Natural deep eutectic systems as alternative nontoxic cryoprotective agents

Natural deep eutectic systems (NADES) are mostly composed of natural primary metabolites such as sugars, sugar alcohols, organic acids, amino acids and amines. These simple molecules have been identified in animals living in environments with extreme temperature amplitudes, being responsible for their survival at negative temperatures during winter. Herein, we report for the first time the use of NADES based on trehalose (Treh) and glycerol (Gly) in cryopreservation, as cryoprotective agents (CPA). The evaluation of the thermal behaviour of these eutectic systems, showed that NADES have a strong effect on the water crystallization/freezing and melting process, being able to reduce the number of ice crystals and hence ice crystal damage in cells, which is a crucial parameter for their survival, upon freezing. Using this NADES as CPA, it is possible to achieve similar or even better cellular performance when compared with the gold standard for cryopreservation dimethyl sulfoxide (DMSO). In this sense, this work relates the physical properties of the NADES with their biological performance in cryopreservation. Our comprehensive strategy results in the demonstration of NADES as a promising nontoxic green alternative to the conventional CPA's used in cryopreservation methods.     

Modeling of cryopreservation of engineered tissues with one-dimensional geometry

Long-term storage of engineered bio-artificial tissues is required to ensure the off-the-shelf availability to clinicians due to their long production cycle. Cryopreservation is likely the choice for long-term preservation. Although the cryopreservation of cells is well established for many cell types, cryopreservation of tissues is far more complicated. Cells at different locations in the tissue could experience very different local environmental changes, i.e., the change of concentration of cryoprotecting chemicals (CPA) and temperature, during the addition/removal of CPA and cooling/warming, which leads to nonuniformity in cell survival in the tissue. This is due to the limitation of mass and heat transfer within the tissue. A specific aim of cryopreservation of tissue is to ensure a maximum recovery of cells and their functionality throughout a tissue. Cells at all locations should be protected adequately by the CPA and frozen at rates conducive to survival. It is hence highly desirable to know the cell transient and final states during cryopreservation within the whole tissue, which can be best studied by mathematical modeling. In this work, a model framework for cryopreservation of one-dimensional artificial tissues is developed on the basis of solving the coupled equations to describe the mass and heat transfer within the tissue and osmotic transport through the cell membrane. Using an artificial pancreas as an example, we carried out a simulation to examine the temperature history, cell volume, solute redistribution, and other state parameters during the freezing of the spherical heterogeneous construct (a single bead). It is found that the parameters affecting the mass transfer of CPA in tissue and through the cell membrane and the freezing rate play dominant roles in affecting the cell volume transient and extracellular ice formation. Thermal conductivity and extracellular ice formation kinetics, on the other hand, have little effect on cell transient and final states, as the heat transfer rate is much faster than mass diffusion. The outcome of such a model study can be used to evaluate the construct design on its survivability during cryopreservation and to select a cryopreservation protocol to achieve maximum cell survival.     

Mechanism of cryoprotection by extracellular polymeric solutes.

To elucidate the means by which polymer solutions protect cells from freezing injury, we cooled human monocytes to -80 degrees C or below in the presence of various polymers. Differential scanning calorimetric studies showed that those polymers which protect cells best have a limiting glass transition temperature (T'g) of approximately -20 degrees C; those with a T'g significantly higher or lower did not protect. Freeze-etch electron micrographs indicated that intracellular ice crystals had formed during this freezing procedure, but remained smaller than approximately 300 nm in the same proportion of cells as survived rapid thawing. We propose that cryoprotection of slowly frozen monocytes by polymers is a consequence of a T'g of -20 degrees C in the extracellular solution. In our hypothesis, the initial concentration and viscosity of protective polymer solutions reduce the extent and rate of cell water loss to extracellular ice and limit the injurious osmotic stress, which cells face during freezing at moderate rates to -20 degrees C. Below -20 degrees C, glass formation prevents further osmotic stress by isolating cells from extracellular ice crystals, virtually eliminating cell water loss at lower temperatures. On the other hand, the protective polymer solutions will allow some diffusion of water away from cells at temperatures above T'g. If conditions are correct, cells will concentrate the cytoplasm sufficiently during the initial cooling to T'g to avoid lethal intracellular freezing between T'g and the intracellular Tg, which has been depressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits.     

Stability of the amorphous state in the system water--glycerol--dimethylsulfoxide

In aqueous solutions containing both glycerol and DMSO, the various states during rewarming after quenching have been identified by X-ray diffraction. The amorphous state of the whole solution has been observed at very low temperatures. The eutectic was seen by X rays after rewarming only in the solutions containing mainly DMSO. In the other solutions only pure ice has been seen. It crystallizes directly in the hexagonal system, if enough DMSO is present. Otherwise, a mixture of cubic and hexagonal ice appears first. The temperature of the end of fusion and the devitrification temperature were measured with a scanning differential calorimeter for a wide range of warming rates. From these measurements was deduced the stability of the amorphous state, defined by the critical heating rate above which no crystallization occurs. That stability presents no maximum, but increases from glycerol to DMSO for a given water concentration in agreement with the fact that Ashwood-Smith considers DMSO a better cryoprotector than glycerol. But a small amount of glycerol in a solution of DMSO greatly enhances the difficulty of crystallization of the eutectic, without decreasing the stability of the amorphous state of the whole solution by much. Then those containing about 10% ($\text{w}\text{w}$) glycerol/(glycerol + DMSO) are perhaps better cryoprotectants than those with only DMSO, at least for low cooling or warming rates where the eutectic may have enough time to crystallize, eventually with deleterious effects, outside or inside the cells.     

An Improved Model for Nucleation-Limited Ice Formation in Living Cells during Freezing

Ice formation in living cells is a lethal event during freezing and its characterization is important to the development of optimal protocols for not only cryopreservation but also cryotherapy applications. Although the model for probability of ice formation (PIF) in cells developed by Toner et al. has been widely used to predict nucleation-limited intracellular ice formation (IIF), our data of freezing Hela cells suggest that this model could give misleading prediction of PIF when the maximum PIF in cells during freezing is less than 1 (PIF ranges from 0 to 1). We introduce a new model to overcome this problem by incorporating a critical cell volume to modify the Toner''s original model. We further reveal that this critical cell volume is dependent on the mechanisms of ice nucleation in cells during freezing, i.e., surface-catalyzed nucleation (SCN) and volume-catalyzed nucleation (VCN). Taken together, the improved PIF model may be valuable for better understanding of the mechanisms of ice nucleation in cells during freezing and more accurate prediction of PIF for cryopreservation and cryotherapy applications.     

Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures

Background and Aims

Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival.

Methods

Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro.

Key Results

Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm² in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure.

Conclusions

The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches.     

A first test of the hypothesis of biogenic magnetite-based heterogeneous ice-crystal nucleation in cryopreservation

An outstanding biophysical puzzle is focused on the apparent ability of weak, extremely low-frequency oscillating magnetic fields to enhance cryopreservation of many biological tissues. A recent theory holds that these weak magnetic fields could be inhibiting ice-crystal nucleation on the nanocrystals of biological magnetite (Fe₃O₄, an inverse cubic spinel) that are present in many plant and animal tissues by causing them to oscillate. In this theory, magnetically-induced mechanical oscillations disrupt the ability of water molecules to nucleate on the surface of the magnetite nanocrystals. However, the ability of the magnetite crystal lattice to serve as a template for heterogeneous ice crystal nucleation is as yet unknown, particularly for particles in the 10–100 nm size range. Here we report that the addition of trace-amounts of finely-dispersed magnetite into ultrapure water samples reduces strongly the incidence of supercooling, as measured in experiments conducted using a controlled freezing apparatus with multiple thermocouples. SQUID magnetometry was used to quantify nanogram levels of magnetite in the water samples. We also report a relationship between the volume change of ice, and the degree of supercooling, that may indicate lower degassing during the crystallization of supercooled water. In addition to supporting the role of ice-crystal nucleation by biogenic magnetite in many tissues, magnetite nanocrystals could provide inexpensive, non-toxic, and non-pathogenic ice nucleating agents needed in a variety of industrial processes, as well as influencing the dynamics of ice crystal nucleation in many natural environments.     

Avoidance of intracellular freezing by the freezing-tolerant New Zealand Alpine weta Hemideina maori (Orthoptera: Stenopelmatidae)

Calorimetric analysis indicates that 82% of the body water of Hemideina maori is converted into ice at 10 degrees C. This is a high proportion and led us to investigate whether intracellular freezing occurs in H. maori tissue. Malpighian tubules and fat bodies were frozen in haemolymph on a microscope cold stage. No fat body cells, and 2% of Malpighian tubule cells froze during cooling to -8 degrees C. Unfrozen cells appeared shrunken after ice formed in the extracellular medium. There was no difference between the survival of control tissues and those frozen to -8 degrees C. At temperatures below -15 degrees C (lethal temperatures for weta), there was a decline in survival, which was strongly correlated with temperature, but no change in the appearance of tissue. It is concluded that intracellular freezing is avoided by Hemideina maori through osmotic dehydration and freeze concentration effects, but the reasons for low temperature mortality remain unclear. The freezing process in H. maori appears to rely on extracellular ice nucleation, possibly with the aid of an ice nucleating protein, to osmotically dehydrate the cells and avoid intracellular freezing. The lower lethal temperature of H. maori (-10 degrees C) is high compared to organisms that survive intracellular freezing. This suggests that the category of 'freezing tolerance' is an oversimplification, and that it may encompass at least two strategies: intracellular freezing tolerance and avoidance.     

Cryopreservation of erythropoietin-responsive cells in murine hematopoietic tissue

The optimal conditions were determined under which maximum survival of murine hematopoietic erythropoietin-responsive cells (ERC) could be ensured during manipulations required for cryopreservation. Cell survival was similar over freezing rates between 2 and 10 °C/min. Optimal cryoprotectants were 10% dimethyl sulfoxide (DMSO) and 20% fetal calf serum; the DMSO was removed by centrifugation after stepwise dilution with 20 vol of medium over a 10-min period. Differing thawing rates for the cell suspensions had minimal effects on survival. “Seeding” the cell suspensions with ice crystals had no effect on ERC recovery. Overall ERC survival varied between 20 and 40%. These results confirm earlier reports that certain ERC populations are more sensitive to damage during cryopreservation than are other hematopoietic progenitor cells.     

Quality improvements to mackerel (Scomber japonicus) muscle tissue frozen using a rapid freezer with the weak oscillating magnetic fields

Although freezing is the most popular long-term food preservation method, the formation of ice crystals during the freezing process often degrades the quality of the product. Recently, several reports have argued that oscillating magnetic fields (OMFs) may affect ice crystallization. In this paper, we investigated the effects of OMFs on fresh mackerel using the Cell Alive System® (CAS®) developed as an additional OMF generator for a rapid freezer. Mackerel fillets were frozen with home freezing (HF), air blast freezing without (ABF) or with CAS (ABF-CAS) (ABI Co. Ltd., Chiba, Japan), and stored them for 2 weeks in the frozen storage between −30 °C and −35 °C. We analyzed the tissue damages of thawed samples histologically. The OMFs has been shown to significantly inhibit tissue damage in mackerel tissue after freezing and thawing (especially, thawing in ice water). And it seems that OMFs suppressed the ice hole counts (p < 0.05), the mean size (p = 0.061), and the increase of interstitial area% (p < 0.05) after freezing/thawing. We also found that it is necessary to avoid re-crystallization during thawing to maintain the quality of the frozen product. The use of OMFs with rapid thawing has the potential to improve cryopreservation in the food industry as well as in the bioscience industry.     

Static magnetic field increases survival rate of dental pulp stem cells during DMSO-free cryopreservation

Abstract

Successful and efficient cryopreservation of living cells and organs is a key clinical application of regenerative medicine. Recently, magnetic cryopreservation has been reported for intact tooth banking and cryopreservation of dental tissue. The aim of this study was to assess the cryoprotective effects of static magnetic fields (SMFs) on human dental pulp stem cells (DPSCs) during cryopreservation. Human DPSCs isolated from extracted teeth were frozen with a 0.4-T or 0.8-T SMF and then stored at ?196?°C for 24?h. During freezing, the cells were suspended in freezing media containing with 0, 3 or 10% DMSO. After thawing, the changes in survival rate of the DPSCs were determined by flow cytometry. To understand the possible cryoprotective mechanisms of the SMF, the membrane fluidity of SMF-exposed DPSCs was tested. The results showed that when the freezing medium was DMSO-free, the survival rates of the thawed DPSCs increased 2- or 2.5-fold when the cells were exposed to 0.4-T or 0.8-T SMFs, respectively (p?<?0.01). In addition, after exposure to the 0.4-T SMF, the fluorescence anisotropy of the DPSCs increased significantly (p?<?0.01) in the hydrophilic region. These results show that SMF exposure improved DMSO-free cryopreservation. This phenomenon may be due to the improvement of membrane stability for resisting damage caused by ice crystals during the freezing procedure.     

Investigating cryoinjury using simulations and experiments: 2. TF-1 cells during graded freezing (interrupted slow cooling without hold time)

Cryopreservation plays a key role in the long-term storage of native and engineered cells and tissues for research and clinical applications. The survival of cells and tissues after freezing and thawing depends on the ability of the cells to withstand a variety of stresses imposed by the cryopreservation protocol. A better understanding of the nature and kinetics of cellular responses to temperature-induced conditions is required to minimize cryoinjury. An interrupted freezing procedure that allows dissection of cryoinjury was used to investigate the progressive damage that occurs to cells during cryopreservation using slow cooling. Simulations of cellular osmotic responses were used to provide interpretation linking states of the cell with events during the freezing procedure. Simulations of graded freezing (interrupted slow cooling without hold time) were correlated with cell recovery results of TF-1 cells. Calculated intracellular supercooling and osmolality, were used as indicators of the probability of cryoinjury due to intracellular ice formation and solution effects, providing direct links of cellular conditions to events in the freezing process. Using simulations, this study demonstrated that both intracellular supercooling and osmolality are necessary to explain graded freezing results.     

An hypothesis for survival of spermatozoa via encapsulation during plane front freezing

Plane front freezing presents the possibility of encapsulating individual cells in the ice phase. The cells may also be pushed ahead of the plane front ice interface, as is always the case for conventional dendritic freezing, where the cells are pushed ahead of the thickening dendrite arms. Cells which are encapsulated during freezing are exposed to hypotonic liquid (pure water) initially upon thawing, while cells which are pushed into the last liquid to freeze are exposed to hypertonic liquid upon thawing. Some exposure to hypertonic intercellular liquid prior to freezing may be required to build up the salt and CPA content in the intracellular liquid and thereby avoid intracellular ice formation at the given cooling rate. Encapsulation of cells by a plane front ice interface should result in three regions of cell survival in the sample: an initial region of cell death due to intracellular ice formation, a final region of cell death due to overexposure to hypertonic intercellular liquid, and an intermediate region of cell survival, where neither damage mechanism has operated to a lethal level. An advantage of plane front freezing over dendritic freezing is that the regions of cell survival and death should be geometrically separate in the sample, rather than mixed at the dendritic microstructural level, as is the case for dendritic freezing. Samples containing populations with very high or very low survival rates for spermatozoa could be obtained by simply cutting up the frozen sample.     

Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation

Cryopreservation of mammalian cells has to date typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled ice nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 °C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below −20 °C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.     

Effect of trehalose as an additive to dimethyl sulfoxide solutions on ice formation,cellular viability,and metabolism

Cryopreservation is the only established method for long-term preservation of cells and cellular material. This technique involves preservation of cells and cellular components in the presence of cryoprotective agents (CPAs) at liquid nitrogen temperatures (−196 °C). The organic solvent dimethyl sulfoxide (Me₂SO) is one of the most commonly utilized CPAs and has been used with various levels of success depending on the type of cells. In recent years, to improve cryogenic outcomes, the non-reducing disaccharide trehalose has been used as an additive to Me₂SO-based freezing solutions. Trehalose is a naturally occurring non-toxic compound found in bacteria, fungi, plants, and invertebrates which has been shown to provide cellular protection during water-limited states. The mechanism by which trehalose improves cryopreservation outcomes remains not fully understood. Raman microspectroscopy is a powerful tool to provide valuable insight into the nature of interactions among water, trehalose, and Me₂SO during cryopreservation. We found that the addition of trehalose to Me₂SO based CPA solutions dramatically reduces the area per ice crystals while increasing the number of ice crystals formed when cooled to −40 or −80 °C. Differences in ice-formation patterns were found to have a direct impact on cellular viability. Despite the osmotic stress caused by addition of 100 mM trehalose, improvement in cellular viability was observed. However, the substantial increase in osmotic pressure caused by trehalose concentrations above 100 mM may offset the beneficial effects of changing the morphology of the ice crystals achieved by addition of this sugar.     

Drug metabolism and viability studies in cryopreserved rat hepatocytes

Rat hepatocytes were cryopreserved optimally by freezing them at 1 degrees C/min to -80 degrees C in cryoprotectant medium containing either 20% (v/v) dimethylsulfoxide (Me2SO) and 25% (v/v) fetal calf serum in Leibowitz L15 medium (Me2SO cryoprotectant) or 25% (v/v) vitrification solution (containing Me2SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz L15 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (24-48 hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-50% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37 degrees C. This decline in viability occurred more rapidly after freezing in Me2SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37 degrees C does not appear to be due to ice-crystal damage as it occurred in cells stored at -10 degrees C (above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80 degrees C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to ice-crystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.     

Basic investigations on the freezing of human lymphocytes

Human lymphocytes were frozen at constant cooling rates in the range 2.4 to 1000 degrees K/min without cryoadditive on the cold stage of a thermally defined cryomicroscope. The volume loss due to water efflux was quantified optically for the cooling rates 2.4, 12, 48, and 120 degrees K/min. The likelihood of the formation of intracellular ice was determined as function of the cooling rate. Intracellular crystallization temperatures were obtained for ice formation during both cooling and rewarming. A theoretical analysis of the cell volume loss during freezing was compared to the experimental data and used for an indirect determination of the water permeability of the cells. A relative optimum of the cooling rate is predicted theoretically under the assumption of a critical level of intracellular salt concentration near the eutectic temperature. The dependence of survival and cooling rate was determined cryomicroscopically by simultaneously applying the FDA/EB fluorescence viability test. The optimal cooling rate of about 35 degrees K/min was also found for 2-ml samples frozen within the range of cooling rates of interest. The results show that for freezing in physiological saline solution (1) the optimum of the cooling rate is theoretically predictable, (2) cryomicroscopical data are significant for freezing of samples of larger volume, and (3) the lethal type of intracellular crystallization is cooling rate dependent and distinguishable from innocuous types.     

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al.¹ and Wilmut ² separately described that mouse embryos could be frozen and stored at -196 °C and, a few years later, Parkening et al. ³ reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding ⁴. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen ⁵. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.Open in a separate windowClick here to view.^{(106M, flv)}     

A new approach for freezing of aqueous solutions under active control of the nucleation temperature

An experimental setup for controlled freezing of aqueous solutions is introduced. The special feature is a mechanism to actively control the nucleation temperature via electrofreezing: an ice nucleus generated at a platinum electrode by the application of an electric high voltage pulse initiates the crystallization of the sample. Using electrofreezing, the nucleation temperature in pure water can be precisely adjusted to a desired value over the whole temperature range between a maximum temperature Tn(max) close to the melting point and the temperature of spontaneous nucleation. However, the presence of additives can inhibit the nucleus formation. The influence of hydroxyethylstarch (HES), glucose, glycerol, additives commonly used in cryobiology, and NaCl on Tn(max) were investigated. While the decrease showed to be moderate for the non-ionic additives, the hindrance of nucleation by ionic NaCl makes the direct application of electrofreezing in solutions with physiological salt concentrations impossible. Therefore, in the multi-sample freezing device presented in this paper, the ice nucleus is produced in a separate volume of pure water inside an electrode cap. This way, the nucleus formation becomes independent of the sample composition. Using electrofreezing rather than conventional seeding methods allows automated freezing of many samples under equal conditions. Experiments performed with model solutions show the reliability and repeatability of this method to start crystallization in the test samples at different specified temperatures. The setup was designed to freeze samples of small volume for basic investigations in the field of cryopreservation and freeze-drying, but the mode of operation might be interesting for many other applications where a controlled nucleation of aqueous solutions is of importance.     

Thermal hysteresis proteins

Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.