Glass-forming tendency in the system water-dimethyl sulfoxide

The glass-forming tendency on cooling and the stability of the wholly amorphous state on warming have been previously reported for many cryoprotective solutions. However, unlike the other solutions, those of dimethyl sulfoxide (Me(2)SO) have not been studied on cooling. In this paper, the glass-forming tendency of Me(2)SO aqueous solutions has been measured for solutions containing 40, 43, 45, and 47.5% (w/w) Me(2)SO. At a concentration of 45% (w/w), the glass-forming tendency decreases in the following order: levo-2, 3-butanediol, 1,3-butanediol, 1,2-propanediol, 1,2,3-butanetriol, dimethyl sulfoxide, dimethylformamide, diethylformamide, 1, 4-butanediol, ethylene glycol, glycerol, 1,3-propanediol. New measurements have also been made on warming the Me(2)SO solutions.     

Effects of solute methoxylation on glass-forming ability and stability of vitrification solutions

The effects of replacing hydroxyl groups with methoxyl (OCH(3)) groups in the polyols ethylene glycol (EG), propylene glycol (PG), glycerol, and threitol were studied by differential scanning calorimetry (DSC) during cooling of aqueous solutions to -150 degrees C and subsequent rewarming. For 35% (w/w) PG, 40% EG, and 45% glycerol, a single substitution of a terminal hydroxyl group with a methoxyl group reduced the critical cooling rate necessary to avoid ice on cooling (vitrify) from approximately 500 to 50 degrees C/min. This reduction was approximately equivalent to increasing the parent polyol concentration by 5% (w/w). The critical warming rate calculated to avoid formation of ice on rewarming (devitrification) was also reduced by methoxyl substitution, typically by a factor of 10(4) for dilute solutions. Double methoxylation (replacement of both terminal hydroxyls) tended to result in hydrate formation, making these compounds less interesting. An exception was threitol, for which substituting both terminal hydroxyls by methoxyls reduced the critical rewarming rate of a 50% solution by a factor of 10(7) without any hydrate formation. These glass-forming and stability properties of methoxylated compounds, combined with their low viscosity, enhanced permeability, and high glass transition temperatures, make them interesting candidate cryoprotective agents for cryopreservation by vitrification or freezing. Copyright 1999 Academic Press.     

Cryoprotectants lead to phenotypic adaptation to freeze-thaw stress in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T

This study was conducted to investigate the ability of cryoprotective chemicals to induce phenotypic cryoadaptation in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T. Tolerance to negative temperature stress (freezing at -20 degrees C and thawing at 37 degrees C) was induced by pretreatment with Me(2)SO, glycerol, lactose, sucrose, and trehalose. Interestingly, Me(2)SO has a significantly greater cryoprotective effect than glycerol. Furthermore, lactose, sucrose, and trehalose, often referred to as osmotica, were shown to have greater cryoadaptive than cryoprotective properties. These results suggest that bacteria such as L. delbrueckii ssp. bulgaricus could be phenotypically adapted to freezing and thawing by an osmotic stress applied prior to freeze-thaw stress.     

Cryopreservation of Plasmodium chabaudi. II. Cooling and warming rates

Various cooling and warming rates were investigated to determine the optimum conditions for cryopreserving the intraerythrocytic stages of Plasmodium chabaudi. Infected blood, equilibrated in 10% v/v glycerol at 37 degrees C or in 15% v/v Me2SO at 0 degree C for 10 min, was cryopreserved using cooling rates between 1 and 5100 degrees C min-1. After overnight storage in liquid nitrogen the samples were warmed at 12,000 degrees C min-1. Warming rates between 1 and 12,000 degrees C min-1 were investigated using samples previously cooled at 3600 degrees C min-1. After thawing, the glycerol and Me2SO were removed by dilution in 15% v/v glucose-supplemented phosphate-buffered saline. Survival was assayed by inoculation of groups of five mice each with 10(6) infected cells and the time taken to reach a level of 2% parasitemia estimated. The optimum cooling rate was 3600 degrees C min-1 for parasites frozen using either 10% glycerol or 15% Me2SO; the pre-2% patent periods were 0.90 and 1.01 days above control values (representing survival levels of 21 and 17.5%, respectively). The optimum warming rate was 12,000 degrees C min-1; the pre-2% patent periods were 1.01 and 1.32 days above control values, respectively (18 and 10% survival), for glycerol and Me2SO. With ethanediol (5% v/v) and sucrose (15% w/v) as cryoprotectants the optimum warming rates were also 12,000 degrees C min-1 while the optimum cooling rates were 330 and 3600 degrees C min-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Water content in an engineered dermal replacement during permeation of Me2SO solutions using rapid MR imaging

The successful cryopreservation of cell and tissues typically requires the use of specialized solutions containing cryoprotective agents. At room temperature, the introduction of a cryopreservation solution can result in cell damage/death resulting from osmotic stresses and/or biochemical toxicity of the solution. For tissues, the permeation and equilibration of a cryoprotective solution throughout the tissue is important in enhancing the uniformity and consistency of the postthaw viability of the tissue. Magnetic resonance (MR) is a common nondestructive technique that can be used to quantitate the temporal and spatial composition of water and cryoprotective agents in a three-dimensional system. We have applied a recently developed rapid NMR imaging technique to quantify the transport of water in an artificial dermal replacement upon permeation of dimethyl sulfoxide (Me2SO) solutions. Results indicate that the rate of water transport is slower in the presence of Me2SO molecules. Furthermore, the transport is concentration-dependent, suggesting that Me2SO tends to retain bound water molecules in the tissue. Moreover, water transport decreases with decreasing temperature, and the presence of cells tends to increase water transport.     

Cryopreservation of Giardia lamblia with dimethyl sulfoxide using a Dewar flask

This study examined the effect of varying freezing conditions on the human intestinal parasite Giardia lamblia (Portland-1 strain) using a constant vacuum in a Dewar flask and an ethanol bath to regulate the cooling rate. The cryopreservation of the trophozoite stage was investigated. Dimethyl sulfoxide (Me2SO), the cryoprotective agent of choice, was added directly to Giardia growth medium. Me2SO toxicity assays were conducted on those concentrations used in the freezing protocol. The results of this study indicated a 6.5% (v/v) Me2SO concentration yields a 90% survival based upon organism motility. A 30.9% cell viability was obtained by freezing in medium without a cryoprotective agent. Recommendations are offered concerning alternate viability criteria.     

A comparison of sucrose,saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) sperm

Previous studies on cane toad (Bufo marinus; Bufonidae; Anura) sperm cryopreservation were extended to compare the effects of cryopreservation in established sucrose (non-ionic) diluents with cryopreservation in ionic diluents containing amphibian Ringer solutions (with and without egg-yolk). In addition, methanol was tested as a cryoprotectant for B. marinus sperm for the first time. Twenty-seven cryoprotective solutions were trialled, with each containing one of the three diluents [10% (w/v) sucrose, simplified amphibian Ringer (SAR) or SAR/egg-yolk], with one of the three cryoprotectants (Me(2)SO, glycerol, or methanol) at one of the three concentrations (10%, 15%, or 20% v/v). Sperm were collected by maceration of testes into cryoprotective solutions with post-thaw recovery assessed as the percentage of motile sperm and the degree (vigour) of motility. Percentage motility was the most sensitive measure of post-thaw recovery. The recovery of motility was lowest in Ringer (SAR) diluents and highest in sucrose diluents, with improved motility in SAR diluents when egg-yolk was added. Methanol was the poorest cryoprotectant and Me(2)SO the most effective. Methanol at high concentrations was shown to support recovery in sucrose diluent but not in SAR, although its effectiveness in SAR was improved by egg-yolk. Overall, the efficacy of diluents in supporting a high percentage of sperm recovery was in declining order: sucrose>SAR/egg-yolk>SAR diluents, and with cryoprotectants: Me(2)SO>glycerol>methanol. In conclusion, SAR offers less potential as a diluent than sucrose, presumably due to the presence of inorganic ions.     

Effect of dimethyl sulfoxide and protein concentration on the viability of two-cell mouse embryos frozen with a rapid freezing technique

Two-cell mouse embryos were frozen by direct plunging into liquid nitrogen after a 3-min exposure to solutions containing 0.25 M sucrose with 1.5, 3, or 4.5 M dimethyl sulfoxide (Me2SO), and 0, 4, 8, 16, or 32 mg/ml bovine serum albumin (BSA). In the absence of BSA, significantly more embryos were lost or damaged during freezing and thawing. Increasing the BSA concentration from 4 to 32 mg/ml had no significant effect on subsequent embryo viability in vivo or in vitro. Blastocyst formation in vitro was greater than 90% in embryos exposed to the cryoprotective solutions only. Although development to blastocysts was not significantly different to nonfrozen controls in most groups frozen in 3 and 4.5 M Me2SO (up to 92% blastocysts), it was significantly reduced when embryos were frozen in 1.5 M Me2SO (up to 65% blastocysts). The development to fetuses of embryos frozen in 3 M Me2SO (64 to 74% fetuses) was not significantly different from nonfrozen controls (68 to 79% fetuses) or embryos frozen by a conventional slow cooling method (70%). Frozen thawed two-cell embryos developed into normal adults which were able to reproduce normally. We conclude that this freezing method can efficiently cryopreserve early cleavage stage mouse embryos.     

Cryopreservation of protozoan parasites

Conventional methods for the propagation and preservation of parasites in vivo or in vitro have some limitations, including the need for labor, initial isolation and loss of strains, bacterial, and fungal contamination, and changes in the original biological and metabolic characteristics. All these disadvantages are considerably reduced by cryopreservation. In this study, we examined the effects of various freezing conditions on the survival of several protozoan parasites after cryopreservation. The viability of Entamoeba histolytica was improved by seeding (p < 0.05, chi2 test), while this was not so effective for Trichomonas vaginalis. Of six cryoprotectants examined, dimethyl sulfoxide (Me(2)SO), and glycerol showed the strongest cryoprotective effects. The optimum conditions for using Me(2)SO were a concentration of 10% with no equilibration, and those for glycerol were a concentration of 15% with equilibration for 2h. The optimum cooling rate depended on the parasite species. Trypanosoma brucei gambiense and Leishmania amazonensis were successfully cryopreserved over a wide range of cooling rates, whereas the survival rates of E. histolytica, T. vaginalis, Pentatrichomonas hominis, and Blastocystis hominis were remarkably decreased when frozen at improper rates. Unlike the cooling rate, exposure of the protozoans to a rapid thawing method produced better motility for all parasites.     

Cryopreservation of granulocytes for transfusion: Studies on human granulocyte isolation,the effect of glycerol on lysosomes,kinetics of glycerol uptake and cryopreservation with dimethyl sulfoxide and glycerol

Existing methods for the cryopreservation of granulocytes employ primarily dimethyl sulfoxide (Me₂SO) rather than glycerol as the cryoprotective additive of choice. Although Me₂SO has been demonstrated to be an effective cryoprotective additive for granulocyte preservation to yield viable cells (dye exclusion, phagocytosis, etc.), the inherent toxicity and clinical objections of Me₂SO as a cryoprotective additive for granulocyte preservation preclude its extensive and routine use in patients. Therefore, glycerol, with its important advantage of nontoxicity, has been investigated for its potential usefulness as a cryoprotective additive for preserving human granulocytes for transfusion.Granulocyte preparations were isolated from impure leukocyte concentrates obtained from the buffy coats of human whole blood. Studies on the isolation and purification of the granulocytes involved separation by sedimentation with dextran, removal of red cells by hypotonic shock with water, resuspension with Plasmatein and further purification by centrifugation. Intact viable granulocytes were obtained with a purity in excess of 90%.Lysosomes were studied as indicators of cryoinjury in granulocytes using β-glucuronidase as the key marker enzyme. This enzyme has been characterized as a sensitive indicator of damage to lysosomes and a direct linear relationship has been established between damage to granulocytes by freezing and amount of lysosomal enzyme released. Addition or presence of the cryoprotectant, glycerol, did not appear to have any adverse effect on lysosomes of intact granulocytes.Studies on the permeation kinetics of glycerol in granulocytes indicated that the additive was freely permeable and did not cause any potentially damaging osmotic changes in cell volume. Granulocytes in various concentrations of glycerol were then frozen at slow, moderate, and rapid cooling rates. Based on the small amount of β-glucuronidase released, good preservation of granulocyte lysosomes has been obtained with a slow cooling rate of 5 °C/min and a concentration of 15% glycerol. Further studies now are necessary to define those conditions of cooling rate and glycerol concentration required to develop a simple method for optimal preservation of granulocytes based on additional functional criteria of viability.     

Cryopreservation of seabream (Sparus aurata) spermatozoa

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.     

1-O-methyl-rac-glycerol: A new agent for the cryopreservation of mononuclear cells

1-O-methyl-rac-glycerol (1-O-MG), also known as 3-methoxy-1,2-propanediol is a lipophilic derivative of glycerol, and has been studied as a new cryoprotective agent (CPA) for mononuclear blood cells (MNC), a well-established experimental model in cryopreservation. The chemical modification of the glycerol molecule results in improved cryobiological properties, such as membrane permeability, thus allowing easier handling in the freeze/thaw process. The optimum preincubation period for MNC and 1-O-MG before freezing is 5 min at 4 degree C, resulting in 86% recovery of viable cells, whereas optimal recovery of glycerol-frozen MNC is only guaranteed after 30 min of preincubation at room temperature (74% viable recovery). The optimal concentration of 1-O-MG is 10% (v/v). Although this new agent offers no improvement in cryoprotective properties over dimethyl sulfoxide (Me2SO) there may be possible pharmacological advantages when used in humans. It is, however, obviously superior to glycerol with regard to its permeation kinetics. 1-O-MG might therefore also be of interest in the cryoprotection of other hematopoietic cells and biological tissues.     

Toxicity and protective efficiency of cryoprotectants to flounder (Paralichthys olivaceus) embryos

With the purpose of finding an ideal cryoprotectant or combination of cryoprotectants in a suitable concentration for flounder (Paralichthys olivaceus) embryo cryopreservation, we tested the toxicities, at culture temperature (16 degrees C), of five most commonly used cryoprotectants-dimethyl sulfoxide (Me2SO), glycerol, methanol (MeOH), 1,2-propylene glycol (PG) and ethylene glycol (EG). In addition, cryoprotective efficiency to flounder embryos of individual and combined cryoprotectants were tested at -15 degrees C for 60 min. Five different concentrations of each of the five cryoprotectants and 20 different combinations of these cryoprotectants were tested for their protective efficiency. The results showed that the toxicity to flounder embryos of the five cryoprotectants are in the following sequence: PG < MeOH < Me2SO < glycerol < EG (P < 0.05); whereas the protective efficiency of each cryoprotectant, at -15 degrees C for a period of 60 min, are in the following sequence: PG > Me2SO approximately MeOH approximately glycerol > EG (greater symbols mean P < 0.05, and approximate symbols mean P > 0.05). Methanol combined with any one of the other cryoprotectants gave the best protection, while ethylene glycol combined with any one of the other cryoprotectants gave the poorest protection at -15 degrees C. Toxicity effect was concentration dependent with the lowest concentration being the least toxic for all five cryoprotectants at 16 degrees C. For PG, MeOH and glycerol, 20% solutions gave the best protection at -15 degrees C; whereas a 15% solution of Me2SO, and a 10% solution of EG, gave the best protection at -15 degrees C.     

Development of optimal techniques for cryopreservation of human platelets. I. Platelet activation during cold storage (at 22 and 8 degrees C) and cryopreservation.

Using the current blood bank storage conditions at 22 degrees C, the viability and function of human platelets can be maintained for only 5 days. This does not allow for the necessary and extensive banking of platelets needed to treat patients afflicted with thrombocytopenia, a side effect of many invasive surgeries such as cardiopulmonary bypass or bone marrow transplantation. The development of optimal techniques for long-term cryopreservation and banking of human platelets would provide the ability to greatly extend the viable life of the platelet and would fulfill an increasing and urgent need in many clinical applications. To determine the optimal techniques for platelet preservation, the expression of an activation marker, phosphatidylserine, on the platelet membrane during storage at 22 and 8 degrees C as well as during the different freezing preservation processes was examined using flow cytometry and annexin V binding assay. Human platelets were identified by both CD41 and light scatter in flow cytometry. In cryopreservation experiments, effects of the following factors on platelet activation were evaluated: (a) cryoprotective agents (CPAs) type: dimethyl sulfoxide (Me2SO), ethylene glycol (EG), and propylene glycol (PG), (b) CPA concentration ranging from 0 to 3 M, and (c) ending temperatures of a slow cooling process at -1 degrees C/min. Our results demonstrated that (a) approximately 50% of platelets were activated on days 7 and 16 at 22 and 8 degrees C, respectively; (b) platelets were not significantly activated after 30-min exposure to 1 M Me2SO, EG, and PG at 22 degrees C, respectively, and (c) there was a significant difference in cryoprotective efficacy among these three CPAs in preventing platelets from cryoinjury. After being cooled to -10 degrees C, 74% of the cryopreserved platelets survived (nonactivated) in 1 M Me2SO solution, while in 1 M EG and 1 M PG solutions, 62 and 42% of the platelets survived, respectively. Using the information that Me2SO consistently yields higher percentages of nonactivated platelets and does not seem to be cytotoxic to platelets for 30-min exposure time, this was found to be the optimal cryoprotective agent for platelets. In addition, significant Me2SO toxicity to platelets was not noted until Me2SO concentrations exceeded 2 M. Finally, a concentration of 1 M Me2SO proved to be the most effective at all cryopreservation ending temperatures tested (-10, -30, -60, and -196 degrees C). In conclusion, under the present experimental conditions, a storage temperature of 8 degrees C appeared to be much better than 22 degrees C. Although the potential chemical toxicity of 1 M Me2SO, EG, or PG is negligible, 1 M Me2SO was found to be optimum for cryopreservation of human platelets. PG has the least cryoprotective function for low-temperature platelet survival.     

Ternary systems with 1,2-propanediol—A new gain in the stability of the amorphous state in the system water-1,2-propanediol-1-propanol

Three ternary systems with water and 1,2-propanediol were investigated, where the third component is 1-propanol, ethanol, or glycerol. 1-Propanol and ethanol give hydrates in their aqueous solutions as well as in these ternary systems, while glycerol gives none. No gain in the stability of the amorphous state and glass-formation tendency is obtained, for the same water contents, when 1,2-propanediol is partially replaced by ethanol. The gain is negligible when it is partially replaced by glycerol. On the contrary, a large maximum in the stability of the amorphous state is obtained, with a critical warming rate dropping from 10⁸ to 10⁴ °C/min in the presence of 65% (w/w) water when 15% (w/w) of the 1,2-propanediol is replaced by 1-propanol. The decrease in the glass formation tendency due to this replacement and corresponding to a few hydrate crystallization is small. Not only the higher stability of the amorphous state, but also in some cases the replacement of ice crystallization by clathrate crystallization at lower temperatures could perhaps contribute to a better cryoprotection of cells for some cooling and warming rates. The similarities observed between the ternary systems investigated gives an idea of the general behaviour of these systems     

Successful Recovery of Motility and Fertility of Cryopreserved Cane Toad (Bufo marinus) Sperm

The recent decline and extinction of amphibian species is a worldwide phenomenon without an identified cause or solution. Assisted reproductive technologies, including sperm cryopreservation, are required to manage endangered amphibian species and preserve their genetic diversity. This study on the Anuran amphibian (Bufo marinus) was undertaken to determine the feasibility of cryopreservation of amphibian sperm. Sperm suspensions for cryopreservation were prepared by macerating testes in cryoprotective additives of 10% (w/v) sucrose or 10% (w/v) sucrose containing either 10, 15, or 20% (v/v) glycerol or 10, 15, or 20% (v/v) dimethyl sulfoxide (Me₂SO). Suspensions were then cooled to −85°C using a controlled rate cooler, stored in LN₂, and thawed in air. The motility and fertilization rate of cryopreserved suspensions and unfrozen control suspensions in Simplified Amphibian Ringer were compared. Sucrose alone had no cryoprotective effect. All other treatments showed varying degrees of recovery of motility and fertilizing capacity. High rates of recovery of motility and fertilizing capacity were observed with 15% Me₂SO (68.9 ± 3.8 and 60.5 ± 4.7%) and 20% glycerol (58.0 ± 5.9 and 81.4 ± 4.3%), respectively. Motility and fertilization rates were similar with Me₂SO but diverged with glycerol as cryoprotectant. The data demonstrate the feasibility of using sperm cryopreservation with amphibian species.     

Cryopreservation of keratinocytes in a monolayer.

The cryopreservation of cells in tissues is one of the major challenges in current cryobiology, especially with regard to the progressively increasing field of tissue engineering. It is very questionable whether protocols which were developed for the cryopreservation of isolated cells are also applicable for cells in more complex structures, such as tissues. As a starting point toward cryopreservation of these three-dimensional structures, the aim of this study was to find an optimum cryopreservation protocol for keratinocytes in a monolayer (two-dimensional structure). These epidermal cells can be transplanted as a monolayer grown on an appropriate matrix for the treatment of deep-dermal burns and leg ulcers. The successful cryopreservation of such transplants would offer the advantage of long-term storage and immediate availability of the transplant. In our study, the variables investigated were the cryoprotective solution and the cooling rate. In order to find a nontoxic cryoprotective agent (CPA) which could be transplanted without an additional washing step, we included hydroxyethyl starch (HES) as a possible CPA in our experimental protocol with the commonly used CPAs Me(2)SO, glycerol, and ethylene glycol. For the evaluation, the cell survival rate was determined by dye exclusion (trypan blue) and the cell metabolism was investigated by cell activity assay (alamarBlue). In conclusion, the cryopreservation protocol with 10 wt.-% HES resulted not only in the highest survival rate (72%) but also in the highest metabolic activity of the cells after thawing; comparable values for the other CPAs were: Me(2)SO, 48%; glycerol, 8%; and ethylene glycol, 10%.     

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.     

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.     

The potential for cryopreserving larvae of the sea urchin, Evechinus chloroticus

Larvae of the sea urchin, Evechinus chloroticus, at varying stages of development, were assessed for their potential to survive cryopreservation. Ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), at concentrations of 1-2 M, were evaluated as cryoprotectants (CPAs) in freezing regimes initially based on methods established for freezing larvae of other sea urchin species. Subsequent work varied cooling rate, holding temperature, holding time, and plunge temperature. Ethylene glycol was less toxic to larvae than Me2SO. However, no larvae survived freezing and thawing in EG. Larvae frozen in Me2SO at the gastrula stage and 4-armed pluteus stage regained motility post-thawing. The most successful freezing regime cooled straws containing larvae in 1.5 M Me2SO from 0 to -35 degrees C at 2.5 degrees C min(-1), held at -35 degrees C for 5 min, then plunged straws into liquid nitrogen. Motility was high 2-4 h post-thawing using this regime but decreased markedly within 24 h. Some 4-armed pluteus larvae that survived beyond this time developed through to metamorphosis and settled. Different Me2SO concentrations and supplementary trehalose did not improve long-term survival. Large variation in post-thaw survival was observed among batches of larvae produced from different females.