Investigating cryoinjury using simulations and experiments. 1: TF-1 cells during two-step freezing (rapid cooling interrupted with a hold time)

There is significant interest in designing a cryopreservation protocol for hematopoietic stem cells (HSC) which does not rely on dimethyl sulfoxide (Me₂SO) as a cryoprotectant. Computer simulations that describe cellular osmotic responses during cooling and warming can be used to optimize the viability of cryopreserved HSC; however, a better understanding of cellular osmotic parameters is required for these simulations. As a model for HSC, the erythroleukemic human cell line TF-1 was used in this study. Simulations, based on the osmotic properties of TF-1 cells and on the solution properties of the intra- and extracellular compartments, were used to interpret cryoinjury associated with a two-step cryopreservation protocol. Calculated intracellular supercooling was used as an indicator of cryoinjury related to intracellular ice formation. Simulations were applied to the two-step cooling protocol (rapid cooling interrupted with a hold time) for TF-1 cells in the absence of Me₂SO or other cryoprotectants and optimized by minimizing the indicator of cryoinjury. A comparison of simulations and experimental measurements of membrane integrity supports the concept that, for two-step cooling, increasing intracellular supercooling is the primary contributor to potential freezing injury due to the increase in the likelihood of intracellular ice formation. By calculating intracellular supercooling for each step separately and comparing these calculations with cell recovery data, it was demonstrated that it is not optimal simply to limit overall supercooling during two-step freezing procedures. More aptly, appropriate limitations of supercooling differ from the first step to the second step. This study also demonstrates why high cell recovery after cryopreservation could be achieved in the absence of traditional cryoprotectants.     

Cryoinjury in human granulocytes and cytoplasts.

Although most isolated cells can be successfully cryopreserved, human granulocytes have little functional recovery after cryopreservation, even under optimized conditions. Cytoplasts, which are vesicles created from human granulocytes by depletion of organelles including granules and the nucleus, can carry out some of the complex functions of the parent granulocyte such as phagocytosis of bacteria, even after cryopreservation. Human granulocytes and cytoplasts were used in this comparative study of low-temperature responses to assess the relative importance of the plasma membrane and the granules in cryoinjury to human granulocytes. Boyle-van't Hoff plots of cell volume as a function of the reciprocal of osmolality showed that granulocytes and cytoplasts have similar osmometric behavior and equivalent osmotically inactive fractions. The hydraulic conductivities were also similar, indicating that the osmotic properties of the plasma membrane and cytoplasm were retained during preparation of the cytoplasts. Assessment of membrane integrity using fluorescein diacetate after graded freezing stresses showed that the low-temperature responses of cytoplasts were similar to those of human lymphocytes and hamster fibroblasts, with recoveries much higher than those of human granulocytes, particularly after post-thaw incubation at 37 degrees C. The results indicate that the plasma membrane is not the primary site of injury to granulocytes during freezing and thawing, and suggest that activation of cytoplasmic elements, such as granules, may constitute the early events in cryoinjury to human granulocytes. These studies have significance in approaches to the cryopreservation of granulocytes and other types of cells, such as platelets, with increased sensitivity to the conditions encountered during freezing and thawing.     

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.     

Factors affecting the survival of mouse embryos cryopreserved by vitrification

Preimplantation stage mouse embryos have been used to examine the response of a simple multicellular system to cryopreservation by the complete vitrification of the suspension. Successful vitrification requires the use of a solution of cryoprotectants that is sufficiently concentrated to supercool and solidify into a glass at practicable cooling rates. Factors that influence the survival of embryos include the concentration and composition of the vitrification solution, the procedure used to equilibrate embryos in this solution, the cooling and warming conditions, and the procedure used to dilute embryos from the vitrification solution. High rates of survival are obtained when embryos are dehydrated prior to vitrification in solutions composed of saline plus multimolar concentrations of either mixtures of permeating cryoprotectants (e.g. dimethyl sulphoxide-acetamide-propylene glycol) or single permeating cryoprotectants (propylene glycol or glycerol). Full permeation of cryoprotectants into the cells is not necessary and may lead to chemical toxicity and osmotic injury. Partial permeation and osmotic shrinkage concentrates the endogenous cytoplasmic macromolecules and greatly increases the likelihood of intracellular vitrification. Vitrification is a practical approach for embryo cryopreservation and offers new opportunities to examine fundamental aspects of cryoprotection and cryoinjury in the absence of freezing.     

Studies of free radical-mediated cryoinjury in the unicellular green alga Euglena gracilis using a non-destructive hydroxyl radical assay: a novel approach for developing protistan cryopreservation strategies

The development of cryoconservation methods for the long-term storage of algal cultures is important for the ex situ preservation of biological diversity and the maintenance of genetic stability within this group of important organisms. However, as many unicellular algae are recalcitrant to cryogenic storage, this study aims to evaluate the role of oxidative stress in cryoinjury. A non-invasive, non-destructive assay method previously applied to animal cells has been developed to evaluate free radical mediated oxidative stress in Euglena gracilis exposed to different cryopreservation treatments. The procedure employs dimethyl sulphoxide as a probe for the hydroxyl radical. Adopting this approach it was possible to identify those components of the cryopreservation protocol which were the most damaging. These were identified as preparative centrifugation and sub-zero freezing treatments. Poststorage survival in E. gracilis was significantly (P < 0.05) enhanced when the chelating agent desferrioxamine was included in the recovery medium whilst methane production was significantly (P < 0.004) reduced, suggesting that the additive was capable of ameliorating oxidative stress. The potential of using novel, exogenous antioxidant treatments developed for medical applications and applying them to enhance cryopreservation tolerance in recalcitrant unicellular algae is discussed.     

Ultrastructure-function correlative studies for cardiac cryopreservation. V. Absence of a correlation between electrolyte toxicity and cryoinjury in the slowly frozen,cryoprotected rat heart

Hearts were frozen to ?17 °C in the initial presence of 2.1 m DMSO. Attempts were made to prevent or minimize the consequences of an osmotic shock based on Lovelock's classical hypothesis of freezing injury. Substitution of mannitol or potassium for NaCl before freezing did not improve the results, nor did perfusion of thawed hearts with hyperosmotic perfusate. It was found that freezing and thawing resulted in a significant attenuation of coronary flow and that, as a result of this, DMSO was apparently retained within the heart after thawing. DMSO was also difficult to remove at 30 °C in the absence of prior freezing and caused a significant drop in coronary flow upon institution of DMSO washout with balanced salt solution. The blanching of freezing and thawing was also seen, in milder form, in nonfrozen hearts. For both frozen-thawed and nonfrozen hearts, the blanching was associated with DMSO washout with balanced salt solution. Flow was improved by perfusion with hyperosmotic perfusate in both nonfrozen and in frozen-thawed hearts, but the improvement was largely temporary. Evidence from earlier studies indicates that electrolyte concentrations during freezing cannot be correlated with cardiac cryoinjury, in support of the present findings. It is suggested instead that cryoprotectant toxicity may be the chief agent of injury under the conditions studied.     

Relation of ice formation to ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

Ultrastructural observations on the frozen state of pancreatic acinar cells were correlated with results of parallel studies before freezing and after thawing, as to cryoinjury and cryoprotection.Data support an hypothesis of freezing injury based upon intracellular ice and solution effects during rapid and slow freezing, respectively. The basis for superiority of extracellular over intracellular glycerol in cryoprotection was demonstrated in terms of these factors.Evidence is offered to explain the ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum (RER) seen after thawing, relative to the combined effects of freezing rate and glycerol. Slow freezing, in combination with the presence of extracellular glycerol, provided sufficient dehydration to almost completely suppress intracellular ice formation, yielding minimal ultrastructural alteration of RER. Greatest cryoinjury, expressed as extensive conversion of RER into sphere-like vesicles, was induced by the extensive intracellular ice formation which accompanied rapid freezing. A mechanism is suggested to explain physical damage of RER by intracellular ice.     

The significance of cooling rates and animal variability for boar sperm cryopreservation: insights from the cryomicroscope

Once the first methods for freezing mammalian semen had been established, research aimed at improving cryopreservation procedures became highly focused on the interactions between cooling rates and the permeability of the plasma membrane to water and cryoprotectants. This was based on the premise that cooling rates could be optimized from a theoretical basis for different species of interest. While this approach has stimulated considerable research, it has not achieved its original aim at the species level, largely because it overlooks inter-individual variation in sperm biochemical composition and physiology. If the underlying hypothesis is valid, however, optimal cooling rates should be identifiable for spermatozoa from individual animals. Experiments with the cryomicroscope revealed that while sperm survival after cryopreservation varied considerably between boars, there was little evidence that optimal freezing rates could be identified for individuals. Based on these findings, we tested the hypothesis that sperm susceptibility to cryoinjury is a consistent feature of each individual, but those individuals differ in susceptibility. This hypothesis was supported by evidence from an experiment with >100 boars; moreover, using genetic analyses, we demonstrated genomic differences between individual boars that correlated with post-thaw sperm quality.     

The thermodynamic principles of isochoric cryopreservation

The goal of this study is to introduce the fundamental thermodynamic principles of isochoric (constant volume) cryopreservation for low temperature preservation of biological materials. Traditionally, cryopreservation is performed in an isobaric process (constant pressure) at 1 atm, because this is our natural environment and it is most convenient experimentally. More than half a century of studies on cryopreservation shows that the major mechanism of damage during isobaric cryopreservation is the increase in intracellular ionic concentration during freezing, which presumably causes chemical damage to the components of cells. Cryoprotectants as well as hyperbaric pressures have been developed as methods to reduce the extent of chemical damage during freezing. The theoretical studies in this paper show that in isochoric cryopreservation, the increase in solution concentration during freezing is lower at each temperature by almost an order of magnitude from that in isobaric cryopreservation. This suggests that isochoric cryopreservation could be a preferential alternative to isobaric cryopreservation. The technology for isochoric cryopreservation is very simple; freezing in a constant volume chamber. Using a simple isochoric cryopreservation device, we confirm the theoretical thermodynamic predictions.     

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.     

Ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

One phase of a study on cryosurvival and cryoprotection of mammalian cells, in terms of ultrastructural alteration of rough endoplasmic reticulum (RER) within rat pancreatic acinar cells, is presented. Small (2–3 mm) squares of tissue, 0.7–0.9 mm in thickness, were compared as unfrozen controls, with (w) and without (wo) glycerol pretreatment (15% $\text{v}\text{v}$ in mammalian Ringer's solution) at 0 °C and 22 °C (to regulate glycerol permeability); as well as parallel frozen-thawed samples, after combinations of slow (3.8 °C/min) freezing (SF) and rapid (38 °C/sec) freezing (RF) with either slow (1.5 °C/min) thawing (ST) or rapid (8 °C/sec) thawing (RT). Regimens compared were SFRT, SFST, RFRT, and RFST, all w and wo glycerol pretreatment at 0 °C and 22 °C. Tissue from each treatment was prepared for electron microscopic observations. The results on rates of freezing and thawing and relative cryoprotection of intracellular and extracellular glycerol under conditions described are intended to serve as a correlative basis for subsequent parallel studies on function (protein synthesis) and ultrastructure of the frozen state. They now indicate the following: (1) Cryoinjury of RER, which occurred during all treatments compared, was manifested in irregularity, dilatation, vesiculation, and altered matrix density of cisternae, and ribosomal derangement or disjunction. Least injury was shown by some disorientation and dilatation with increasing degrees of damage involving accentuation of these and other alterations. Such ultrastructural alterations to RER are not unique to cryoinjury, since they have been induced by treatments and agents other than freeze-thawing in experimental pathology. (2) Cryoinjury is unique, however, in that it can be regulated to demonstrate a spectrum of degrees of injury to cells and their organelles, immediately after cryoexposure. Controlled cryoinjury is suggested as a research tool for studies on injury, in general, on an ultrastructural-functional level. (3) Glycerol is injurious or toxic during pretreatment. Toxicity, which resembles cryoinjury, is greater during 22 ° C (intracellular) than 0 °C (extracellular) glycerol pretreatment, especially with respect to dilatation of cisternae. (4) Extra-cellular glycerol is cryoprotective during both slow and rapid freezing followed by either slow or rapid thawing, while little or no cryoprotection is afforded when glycerol is located simultaneously in the intracellular and extracellular location. (5) Rate of freezing is more important than rate of thawing as a factor in cryosurvival. Rapid freezing is more injurious than slow freezing, in the absence of glycerol or in the presence of extracellular glycerol, with slight or no differences seen as a function of thawing rate. Neither rate of freezing nor rate of thawing is of serious consequence when glycerol is intracellular. (6) Rate of thawing has importance after slow freezing, when slow thawing is more injurious than rapid, but not after rapid freezing, either in the presence or absence of extracellular glyeerol.     

Studies of free radical-mediated cryoinjury in the unicellular green alga Euglena gracilis using a non-destructive hydroxyl radical assay: A novel approach for developing protistan cryopreservation strategies

The development of cryoconservation methods for the long-term storage of algal cultures is important for the ex situ preservation of biological diversity and the maintenance of genetic stability within this group of important organisms. However, as many unicellular algae are recalcitrant to cryogenic storage, this study aims to evaluate the role of oxidative stress in cryoinjury. A non-invasive, non-destructive assay method previously applied to animal cells has been developed to evaluate free radical mediated oxidative stress in Euglena gracilis exposed to different cryopreservation treatments. The procedure employs dimethyl sulphoxide as a probe for the hydroxyl radical. Adopting this approach it was possible to identify those components of the cryopreservation protocol which were the most damaging. These were identified as preparative centrifugation and sub-zero freezing treatments. Post-storage survival in E. gracilis was significantly (P < 0.05) enhanced when the chelating agent desferrioxamine was included in the recovery medium whilst methane production was significantly (P < 0.004) reduced, suggesting that the additive was capable of ameliorating oxidative stress. The potential of using novel, exogenous antioxidant treatments developed for medical applications and applying them to enhance cryopreservation tolerance in recalcitrant unicellular algae is discussed.     

Cryobiological determinants of frozen semen quality, with special reference to stallion

Success in cryopreserving stallion semen has been very variable. Several different freezing regimes have been published. However, because extenders and procedures used in each regime have differed, direct comparison of these techniques has been very difficult, and controlled studies comparing different techniques have not been reported. A number of different factors affect sperm cryosurvival. In this article we review briefly current cryopreservation procedures for stallion semen, and then in more detail cryobiological determinants of sperm function, and mechanisms of cryoinjury and cryoprotectant action. Specific attention is given to data relating to stallion sperm. The complexity of sperm cell biology is believed to be an important factor when developing improvements in stallion semen cryopreservation. It may be assumed that impairment of cell function resulting from cold and osmotic shock is a main source of stallion sperm sensitivity to conventional freezing procedures. Further physiological studies on stallion sperm are required to understand the mechanisms by which cryopreservation alters sperm function and influences selection of sperm with higher fertilizing potential. Such studies should focus especially on the processes involved in sperm volume regulation, sperm-oviduct interaction, capacitation and cellular signalling, and on the alterations in these processes caused by cryopreservation.     

Cryopreservation of Human Oocytes and Ovarian Tissue

Oocyte cryopreservation has the potential to be an important adjunct to assisted reproductive technologies and bypasses some ethical, moral, and religious dilemmas posed by human embryo cryopreservation. The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Among the morphological factors, the maturity, quality, size of the oocyte, the presence or the absence of the cumulus oophorus seems to play an important role in oocyte survival after thawing. The main biophysical factor of cellular disruption during cryopreservation process in the intracellular ice formation that can be avoided by an adequate cell dehydration; thus reducing the intracellular water by increasing the dehydration process we can limit the damages of the cryopreservation procedure. The dehydration process can be affected by the presence and concentration of the cryoprotectants in the freezing solutions (equilibration and loading solutions), and by the freezing and thawing rate. Two additional properties of cryoprotectants help to protect cells during slow cooling, when the cells are very dehydrated and are surrounded by concentrated salts. The cryoprotectants appear to reduce damage caused by high levels of salt, a property known as salt buffering. Some events occurring to the oocyte during cryopreservation procedure has been found to be a premature exocitosis of cortical granules, leading to an intempestive zona hardening and consequently to a reduction of fertilization rate, and the cryoinjury to the zona pellucida leading to a polispermic fertilization. ICSI is an efficient method to by pass these two events and to achieve a satisfactory outcome in terms of normal fertilization of cryopreserved oocytes. The application of the ICSI to cryopreserved oocytes did not seem to increase the degeneration rate after insemination with respect to fresh oocytes. The increased oocyte survival rate and the use of ICSI have facilitated the recent increase in the number of pregnancies and live birth.     

Improved recovery of post-thaw motility and vitality of human spermatozoa cryopreserved in the presence of dithiothreitol

Semen was collected in the laboratory from nine healthy donors. The concentrations and the percentages of live and motile spermatozoa in all semen samples were within the normal range. Each sample was diluted with citrate-egg yolk-glycerol medium with and without 5 mM dithiothreitol (DTT). Samples were frozen in liquid nitrogen vapor (?70 °C) for 7 min and subsequently stored in liquid nitrogen. The effect of DTT in cryopreservation of sperm was determined by comparing percentage of motile and live spermatozoa between controls and DTT-treated post-thaw samples. Percentage of motile spermatozoa was determined by two techniques, laser Doppler velocimetry (LDV) and light microscopy. The percentage of live spermatozoa was measured by microscopic evaluation after staining with eosin-nigrosin. It was shown that the addition of DTT to the freezing medium significantly improved the recovery of motile and live spermatozoa in the post-thaw samples. The mean motility recovery, as measured by LDV, was 44.9% in the controls as compared to 73.9% in the DTT-treated samples. Similarly the mean recovery of live spermatozoa in the controls and DTT-treated samples was 66.5 and 86.6%, respectively. Based on these results, a new hypothesis implicating lipid peroxidation in cryoinjury is proposed. It is also suggested that the use of DTT in the freezing medium may offer an advantage over the commonly used techniques of human sperm cryopreservation.     

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.     

Cryopreservation of sea urchin embryos (Paracentrotus lividus) applied to marine ecotoxicological studies

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

Effects of synthetic antifreeze glycoprotein analogue on islet cell survival and function during cryopreservation

The antifreeze glycoprotein (AFGP), found in the blood of polar fish, is known to prevent ice crystal growth and to depress the freezing temperature, which may in turn protect tissues from freezing injury. The chemical synthesis of AFGP is an attractive alternative to its difficult isolation from natural sources, and this would permit quality control and mass production. In spite of recent success in islet transplantation for the treatment of type 1 diabetes mellitus, existing methods for the long-term preservation of islets are considered to be suboptimal and inadequate, which indicates the need for the development of improved methods. Rat islets were isolated from male Wistar rats, using intraductal collagenase distention, mechanical dissociation, and Ficoll-Conray gradient purification. Islets were cultured overnight and then cryopreserved in RPMI1640 in the presence of dimethyl sulfoxide (Me2SO) and 10% FCS with various concentrations of syAFGP, followed by slow cooling (0.3 degrees C/min) and rapid thawing (200 degrees C/min) as described by Rajotte. The freezing process was observed by cryomicroscopy. Islet recovery post-cryopreservation was 85.0 +/- 6.2% with syAFGP and 63.3 +/- 14.2% without syAFGP, both compared with the pre-cryopreservation counts (P < 0.05). The in vitro islet function measured by insulin release was equivalent to a static stimulation index of 3.86+/-0.43 for the islets that were frozen-and-thawed with syAFGP, compared to 2.98 +/- 0.22 without syAFGP (P < 0.05). At a concentration of around 500 microg/ml syAFGP, a strong attenuation of ice crystal growth and formation was observed by cryomicroscopy and these ice crystals did not cause cryoinjury. In conclusion, the attenuation of ice crystallization by syAFGP improves islet survival and function following cryopreservation and thawing.     

深低温冷冻技术的研究进展

细胞及组织的深低温保存有较高的临床应用和研究价值,在冷冻保存技术中两个关键领域首先发展的是冷冻控制率。冷冻保护剂能增加溶液粘性,提高冷冻速率从而保护细胞及组织免受冷冻损伤。对最佳冷冻保护剂的研究为保存临床应用的组织工程产品提供了理论依据。而玻璃化冷冻近年来越来越受到人们的关注,玻璃化冷冻技术具有冷冻速度快、冻融损伤小,操作简单等优点,能够提高复苏后的存活率。本文对深低温保护剂的组成、分类、应用及冷冻保存的重大进展和障碍进行了综述。     

Effect of egg yolk and seminal plasma heparin binding protein interaction on the freezability of buffalo cauda epididymal spermatozoa

Egg yolk is routinely used in most of the extenders for cryopreservation of semen, but mechanisms of protection of spermatozoa by egg yolk are not very clear. Investigations with buffalo cauda epididymal sperm have shown that seminal plasma heparin binding proteins have detrimental effects during semen cryopreservation. The present study was conducted to investigate the effect of egg yolk on the detrimental effects of heparin binding proteins during cryopreservation of buffalo cauda epididymal spermatozoa. The results indicated that egg yolk was able to reduce the heparin binding proteins mediated cryoinjury in spermatozoa. One of the mechanisms of protection of spermatozoa from cryoinjury by egg yolk may be due to the inhibition of deleterious actions of heparin binding proteins on the spermatozoa.