Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi

One hundred and ninety five strains of fungi were observed during freezing and thawing using a cryogenic light microscope. There was no obvious link between taxonomic position and their morphological response to freezing and thawing. The viability of seven of these strains was examined following freezing and thawing in the presence or absence of the cryoprotectants glycerol and dimethyl sulphoxide. Intracellular ice and hyphal shrinkage were not necessarily lethal events, but in many cases they affected the rate and quality of growth. Both cryoprotectants reduced shrinkage, shifted the cooling rate where intracellular ice formed in many cases, and improved the recovery of strains. The results presented aid the development of successful cryopreservation protocols.     

A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing

The changes in morphology of Penicillium expansum Link and Phytophthora nicotianae Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage. Viability of 0.5-1.0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 degrees C in the presence or absence of glycerol. In P. expansum shrinkage occurred in all hyphae at rates of cooling of less than 15 degrees C min-1; at faster rates intracellular ice nucleation occurred. The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18 degrees C min-1 to 55 degrees C min-1. This species was particularly resistant to freezing injury and recovery was greater than 60% at all rates of cooling examined. At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated. In contrast, during the cooling of Ph. nicotianae in the growth medium, shrinkage occurred and no samples survived on thawing from -196 degrees C. However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 degrees C min-1 the loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.     

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

Skin ice nucleators and glycerol in the freezing-tolerant frog <Emphasis Type="Italic">Litoria ewingii</Emphasis>

The brown tree frog (Litoria ewingii) is the only known Southern Hemisphere vertebrate that can survive full-body freezing. Freezing challenges living organisms in many ways, with ice formation in the body producing a suite of physical and metabolic stresses which can damage cells and tissues. The present study looked at two mechanisms that address some of these stresses: cryoprotectants and ice nucleating agents (INAs). Skin secretions from L. ewingii were sampled along with microhabitat substrate and tested for the presence of INAs, which help control ice formation in the body. L. ewingii plasma was tested for seasonal and freezing-induced changes in both glucose and glycerol, which may have a cryoprotective role in freezing-tolerant frogs. Glycerol levels increased on freezing and decreased on thawing, while glucose levels did not change on freezing but increased on thawing. This suggests that glycerol may be acting as a cryoprotectant, although levels are low compared to other frogs. A clear seasonal change was seen in INA activity, with greater activity in winter than in summer. While potent INAs came from the microhabitat substrate, this work has shown for the first time that skin secretions also contain active INAs.     

Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice

Successful cryopreservation demands there be little or no intracellular ice. One procedure is classical slow equilibrium freezing, and it has been successful in many cases. However, for some important cell types, including some mammalian oocytes, it has not. For the latter, there are increasing attempts to cryopreserve them by vitrification. However, even if intracellular ice formation (IIF) is prevented during cooling, it can still occur during the warming of a vitrified sample. Here, we examine two aspects of this occurrence in mouse oocytes. One took place in oocytes that were partly dehydrated by an initial hold for 12 min at -25 degrees C. They were then cooled rapidly to -70 degrees C and warmed slowly, or they were warmed rapidly to intermediate temperatures and held. These oocytes underwent no IIF during cooling but blackened from IIF during warming. The blackening rate increased about 5-fold for each five-degree rise in temperature. Upon thawing, they were dead. The second aspect involved oocytes that had been vitrified by cooling to -196 degrees C while suspended in a concentrated solution of cryoprotectants and warmed at rates ranging from 140 degrees C/min to 3300 degrees C/min. Survivals after warming at 140 degrees C/min and 250 degrees C/min were low (<30%). Survivals after warming at > or =2200 degrees C/min were high (80%). When warmed slowly, they were killed, apparently by the recrystallization of previously formed small internal ice crystals. The similarities and differences in the consequences of the two types of freezing are discussed.     

Studies on the vitality of human bone marrow during collection and deep-freeze preservation

The cryoprotective effect of dimethylsulfoxide, glycerol and polyethylenglycol during freezing and thawing of human bone marrow was investigated by eosin staining test, an acridinorange fluochrome staining test and by RNA- and DNA-synthesis tests. In these tests the overall yield of vital nucleated cells, referred to the number in the absence of cryoprotectants and freezing and thawing, amounted to 50% with dimethylsulfoxide, 30% with glycerol, and 10% with polyethylenglycol. With dimethylsulfoxide and glycerol the loss of vital nucleated cells is almost entirely due to the addition of cryoprotectants. Polyethylenglycol freezing and thawing also leads to a great loss of vital nucleated cells. The results with dimethylsulfoxide show that the currently employed techniques of punction, preparation, freezing and thawing of bone marrow are suitable for clinical application.     

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.     

An isolated perfused rat mesentery model for direct observation of the vasculature during cryopreservation

An intact vasculature is essential for successful hypothermic perfusion and cryopreservation of solid organs, but few studies have specifically assessed the vascular effects of these procedures. A technique was therefore developed for continuous, direct observation of an isolated vascular bed during hypothermic perfusion with cryoprotectants, and during freezing and thawing. The isolated rat mesentery was spread across a controlled low temperature microscope stage and perfused with solutions containing fluorescein isothiocyanate (FITC)-Dextran 70 as an indicator of macromolecular permeability of the vessels. Hypertonic citrate washout, HP-5 perfusion (23), rapid and slow addition and removal of glycerol, and freezing/thawing were studied. Control perfusion with HP-5 produced slow FITC-Dextran leakage, reflecting normal physiological macromolecular permeability of vessels. Rapid addition of glycerol dramatically increased vascular permeability, consistent with osmotic damage to vessels. Rapid removal stopped flow through capillaries and decreased vascular dimensions, suggesting overhydration of endothelial cells and extravascular tissue. Venules and capillaries were the most susceptible vessels to osmotic stress. Slow addition and removal of glycerol (80 mmol/liter/min) produced results similar to control perfusions. During slow freezing (0.5 degree C/min to -5 degrees C) extravascular ice compressing vessels was more obvious than intravascular ice. Glycerol afforded some protection to the microvasculature during freeze/thaw cycles since flow was reestablished in venules and arterioles after thawing, although FITC-Dextran leakage indicated that damage had occurred.     

Freezing of nonwoody plant tissues: IV. Nucleation sites for freezing and refreezing of onion bulb epidermal cells

A freezing microscope and videotape recorder were used to study ice formation in onion bulb epidermal cells during repeated freezing. Behavior during refreezing after freezing and thawing indicated that the barrier to ice crystal propagation previously reported in living cells was not damaged by freezing. Refreezing in many cells began at a site other than that responsible for the initial freezing.     

Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm

The process of freezing and thawing induces certain cellular damage in rainbow trout (Oncorhynchus mykiss) spermatozoa. We have previously demonstrated that after freezing and thawing decreased fertility in rainbow trout (Oncorhynchus mykiss) spermatozoa, is related to sublethal damage to the plasma membrane. External cryoprotectants are known to stabilize the sperm cell membrane against such damage. In the current study, we used a basic freezing extender containing #6 Erdahl and Graham and 7% DMSO and added egg yolk, BSA, and a soybean-protein complex (DanPro S760) singly and in various combinations. To assess the effect of these cryoprotectants we evaluated the percentage of cells with progressive motility, permeability of cells to propidium iodide (viability) after exposure for 30 sec, 2, 5, 10 and 15 min. to hypo- and isoosmotic solutions of 10 and 300 mOsm, and the in vitro fertility rate. Fertility trials were performed using 1.87 x 10(7) spermatozoa/egg. Some of the tested stabilizers increased motility, increased viability, or reduced cell fragility after freezing and thawing. Nevertheless these quality improvements demonstrated by the "in vitro" tests do not always correlate with high fertility. The best membrane protection in terms of resistance to hypoosmotic shock was achieved when BSA and egg yolk were added to the extender. The highest fertility rates were obtained with DanPro S760 alone or in combination with BSA; the use of BSA with egg yolk did not improve this parameter. Our results demonstrated that some external cryoprotectants effectively increased membrane resistance during freezing and thawing, but some of the tested mixtures interfered with fertilization. Soybean protein concentrate provided good protection and increased fertility rates in cryopreserved trout spermatozoa.     

Ultrastructure before freezing,while frozen,and after thawing in assessing cryoinjury of mouse epididymal spermatozoa

Tails of mouse epididymides were treated as follows: control, unfrozen with and without cryoprotective agents (CPA); frozen (to below ?80 °C), slowly (8 °C/min), and rapidly (18 °C/sec), with and without CPA. Intracellular and/or extracellular location of CPA, at least glycerol, was influenced, respectively, by high (22 °C) or low (0 °C) exposure temperature. Standard procedures in electron microscopy were employed and the frozen state preserved by freeze-substitution. Motility before freezing and after thawing was the criterion of cryosurvival.Results showed no evidence of deleterious ultrastructural effects of freezing at rates compared, or of benefits of CPA, regardless of their cellular location. Differences were noted, however, in the appearance of spermatozoa in the frozen state, as a function of the rate of freezing but not as a function of the presence, absence, or location of either glycerol of DMSO. Rapidly frozen cells showed intracellular ice formation in the acrosome, neck, midpiece, and tail regions; there was no intranuclear ice, and extracellular ice artifacts were small. Slowly frozen cells showed large extracellular ice artifacts with evidence of shrinkage distortion due to the dehydration induced by extracellular ice. No spermatozoa survived any of the freezing treatments, showing the lethal effect of both extracellular ice during slow freezing and of intracellular and/or extracellular ice during rapid freezing.     

Thermodynamic modeling and cryomicroscopy of cell-size, unilamellar, and paucilamellar liposomes

Cryomicroscope studies of large unilamellar liposomes indicate that liposomes are an excellent model for studying membrane response to freezing and thawing. Liposomes are attractive for such use because they can be custom-manufactured for a particular investigation. In addition, liposome responses to freezing and thawing mimic real cell behavior in a number of significant ways. Analogous behavior includes osmotic shrinkage at slow cooling rates, internal ice formation at fast cooling rates, comparable nucleation temperatures, and a variety of comparable thawing responses. Experimental determination has been made of the equilibrium osmotic properties and the nonequilibrium water transport properties of the egg lecithin liposomes used in the freezing studies. These properties have been used in a computer model to simulate volume changes resulting from water transport during freezing and thawing. Comparison between computer model predictions and experimental data for the liposome volume response during freezing indicates reasonable agreement whereas computer simulations of volume response during thawing do not match experimental data well.     

Natural cryoprotectants combinations of l-proline and trehalose for red blood cells cryopreservation

Cryopreservation of red blood cells (RBCs) holds great potential benefits for supplying transfusion timely in emergencies. Currently, glycerol is the main cryoprotectant permitted in clinical therapy for RBCs cryopreservation, but its broad application is limited by the toxicity and complex deglycerolization process. Successful cryopreservation of RBCs using more effective materials should be studied to reduce freezing damage, increase biocompatibility, and save processing time. Herein, a simple protocol using natural cryoprotectants combinations of l-proline and trehalose attains a low degree of hemolysis (11.2 ± 2.73%) after thawing compared to glycerol. Furthermore, the morphology of RBCs and the activities of Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase maintain well. Further mechanism study shows that l-proline plays an important role in decreasing the freezing points and inhibiting the growth of ice crystal by permeating into cells during the freezing process. While trehalose works as an inhibitor of ice growth in the freezing process and ice recrystallization in the thawing process. This simple l-proline & trehalose combinations protocol is a promising method to replace current time-consuming and labor-intensive cryopreservation methods of RBCs.     

Cryopreservation of seabream (Sparus aurata) spermatozoa

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

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 development of a cryopreservation method suitable for a large cyanobacteria collection

Laboratories worldwide working with cyanobacteria have created culture collections to carry out related studies. However, the lack of manpower (especially after the studies were finished), maintenance costs and proper preservation methods often results in the loss of research materials and a waste of isolation effort. Several parameters are generally considered very important in cryopreservation, including the choice of the cryoprotectant, cryoprotectant concentration, freezing rate, physiological status of the culture and thawing procedure. This makes it very difficult to establish universal guidelines for cyanobacteria cryopreservation. Herein, we present a cryopreservation method suitable for a range of strains, using two cryoprotectants (methanol and dimethyl sulfoxide at a final concentration of 5 and 3 %, respectively) along with a combined vital staining and reproductive viability criteria for the post-thawing recovery. The obtained results are very encouraging as more than 83 % of tested cyanobacteria were amenable for cryopreservation, 80 % of strains (111 in total) showing more than 90 % recovery with at least one of the cryoprotectants used.     

An Adiabatic Calorimetry Method to Determine the Thermodynamic Characteristics of Cryoprotectants

Biophysics - Abstract—The efficiency of cryoprotectants used to protect cells from damage is usually evaluated by the changes in vital cell parameters after a freezing–thawing cycle....     

Thermal shock and dilution shock as the causes of freezing injury

We suggest that during slow freezing, cellular membranes are altered by the hypertonic solutions produced. This alteration in itself does not cause membrane leakage of normally impermeant solutes but it renders the cells susceptible to solute leakage on the application of a stress, which is provided during freezing by the reduction in temperature (thermal shock) and during thawing by dilution (dilution shock).During slow freezing the effects of cooling rate changes are due to the different times available for the hypertonic solutions to affect the membrane. At a given cooling rate cryoprotective agents reduce the effect on the cells at each temperature during freezing perhaps by reducing the ionic strength. The thermal shock stress during cooling and the dilution shock during thawing thus damages the cells less. With rapid freezing, there is insufficient time for these effects to take place during cooling, which allows the cells to reach low temperatures without thermal shock damage. However, the presence of extracellular ice and the formation of intracellular ice provide hypertonic conditions that render the cells liable to dilution shock on thawing. The slower the rate of thawing of rapidly cooled cells the greater will be the damage from this dilution shock.     

Effects of extender composition, cooling rate and freezing on the fertilisation viability of spermatozoa of the Pacific oyster (Crassostrea gigas)

The aim of this research was to optimise protocols for freezing spermatozoa of the Pacific oyster. All the phases of the cryopreservation procedure (choice of cryoprotectant, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa to restore on thawing the morphological and physiological characteristics of fresh semen. The choice of type and concentration of cryoprotectant in which semen is incubated before freezing is fundamental for a successful cryopreservation: the cryoprotectants (dimethylsulfoxide--Me(2)SO, ethylene glycol--EG, propylene glycol-PG, and glycerol in concentrations between 5 and 15%) were tested for their toxicity on the semen exposed up to 30 min at +26 degrees C (room temperature) by evaluating its ability to fertilise and the embryo development to the regular D larval stage. The best cryoprotectants, Me(2)SO, EG, and PG 5, 10, and 15% respectively, were used for the pre-cooling (adaptation/cooling) tests. Two different adaptation/cooling procedures were tested: (A) from +26 degrees C to 0-2 degrees C (2.6 degrees C/min) and (B) at +26 degrees C for 15 min. Lastly, using the cryoprotectants and the adaptation procedure (B) that had given the best results in the preceding stages of the experiment, four cooling rates were tested: 6, 11, 16, and 21 degrees C/min. It was seen that the semen that was incubated with EG 10%, adapted at +26 degrees C for 15 min, and then cooled at a rate of 6 degrees C/min showed a percentage of regular D larvae on thawing comparable to that of fresh semen (p > 0.05).     

Infective Juveniles of the Entomopathogenic Nematode,Steinernema feltiae Produce Cryoprotectants in Response to Freezing and Cold Acclimation

Steinernema feltiae is a moderately freeze-tolerant entomopathogenic nematode which survives intracellular freezing. We have detected by gas chromatography that infective juveniles of S. feltiae produce cryoprotectants in response to cold acclimation and to freezing. Since the survival of this nematode varies with temperature, we analyzed their cryoprotectant profiles under different acclimation and freezing regimes. The principal cryoprotectants detected were trehalose and glycerol with glucose being the minor component. The amount of cryoprotectants varied with the temperature and duration of exposure. Trehalose was accumulated in higher concentrations when nematodes were acclimated at 5°C for two weeks whereas glycerol level decreased from that of the non-acclimated controls. Nematodes were seeded with a small ice crystal and held at -1°C, a regime that does not produce freezing of the nematodes but their bodies lose water to the surrounding ice (cryoprotective dehydration). This increased the levels of both trehalose and glycerol, with glycerol reaching a higher concentration than trehalose. Nematodes frozen at -3°C, a regime that produces freezing of the nematodes and results in intracellular ice formation, had elevated glycerol levels while trehalose levels did not change. Steinernema feltiae thus has two strategies of cryoprotectant accumulation: one is an acclimation response to low temperature when the body fluids are in a cooled or supercooled state and the infective juveniles produce trehalose before freezing. During this process a portion of the glycerol is converted to trehalose. The second strategy is a rapid response to freezing which induces the production of glycerol but trehalose levels do not change. These low molecular weight compounds are surmised to act as cryoprotectants for this species and to play an important role in its freezing tolerance.