samples cooled at 40 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.04 to 0.61 microm/min.atm and E(Lp) = 8.2 to 54.2 kcal/mol. Calculations made of the theoretical response of tissue at subzero temperatures suggest that the optimal cooling rates to cryopreserve ovarian tissue are significantly dependent upon suprazero cooling conditions.

Cellular Response of Adipose Derived Passage-4 Adult Stem Cells to Freezing Stress

A differential scanning calorimeter technique was used to generate experimental data for volumetric shrinkage during cooling at 20 degrees C/min in adipose derived adult stem cells (ASCs) in the presence and absence of cryoprotective agents (CPAs). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the membrane permeability parameters of ASCs were obtained. For passage-4 (P4) ASCs, the reference hydraulic conductivity Lpg and the value of the apparent activation energy ELP were determined to be 1.2 X 10(-13) m3/Ns and 177.8 kJ/mole, respectively. We found that the addition of either glycerol or dimethylsulfoxide (DMSO) significantly decreased the value of the reference hydraulic conductivity Lpg(cpa) and the value of the apparent activation energy ELp(cpa) in P4 ASCs. The values of Lpg(cpa) in the presence of glycerol and DMSO were determined as 0.39 x 10(-13) and 0.50 X 109-13) m3/Ns, respectively, while the corresponding values of ELp(cpa) were 51.0 and 61.5 kJ/mole. Numerical simulations of water transport were then performed under a variety of cooling rates (5-100 degreesC/min) using the experimentally determined membrane permeability parameters. And finally, the simulation results were analyzed to predict the optimal rates of freezing P4 adipose derived cells in the presence and absence of CPAs.

A theoretically estimated optimal cooling rate for the cryopreservation of sperm cells from a live-bearing fish, the green swordtail Xiphophorus helleri

Sperm cryopreservation of live-bearing fishes, such as those of the genus Xiphophorus is only beginning to be studied, although these fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. To explore optimization of techniques for sperm cryopreservation of these fishes, this study measured the volumetric shrinkage response during freezing of sperm cells of Xiphophorus helleri by use of a shape-independent differential scanning calorimeter (DSC) technique. Volumetric shrinkage during freezing of X. helleri sperm cell suspensions was obtained in the presence of extracellular ice at a cooling rate of 20 °C/min in three different media: (1) Hanks’ balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol; and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of 33.3 μm in length and 0.59 μm in diameter with an osmotically inactive cell volume ( V b) of 0.6 V o, where V o is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water, L pg or L pg[cpa] and the activation energy, E Lp or E Lp[cpa]) of the Xiphophorus helleri sperm cell membrane were determined. The best-fit membrane permeability parameters at 20 °C/min in the absence of CPAs were: L pg = 0.776 × 10 −15 m 3/Ns (0.0046 μm/min atm), and E Lp = 50.1 kJ/mol (11.97 kcal/mol) ( R 2 = 0.997). The corresponding parameters in the presence of 14% glycerol were L pg[cpa] = 1.063 × 10 −15 m 3/Ns (0.0063 μm/min atm), and E Lp[cpa] = 83.81 kJ/mol (20.04 kcal/mol) ( R 2 = 0.997). The parameters in the presence of 10% DMSO were L pg[cpa] = 1.4 × 10 −15 m 3/Ns (0.0083 μm/min atm), and E Lp[cpa] = 90.96 kJ/mol (21.75 kcal/mol) ( R 2 = 0.996). Parameters obtained in this study suggested that the optimal rate of cooling for X. helleri sperm cells in the presence of CPAs ranged from 20 to 35 °C/min and were in close agreement with recently published, empirically determined optimal cooling rates.

Variation in the membrane transport properties and predicted optimal rates of freezing for spermatozoa of diploid and tetraploid Pacific oyster, Crassostrea gigas.

In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment "ball-on-stick" model with a "ball" 1.66 microm in diameter and a "stick" 41 microm in length and 0.14 microm wide. Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 microm in diameter and a "stick" 53 microm in length and 0.17 microm wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 Vo, where Vo is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: Lpg = 0.30 x 10(-15) m(3)/Ns (0.0017 microm/min-atm) and ELp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.27 x 10(-15) m(3)/Ns (0.0015 microm/min-atm) and ELp[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: Lpg = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp[cpa] = 37.6 kJ/mole (9.0 kcal/mole). The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70 degrees C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C. gigas.

Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents

In the present study a shape independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of ejaculated canine sperm cells. Volumetric shrinkage during freezing of canine sperm cell suspensions was obtained at cooling rates of 5 and 10 °C/min in the presence of extracellular ice and CPAs (6 different combinations of freezing media were used, ranging from a media with no CPAs, and those with 0.5%, 3%, and 6% glycerol and with 0.5% and 3% Me 2SO). Using previously published data, the canine sperm cell was modeled as a cylinder of length 105.7 μm and a radius of 0.32 μm with an osmotically inactive cell volume, V b, of 0.6 V o, where V o is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data the best fit membrane permeability parameters ( L pg and E Lp) were determined. The “combined best fit” membrane permeability parameters at 5 and 10 °C/min for canine sperm cells in the absence of CPAs are: L pg=0.52×10 −15 m 3/Ns (0.0029 μm/min-atm) and E Lp=64.0 kJ/mol (15.3 kcal/mol) ( R 2=0.99); and the corresponding parameters in the presence of CPAs ranged from L pg[cpa]=0.46 to 0.53 × 10 −15 m 3/Ns (0.0027–0.0031 μm/min-atm) and E Lp[cpa]=46.4–56.0 kJ/mol (11.1–13.4 kcal/mol). These parameters are significantly different than previously published parameters for canine and other mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that optimal rates of freezing canine sperm cells ranges from 10 to 30 °C/min; these theoretical cooling rates are found to be in close conformity with previously published but empirically determined optimal cooling rates.

Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry.

Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5 degrees C/min and 20 degrees C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 microm and a radius of 0.66 microm with an osmotically inactive cell volume (V(b)) of 0.6V(o), where V(o) is the isotonic cell volume. Sperm samples were collected using water-insoluble Vaseline in the artificial vagina and slow cooled at < or = 0.3 degrees C/min in an Equitainer-I from 37 degrees C to 4 degrees C. By fitting a model of water transport to the experimentally obtained DSC volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The combined best-fit parameters of water transport (at both 5 degrees C/min and 20 degrees C/min) in Kenney extender (absence of CPAs) are L(pg) = 0.02 microm min(-1) atm(-1) and E(Lp) = 32.7 kcal/mol with a goodness-of-fit parameter R(2) = 0.96, and the best-fit parameters in the lactose-EDTA extender (the CPA medium) are L(pg)[cpa] = 0.008 microm min(-1) atm(-1) and E(Lp)[cpa] = 12.1 kcal/mol with R(2) = 0.97. These parameters suggest that the optimal cooling rate for equine sperm is approximately 29 degrees C/min and is approximately 60 degrees C/min in the Kenney extender and in the lactose-EDTA extender. These rates are predicted assuming no intracellular ice formation occurs and that the approximately 5% of initial osmotically active water volume trapped inside the cells at -30 degrees C will form innocuous ice on further cooling. Numerical simulations also showed that in the lactose-EDTA extender, equine sperm trap approximately 3.4% and approximately 7.1% of the intracellular water when cooled at 20 degrees C/min and 100 degrees C/min, respectively. As an independent test of this prediction, the percentage of viable equine sperm was obtained after freezing at 6 different cooling rates (2 degrees C/min, 20 degrees C/min, 50 degrees C/min, 70 degrees C/min, 130 degrees C/min, and 200 degrees C/min) to -80 degrees C in the CPA medium. Sperm viability was essentially constant between 20 degrees C/min and 130 degrees C/min.

Stabilization of liposome bilayers to freezing and thawing: Effects of cryoprotective agents and membrane proteins

Lipid bilayer vesicles (liposomes) with and without glycoprotein incorporated into the membranes were tested for stability during freezing and thawing, in presence and absence of the cryoprotective agents (CPA) glycerol and dimethyl sulfoxide. Changes in turbidity and loss of energy transfer between fluorescent probes present in the bilayers were used to estimate membrane integrity. Freezing caused a 30 to 40% destruction of protein-free liposomes, in absence of CPA. CPA at 10 to 20% concentration prevented such losses, but at higher concentrations destabilized liposomes even without freezing. Protein-containing liposomes suffered no loss on freezing in absence or presence of CPA at moderate concentrations. Lowering of the storage temperature of frozen samples within the range of −5 to −27 °C increased the freeze damage. Slower rates of cooling and warming caused a slightly greater loss. The results are interpreted in terms of the liquid mosaic model for lipid bilayers. CPA at higher concentrations destabilize bilayers by dissolving phospholipids. At moderate concentrations, however, they prevent the damaging effect of dehydration of the lipid on freezing. Proteins appear to stabilize bilayers by providing increased hydration at the membrane surface, and by additional hydrophobic binding in the membrane interior.

Conditions for recovery of hamsters' hearts after freezing at high subzero temperatures in the absence of cryoprotective agents

In a study of the relationship between the length of time allowed for ice to form at a given freezing temperature and the ability of frozen tissues to recover after thawing, excised hamster hearts were frozen at −1.5, −2, or −3 °C, in the absence of cryoprotective agents, for times corresponding to 1 4 , 1 2 , 3 4 , or full length of the plateau of the freezing curve. As judged by the strength of the beats and the survival time after thawing, freezing at −3 °C for 11 min (full plateau) did not permit any recovery, freezing at −1.5 °C for 6.25 min (one-fourth plateau) gave a close-to-normal recovery, freezing under intermediate conditions of temperature and time gave intermediate results. Thus, it seems established that there is a lower limit of temperature at which the congelation of all the freezable water results, in a relatively short time, in the production of lethal injury. But, we still don't know if there is an upper limit of temperature at which hearts can be frozen for long times without injury (as in the case of erythrocytes).

42. On the possible effect of the rates of nucleation and of crystal growth on hemolysis when blood is frozen in the presence and absence of cryoprotective agents: B. Luyet, C. Kroener, and G. Rapatz (American Foundation for Biological Research, Madison, Wisconsin)

42. On the possible effect of the rates of nucleation and of crystal growth on hemolysis when blood is frozen in the presence and absence of cryoprotective agents: B. Luyet, C. Kroener, and G. Rapatz (American Foundation for Biological Research, Madison, Wisconsin)