Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest

Phase diagrams are of great utility in cryobiology, especially, those consisting of a cryoprotective agent (CPA) dissolved in a physiological salt solution. These ternary phase diagrams consist of plots of the freezing points of increasing concentrations of solutions of cryoprotective agents (CPA) plus NaCl. Because they are time-consuming to generate, ternary diagrams are only available for a small number of CPAs. We wanted to determine whether accurate ternary phase diagrams could be synthesized by adding together the freezing point depressions of binary solutions of CPA/water and NaCl/water which match the corresponding solute molality concentrations in the ternary solution. We begin with a low concentration of a solution of CPA+salt of given R (CPA/salt) weight ratio. Ice formation in that solution is mimicked by withdrawing water from it which increases the concentrations of both the CPA and the NaCl. We compute the individual solute concentrations, determine their freezing points from published binary phase diagrams, and sum the freezing points. These yield the synthesized ternary phase diagram for a solution of given R. They were compared with published experimental ternary phase diagrams for glycerol, dimethyl sulfoxide (DMSO), sucrose, and ethylene glycol (EG) plus NaCl in water. For the first three, the synthesized and experimental phase diagrams agreed closely, with some divergence occurring as wt% concentrations exceeded 30% for DMSO and 55% for glycerol, and sucrose. However, in the case of EG there were substantial differences over nearly the entire range of concentrations which we attribute to systematic errors in the experimental EG data. New experimental EG work will be required to resolve this issue.     

Postfreeze viability of human renal epithelial carcinoma cells related to phase transformations in ternary solutions

The postfreeze viability of human renal epithelial carcinoma cells frozen in solutions based on a complex physiologic support medium to which additions of NaCl and a cryoprotective agent, either glycerol or dimethyl sulfoxide (DMSO) were made, have been determined by a dye exclusion technique. The support medium consisted of either Eagle's Minimum Essential Medium with Hanks' salts added (MEM) or this same medium supplemented with 20 vol% heat-inactivated fetal calf serum (MEM + FCS). Glycerol was found to be an ineffective cryoprotective agent for these cells, while DMSO was highly effective. Addition of NaCl along with the DMSO further improved the viability of cells frozen at −196 °C. Freezing and thawing rates were found to be important with a slow freezing rate, 2.5 °C/min, and a rapid thawing rate, 240°C/min, yielding the best results.Maximum viability occurred in solutions containing 80 to 95 wt.% (MEM + FCS) with the balance being DMSO and NaCl in the weight ratio of 9:1. In addition to primary ice formation, two nonequilibrium glassy phases were observed during DTA studies of these solutions (10). The exintence of these vitreous states reduces the chances thet cells will be exposed to hypertonic concentrations of salt in the extracellur fluids during freezing-out of primary ice.     

Peculiarities of state diagrams of aqueous solutions of cryoprotective agents

The phase transitions in aqueous solutions of glycerol and PEO-1500 within the temperature range of +30 to −150 °C have been studied using the methods of thermoplastic analysis and volumetric scanning tensodilatometry. We present the revealed phenomenon of cluster cyrystallization of these solutions as well as principles of describing this phenomenon using state diagrams, containing the intervals of concentration corresponding to the existence of amorphous and cryocolloid fractions. We note that for the cryocolloid fraction, a low temperature association of molecules of cryoprotective agents leads the formation of ice nanocrystals either close to or directly inside the aggregations. These fractions exist in cooled cryoprotective solutions near the vitrification temperatures of the liquid phase and may contribute to the initiation of damaging events in cryopreserved biological systems. Our data may be helpful in explaining the peculiarities observed during crystallization of cryoprotective solutions and may further contribute to a broader understanding of the principles of protection and protocol optimization of biological materials at temperatures approaching vitrification.     

Postfreeze viability of human renal epithelial carcinoma cells in quaternary solutions

The postfreeze survival of human renal epithelial carcinoma cells frozen in suspensions based on (MEM + FCS) (Eagle's Minimum Essential Medium with Hanks' salts added and supplemented with 20 vol% heat-inactivated fetal calf serum) to which additions of NaCl and the cryoprotective compounds, dimethyl sulfoxide and glycerol, were made, have been determined by a vital dye exclusion technique. A significant range of aqueous-rich quaternary solutions were found to be highly effective cryoprotective agents for these cells under optimum freezing and thawing conditions.High survival occurs in a composition series emanating from the region of high postfreeze cell viability in the ternary (MEM + FCS)-NaCl-DMSO system, between 80 and 95% (MEM + FCS), along isopleths that substitute glycerol for NaCl in various proportions. In addition, viability remains sufficiently high as a few percentage of glycerol is substituted for DMSO alone.     

The nonequilibrium phase and glass transition behavior of beta-lactoglobulin

Concentrated solutions of bovine beta-lactoglobulin were studied using osmotic stress and rheological techniques. At pH 6.0 and 8.0, the osmotic pressure was largely independent of NaCl concentration and could be described by a hard sphere equation of state. At pH 5.1, close to the isoelectric point, the osmotic pressure was lower at the lower NaCl concentrations (0 mM, 100 mM) and was fitted by an adhesive hard sphere model. Liquid-liquid phase separation was observed at pH 5.1 at ionic strengths of 13 mM and below. Comparison of the liquid-liquid and literature solid-liquid coexistence curves showed these solutions to be supersaturated and the phase separation to be nonequilibrium in nature. In steady shear, the zero shear viscosity of concentrated solutions at pH 5.1 was observed at shear rates above 50 s(-1). With increasing concentration, the solution viscosity showed a progressive increase, a behavior interpreted as the approach to a colloidlike glass transition at approximately 60% w/w. In oscillatory shear experiments, the storage modulus crossed the loss modulus at concentrations of 54% w/w, an indication of the approaching glass transition. Comparison of the viscous behavior with predictions from the Krieger-Dougherty equation indicates the hydrodynamic size of the protein decreases with increasing concentration, resulting in a slower approach to the glass transition than a hard sphere system.     

Thermodynamic nonequilibrium phase change behavior and thermal properties of biological solutions for cryobiology applications

Understanding the phase change behavior of biomaterials during freezing/thawing including their thermal properties at low temperatures is essential to design and improve cryobiology applications such as cryopreservation and cryosurgery. However, knowledge of phase change behavior and thermal properties of various biomaterials is still incomplete, especially at cryogenic temperatures (< or = -40 degrees C). Moreover, in these applications, chemicals are often added to improve their outcome, which can result in significant variation in the phase change behavior and thermal properties from those of the original biomaterials. These chemical additives include cryoprotective agents (CPAs), antifreeze protein (AFP), or cryosurgical adjuvants like sodium chloride (NaCl). In the present study, phase change behavior and thermal properties of saline solutions--either water-NaCl or phosphate buffered saline (PBS)--with various chemical additives were investigated. The chemical additives studied are glycerol and raffinose as CPAs, an AFP (Type III, molecular weight = 6500), and NaCl as a cryosurgical adjuvant. The phase change behavior was investigated using a differential scanning calorimeter (DSC) and a cryomicroscope. The specific and latent heat of these solutions were also measured with the DSC. The saline solutions have two distinct phase changes--water/ice and eutectic phase changes. During freezing, eutectic solidification of both water-NaCl and PBS are significantly supercooled below their thermodynamic equilibrium eutectic temperatures. However, their melting temperatures are close to thermodynamic equilibrium during thawing. These eutectic phase changes disappear when even a small amount (0.1 M glycerol) of CPA was added, but they are still observed after the addition of an AFP. The specific heats of these solutions are close to that of ice at very low temperatures (< or = -100 degrees C) regardless of the additives, but they increase between -100 degrees C and -30 degrees C with the addition of CPAs. The amount of latent heat, which is evaluated with sample weight, generally decreases with the addition of the additives, but can be normalized to approximately 300 J/g based on the weight of water which participates in the phase change. This illustrates that thermal properties, especially latent heat, of a biomaterial should be evaluated based on the understanding of its phase change behavior. The results of the present study are discussed in the context of the implications for cryobiology applications.     

Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection

Plant shoot tips do not survive exposure to liquid nitrogen temperatures without cryoprotective treatments. Some cryoprotectant solutions, such as plant vitrification solution 2 (PVS2), dehydrate cells and decrease lethal ice formation, but the extent of dehydration and the effect on water freezing properties are not known. We examined the effect of a PVS2 cryoprotection protocol on the water content and phase behavior of mint and garlic shoot tips using differential scanning calorimetry. The temperature and enthalpy of water melting transitions in unprotected and recovering shoot tips were comparable to dilute aqueous solutions. Exposure to PVS2 changed the behavior of water in shoot tips: enthalpy of melting transitions decreased to about 40 J g H2O(-1) (compared to 333 J g H2O(-1) for pure H2O), amount of unfrozen water increased to approximately 0.7 g H2O g dry mass(-1) (compared to approximately 0.4 g H2Og dry mass(-1) for unprotected shoot tips), and a glass transition (T(g)) at -115 degrees C was apparent. Evaporative drying at room temperature was slower in PVS2-treated shoot tips compared to shoot tips receiving no cryoprotection treatments. We quantified the extent that ethylene glycol and dimethyl sulfoxide components permeate into shoot tips and replace some of the water. Since T(g) in PVS2-treated shoot tips occurs at -115 degrees C, mechanisms other than glass formation prevent freezing at temperatures between 0 and -115 degrees C. Protection is likely a result of controlled dehydration or altered thermal properties of intracellular water. A comparison of thermodynamic measurements for cryoprotection solutions in diverse plant systems will identify efficacy among cryopreservation protocols.     

Thermal properties of ethylene glycol aqueous solutions

Preventing ice crystallization by transforming liquids into an amorphous state, vitrification can be considered as the most suitable technique allowing complex tissues, and organs cryopreservation. This process requires the use of rapid cooling rates in the presence of cryoprotective solutions highly concentrated in antifreeze compounds, such as polyalcohols. Many of them have already been intensively studied. Their glass forming tendency and the stability of their amorphous state would make vitrification a reality if their biological toxicity did not reduce their usable concentrations often below the concentrations necessary to vitrify organs under achievable thermal conditions. Fortunately, it has been shown that mixtures of cryoprotectants tend to reduce the global toxicity of cryoprotective solutions and various efficient combinations have been proposed containing ethanediol. This work reports on the thermal properties of aqueous solutions with 40, 43, 45, 48, and 50% (w/w) of this compound measured by differential scanning calorimetry. The glass forming tendency and the stability of the amorphous state are evaluated as a function of concentration. They are given by the critical cooling rates v(ccr)above which ice crystallization is avoided, and the critical warming rates v(cwr) necessary to prevent ice crystallization in the supercooled liquid state during rewarming. Those critical rates are calculated using the same semi-empirical model as previously. This work shows a strong decrease of averaged critical cooling and warming rates when ethanediol concentration increases, V(ccr) and V(cwr) = 1.08 x 10 (10) K/min for 40% (w/w) whereas V(ccr) = 11 and V(cwr) = 853 K/min for 50% (w/w). Those results are compared with the corresponding properties of other dialcohols obtained by the same method. Ethylene glycol efficiency is between those of 1,2-propanediol and 1,3-propanediol.     

The influence of hydroxyethyl starch on ice formation in aqueous solutions

Differential scanning calorimetry, and, in some supplementary experiments, X-ray diffractometry and cryomicroscopy, were applied to study the influence of concentration (< 70 wt%) and cooling/warming rates (< 320 K/min) on ice formation in aqueous solutions of HES. The calorimetric measurements of the quantity of crystallizing water indicated that a mass fraction ? = 0.522 (i.e., grams water per gram HES) remained unfrozen. These results are in good agreement with our earlier extrapolations from ternary phase diagram data and tend to support the proposed cryoprotective mechanism. The value of ? determined during warming was essentially independent of composition up to the corresponding saturation concentration. It was observed that solutions containing 60 wt% HES or more remained wholly amorphous during cooling even at rates as low as 2.5 K/min (down to 120 K). Such glassy solutions are subject to devitrification at temperatures T_d which depend on the warming rate. The concentrations close to 55 wt% HES mark a transitional range exhibiting two crystallization peaks, probably due to different mechanisms of nucleation, the portion of ice formed during cooling being related to the imposed cooling rate. All samples showed a recrystallization transition at 257.5 K which was also observed cryomicroscopically. Glass transitions, however, could not be detected by the methods applied in this study. The X-ray diffraction patterns contained the structure of only one solid phase, namely hexagonal ice. A comparison of various modifications of HES, PEG, and PVP involving bound water and melting temperature did not reveal marked differences. Minimum initial HES concentrations preventing lethal salt enrichment were computed for both binary and ternary mass fractions of NaCl as biologically relevant parameters, yielding 24.1 and 10.8 wt% HES, respectively.     

Influence of lipids on ice formation during the freezing of cryoprotective medium

The influence of lipids on ice formation during the freezing of cryoprotective medium for the semen of rainbow trout has been studied by the cryomicroscopy technique. It was shown that the lipids extracted from marine vertebrates and liposomes from the lipids of trout sperm effectively inhibit the ice formation in cryoprotective solutions during freezing, fundamentally changing the form and size of ice crystals. At high concentrations of lipids, either the crystallization does not occur in the cryoprotective medium or, even if ice crystals are formed, they have a broken shape and blurred borders. The addition of egg yolk sligthly increases the size and essentially changes the shape of ice crystals during the freezing of solution.     

Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions

Vitrification solutions are aqueous cryoprotectant solutions which do not freeze when cooled at moderate rates to very low temperatures. Vitrification solutions have been used with great success for the cryopreservation of some biological systems but have been less successful or unsuccessful with other systems, and more fundamental knowledge about vitrification solutions is required. The purpose of the present survey is to show that a general understanding of the physical behavior and biological effects of vitrification solutions, as well as an understanding of the conditions under which vitrification solutions are required, is gradually emerging. Detailed nonequilibrium phase diagram information in combination with specific information on the tolerance of biological systems to ice and to cryoprotectant at subzero temperatures provides a quantitative theoretical basis for choosing between vitrification and freezing. The vitrification behavior of mixtures of cryoprotective agents during cooling is predictable from the behavior of the individual agents, and the behavior of individual agents is gradually becoming predictable from the details of their molecular structures. Progress is continuing concerning the elucidation of mechanisms and cellular sites of toxicity and mechanisms for the reduction of toxicity. Finally, important new information is rapidly emerging concerning the crystallization of previously vitrified cryoprotectant solutions during warming. It appears that vitrification tendency, toxicity, and devitrification all depend on subtle variations in the organization of water around dissolved substances.     

Impacts of trehalose and l-proline on the thermodynamic nonequilibrium phase change and thermal properties of normal saline

Understanding the phase change behavior and thermal properties of cryoprotective agents (CPAs) in biological solutions is essential for enhancing the success of cryopreservation and biobanking. In this study, the phase change behavior and thermal properties of normal saline added with trehalose or l-proline were investigated using differential scanning calorimeter (DSC) and cryomicroscope during freezing and warming. The addition of trehalose or l-proline can eliminate the eutectic formation in normal saline. Trehalose had significantly lower latent heat release than l-proline does at a high concentration of 1 M (P < 0.05), while unfrozen water content of trehalose is significantly lower than that of l-proline at all the concentrations (P < 0.05). It was also found that addition of 0.2 M, 0.3 M and 1 M trehalose can achieve partial vitrification in normal saline and that the glass transition temperature rises along with the increase in concentrations of trehalose. However, no vitrification was observed in normal saline with l-proline at any concentrations. Besides, rates of ice crystal growth in normal saline added with trehalose are slower than those in normal saline with l-proline at the same concentrations. These results suggest that both trehalose and l-proline can act as CPAs by avoiding eutectic formation and inhibiting ice formation in normal saline for cell cryopreservation. It could be useful for CPA selection and designing in the future.     

Phase diagram relationships in cryobiology

The reactions which occur during freezing in biological systems employing DMSO as a cryoprotective agent may well involve information given by a near equilibrium ternary H₂O-DMSO-NaCl phase diagram. The initial freezing point depressions for solutions with three different DMSO-NaCl initial ratios (R) have been determined over the onefold surface of ice saturation. DMSO has been shown to be more effective in reducing NaCl concentration in the residual liquid than had been previously predicted. The temperature and the fraction solid which must be reached for the occurrence of second phase coprecipitation with ice have been shown to be a strong function of initial R value. Ternary invariant reactions have been identified at ?35 °C, and tentatively identified at ?115 and ?105 °C for solutions having DMSO/NaCl ratios of R = 9, 5, and 1, respectively. Metastable nonequilibrium phase formation has been observed for slow cooling of a solution with R = 1. This metastable condition results in different phase relationships upon thawing than upon the initial freezing. By quenching the system after partial rewarming, it has been demonstrated that this metastable condition can be eliminated.     

Formation of Ice Microparticles in the Ovarian Fluid and Homogenates of Unfertilized Russian Sturgeon Eggs during Cooling to –196°C

Cryopreservation of fish and amphibian eggs is still an unsolved problem. The formation of ice crystals inside and outside cells acts as a main detrimental factor during a deep freezing of fish eggs, as well as crystal growth (recrystallization and repeated crystallization). Designing efficient cryoprotective media is necessary in order to avoid egg injury from freezing. Additional components that are present in a cryoprotective medium and reduce the thermomechanical stress and cracks of frozen tissues might increase oocyte survival after freezing–thawing. Natural components of eggs and the ovarian fluid are promising as such additives. The formation of ice microparticles was studied in thin layers (0.2 mm) of the ovarian fluid and components of Russian sturgeon egg homogenates upon their cooling to a liquid nitrogen temperature (–196°C). The processes of freezing, ice cracking, and microparticle formation were observed as the temperature was decreased gradually. The shape and size of ice microparticles were found to depend on the composition of the freezing solution. Certain fractions of egg homogenate were assumed to be suitable as components of a cryoprotective medium.

     

Electrolytes and plasma expanders

Summary The effect of Gey's balanced salt solution deficient in NaCl on cells in tissue culture led to a study of various solutions employed as vehicles for plasma expanders.The behavior of epithelium from the adult human mucosa, tonsils from children, and mouse kidney papilla was recorded in a perfusion chamber with phase contrast, time-lapse cinematography. After perfusion for one to two hours with fluid nutrient medium, test solutions were brought in contact with the cells for one hour. The fluid system was then generally replaced with Gey's balanced salt solution and, or, the proteinaceous nutrient medium.So-called normal saline produced marked injury both when employed alone or in combination with 6% Dextran. Damage resulting from 5% dextrose either alone or in combination with 6% Dextran was characterized by vacuole formation. Dextran at 6% in Gey's balanced salt solution or in Ringer's solution appeared to produce little injury to cells under the conditions of these experiments.Work in progress is designed to examine the lifesaving value of the various solutions in the treatment of partially exsanguinated dogs.This investigation was supported by the Medical Research and Development Board, Office of the Surgeon General, Department of the Army, under Contract No. DA-49-007-MD-32.     

Isolation and Characterization of Cyanobacteria from Microbial Mats of the Ebro Delta,Spain

Isolation and identification of several strains of cyanobacteria from microbial mats of the Ebro Delta, Spain, are described. A series of tenfold dilutions was the first step of isolation. Self-isolation techniques, which use one or several physiological characteristics of a cyanobacterium, were applied in some cases to obtain enrichment cultures. Twelve filamentous strains were isolated and stored in axenic culture. As only a few cyanobacterial species can be frozen and revived without any cryoprotective agent, preservation of isolated strains was assayed with several cryoprotective solutions. Methanol and glycerol were not suitable as cryoprotective agents for most of the isolates. Dimethyl sulfoxide (DMSO) was apparently the best cryoprotector. A new method, which used a filter paper as a growing substratum that later could be directly stored at −80°C, was successfully used. A morphological study of each strain under light and electron microscopy was made to classify them. All isolated strains belong to phylum BX, Class 1, subsection III of the Bergey's manual of systematic bacteriology, 2nd ed., vol. 1. Most genera are included in the LPP group as Lyngbya aestuarii and Microcoleus chthonoplastes. Received: 30 April 2002 / Accepted: 31 May 2002     

Glass-forming property of the system diethyl sulphoxide/water and its cryoprotective action on Escherichia coli survival

In this work the thermal properties of diethyl sulphoxide (Et2SO), as well as its cryoprotective ability are studied and related to other well-known cryoprotectant substances, like dimethyl sulphoxide (Me2SO). We have investigated the thermal properties of Et2SO/water systems using Differential Scanning Calorimetry at a very low heating/cooling rate (2 degrees C/min). Liquid/solid or glassy/crystalline transitions have been observed only for the solutions with content of Et2SO ranging from 5 up to 40% w/w and/or greater than 85%. In the 45-75% w/w Et2SO range we have found a noticeable glass-forming tendency and a great stability of the amorphous state to the reheating. In samples with Et2SO content ranging from 80 to 85%, we observed a great stability of the glass forming by cooling, but a lesser stability to the subsequent reheating. The glass-forming tendency of these solutions is discussed in terms of existing competitive interactions between molecules of Et2SO, on the one hand, and Et2SO and water molecules, on the other hand. The results are well explainable on the basis of the model structure of water/Et2SO solutions, deduced by Raman and infrared studies [J. Mol. Struct. 665 (2003) 285-292]. The cryoprotective ability of Et2SO on Escherichia coli survival has been also investigated, and a comparison among Et2SO and other widely used cryoprotectants, like Me2SO and glycerol has been done. Survival of E. coli, determined after freezing-thawing process, was maximal at 45% w/w Et2SO (more than 85% viability). It should be noted that at the same concentration the survival is only about 35% in the presence of Me2SO and not more than 15% in the presence of glycerol. These features are well consisted with the glass-forming properties of Et2SO.     

Inhibiting ice recrystallization and optimization of cell viability after cryopreservation

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220?mM solutions of disaccharides; however, the best cell viability was obtained when a 200?mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.