Cryobiology

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We exposed human corneas to various concentrations of four cryoprotectants by one of two methods: a gradual increase to the final concentration (ramp method) and a series of steps to the final concentration (step method). Endothelial damage was manifest as a decrease in the number of endothelial cells per unit area. The highest concentrations that did not cause a loss of endothelial cells by the ramp and step methods, respectively, were 4.3 and 2.0 M glycerol, 2.0 and 4.3 M dimethylsulfoxide, 2.0 and 3.0 M 1,2-propanediol, and 2.0 and 2.5 M 2,3-butanediol. The ramp method achieved higher final concentrations with the more slowly permeating glycerol, but required low toxicity. The step method achieved higher final concentrations with the more toxic cryoprotectants by limiting the exposure time, but required more rapid permeation. None of the four cryoprotectants was tolerated at concentrations sufficient for vitrification at practical cooling and warming rates.
 
We tested the tolerance of human corneas to a vitrification solution, modified VS41A, containing 3.1 M dimethyl sulfoxide, 3.1 M formamide, and 2.2 M 1,2-propanediol in a carrier solution consisting of the corneal storage medium CPTES with 2.5% w/v chondroitin sulfate. Seven human corneas were exposed for 10 min each to graded concentrations of the solution at 0 degree C, remaining in the full-strength solution for 10 min. The corneas had significantly more endothelial cell damage (P < 0.05) than seven mated control corneas, but it was minimal (4.3% cell loss). Attempts at vitrification and rewarming of three corneas exposed to the solution by this protocol, however, resulted in ice formation in the peripheral corneal stroma and severe endothelial damage. Presumably, equilibration with the cryoprotectant in the thicker periphery of the human cornea had not occurred. Ice did not form on the center of one cornea, and substantial numbers of central endothelial cells survived after vitrification in this case. Immersion of the human corneas for 25 min in each of the four graded solutions at 0 degree C was required for sufficient penetration of the cryoprotectant to allow total corneal vitrification and rewarming without ice formation. This prolonged exposure to modified VS41A caused unacceptable damage to the corneal endothelium, however. Successful vitrification of human corneas with this solution will require a safe method for obtaining corneal equilibration with the cryoprotectant.
 
The preceding papers in this series have described techniques that permit the introduction and removal of propane-1,2-diol (propylene glycol, PG) with human platelets, in concentrations up to 2 M, without producing serious damage. These methods have now been used in attempts to cryopreserve platelets, with assessment of survival by the hypotonic stress response and ADP-induced aggregation. PG concentrations of 0.5, 1.0, 2.0, and 2.5 M and cooling rates between 0.4 and 100 degrees C/min were studied. The maximum response in the hypotonic stress test was no better than 17% and the greatest ADP-induced aggregation was only 6%; these results were obtained with 0.5 M PG, a cooling rate of 14 degrees C/min, and rapid warming (approximately 150 degrees C/min). The failure of PG concentrations greater than 0.5 M to improve survival was unexpected. When cooling was interrupted at progressively lower temperatures and function assessed, it was possible to relate the extent of damage to temperature and then, with the aid of phase diagrams, it was possible to show that, irrespective of the initial concentration of PG, the extent of damage was closely correlated with the concentration of PG produced at the minimum temperature used. It is concluded that the toxicity of PG increases so steeply with the increasing concentration produced by the separation of ice during freezing that this effect is sufficient to counteract the cryoprotective action of this solute for platelets.
 
Any method of cryopreservation of the cornea must maintain integrity of the corneal endothelium, a monolayer of cells on the inner surface of the cornea that controls corneal hydration and keeps the cornea thin and transparent. During freezing, the formation of ice damages the endothelium, and vitrification has been suggested as a means of achieving ice-free cryopreservation of the cornea. To achieve vitrification at practicable cooling rates, tissues must be equilibrated with high concentrations of cryoprotectants. In this study, the effects of propane-1,2-diol on the structure and function of rabbit corneal endothelium were studied. Corneas were exposed to concentrations of propane-1,2-diol ranging from 10 to 30% v/v in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, and 6% w/v bovine serum albumin. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 degrees C for 6 hr. Exposure to 10-15% v/v propane-1,2-diol was well tolerated for 20 min at 4 degrees C when the cryoprotectant was removed in steps or by sucrose dilution. However, exposure to 25% v/v propane-1,2-diol for 20 min at 0 or -5 degrees C was consistently tolerated only when 2.5% w/v chondroitin sulfate was included in the vehicle solution. Exposure to 30% v/v propane-1,2-diol was harmful at -5 and -10 degrees C. The endothelial damage following exposure to 30% v/v propane-1,2-diol was probably the result of a toxic effect rather than osmotic stress. Although 25% v/v propane-1,2-diol does not vitrify at cooling rates that are practicable for corneas, it could at this concentration form a major component of a vitrification solution comprising a mixture of cryoprotectants.
 
The merit of corneal cryopreservation by vitrification as opposed to conventional freezing is the avoidance of ice damage which is believed to disrupt the integrity of the corneal endothelium resulting in loss of corneal transparency. The cornea must be equilibrated with high concentrations of cryoprotectant in order to achieve vitrification at practicable cooling rates. In an earlier study, corneas were exposed to 3.4 mol/liter propane-1,2-diol (Rich and Armitage (1990) Cryobiology 27, 42-54). The present study exposed rabbit corneas to concentrations of propane-1,2-diol between 3.4 and 5.4 mol/liter in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, 6% (w/v) bovine serum albumin, and 2.5% (w/v) dextran sulfate. Dextran sulfate was as effective as chondroitin sulfate at improving endothelial tolerance of 3.4 mol/liter propane-1,2-diol. This beneficial effect may be linked to the polyanionic nature of these molecules. Corneas exposed to 5.4 mol/liter propane-1,2-diol were cooled in liquid nitrogen vapor at a temperature of -140 degrees C for 2 h. Warming was achieved by direct transfer to a dilution solution at -10 degrees C. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 degrees C for 6 h. Endothelial structure was observed by specular microscopy during the perfusion and by scanning electron microscopy after perfusion. Corneas tolerated exposure to 3.4 mol/liter propane-1,2-diol for 20 min at 0 degrees C and to 4.1 mol/liter for 10 min at -10 degrees C. Exposure to 4.8 and 5.4 mol/liter for 10 min at -10 degrees C caused endothelial damage, although a degree of endothelial function was retained. Function following exposure to 5.4 mol/liter was improved by reducing the temperature of exposure to -15 degrees C. Corneas cooled after exposure to 5.4 mol/liter propane-1,2-diol for 10 min at -15 degrees C apparently vitrified, but devitrified on warming. The corneas swelled to such an extent during perfusion that the endothelium could not be viewed by specular microscopy, subsequent scanning electron microscopy showed a severely disrupted endothelium.
 
In previous studies, we demonstrated that 1,2-propanediol induces shortening and bundling of actin filaments, both in vitro and in vivo, and that it enhances actin/alpha-actinin interaction, especially at low temperature. 1,2-Propanediol also promotes homogeneous microporous networks which can be vitrified by rapid cooling. In the present study, dynamical rheological measurements were performed under various sets of experimental conditions including temperature (4 or 20 degrees C), protein concentrations (actin and alpha-actinin), and 1,2-propanediol presence or absence. Gelation kinetics were monitored, and the resulting actin mechanical properties investigated, in order to untangle the respective effects of the experimental parameters. Whether in the presence or absence of solvent, low temperature brings about a rigidification of the sample, as does high protein concentration, as expected. However, 1,2-propanediol itself involves either softening of the sample (at high temperature and low protein concentration or at low temperature and high protein concentration) or rigidification in the case of low temperature and low protein concentration. These effects result from the competition between actin/alpha-actinin affinity (enhanced by both low temperature and 1,2-propanediol), bundling of filaments (fostered by alpha-actinin for alpha-actinin/actin ratios used), rate of actin polymerization (higher at high temperature), shortening effect of 1,2-propanediol on actin filaments, and chain mobility (lower at high protein concentration). As discussed, only the combination of low temperature and low protein concentration induces full crosslinking of the system into a viscoelastic solid under the influence of 1,2-propanediol.
 
Using high-pressure liquid chromatography two cryoprotectant solvents, dimethyl sulphoxide (four manufacturers) and 1,2-propanediol (one manufacturer) were investigated for aldehyde content. Fractionation of the aldehydes by high pressure liquid chromatography identified up to 11 aldehydes and two ketones in both cryoprotectant solvents in varying concentrations, which differed between manufacturer and container type. Of the 11 aldehydes identified, formaldehyde and acetylaldehyde were consistently in the greatest concentrations. As the low molecular weight aldehydes identified contain reactive polarised carbonyl groups they represent a potential source of intracellular damage when used in oocyte cryopreservation.
 
It is generally assumed that when cells are cooled at rates close to those corresponding to the maximum of survival, once supercooling has ceased, above the eutectic melting temperature the extracellular ice is in equilibrium with the residual solution. This did not seem evident to us due to the difficulty of ice crystallization in cryoprotective solutions. The maximum quantities of ice crystallized in glycerol and 1,2-propanediol solutions have been calculated from the area of the solidification and fusion peaks obtained with a Perkin-Elmer DSC-2 differential scanning calorimeter. The accuracy has been improved by several corrections: better defined baseline, thermal variation of the heat of fusion of the ice, heat of solution of the water from its melting with the residual solution. More ice crystallizes in the glycerol than in the 1,2-propanediol solutions, of which the amorphous residue contains about 40 to 55% 1,2-propanediol. The equilibrium values are unknown in the presence of 1,2-propanediol. With glycerol, in our experiments, the maximum is first lower than the equilibrium but approaches it as the concentration increases. It is not completely determined by the colligative properties of the solutes.
 
Three ternary systems with water and 1,2-propanediol were investigated, where the third component is 1-propanol, ethanol, or glycerol. 1-Propanol and ethanol give hydrates in their aqueous solutions as well as in these ternary systems, while glycerol gives none. No gain in the stability of the amorphous state and glass-formation tendency is obtained, for the same water contents, when 1,2-propanediol is partially replaced by ethanol. The gain is negligible when it is partially replaced by glycerol. On the contrary, a large maximum in the stability of the amorphous state is obtained, with a critical warming rate dropping from 108 to 104 °C/min in the presence of 65% (w/w) water when 15% (w/w) of the 1,2-propanediol is replaced by 1-propanol. The decrease in the glass formation tendency due to this replacement and corresponding to a few hydrate crystallization is small. Not only the higher stability of the amorphous state, but also in some cases the replacement of ice crystallization by clathrate crystallization at lower temperatures could perhaps contribute to a better cryoprotection of cells for some cooling and warming rates. The similarities observed between the ternary systems investigated gives an idea of the general behaviour of these systems
 
We have studied the effect of 1,2-propanediol (PROH) on cumulus-oocyte complexes from the mouse. We determined the morphological survival rate, the pattern of parthenogenetic activation, and the microtubular and chromosomal organization. Cumulus-oocyte complexes were collected at 16 h post hCG from superovulated female hybrid mice. These cumulus-intact oocytes were exposed to 1.5 or 3 M PROH for 6, 12, or 18 min at 0, 22, or 37 degrees C. The cryoprotectant was diluted out in a 1 M sucrose solution at 22 degrees C. After 5-6 h at 37 degrees C, oocytes were denuded and examined under Nomarski optics. The results show that PROH can induce degeneration and parthenogenetic activation in the mouse oocyte in a concentration, temperature, and time-dependent way. As the activation stimulus was strengthened, an increasing proportion of oocytes shifted from parthenogenetic activation with polar body extrusion to parthenogenetic activation with polar body retention and even to immediate cleavage. Nontoxic and nonactivating conditions involved mainly exposure to 1.5 M PROH at 0 degrees C. Spindle integrity and chromosomal organization were analyzed for exposure to 1.5 and 3 M PROH for 12 min at 0 degrees C. The separate effect of cooling and exposure to 1 M sucrose were also evaluated. Microtubules were visualized by monoclonal anti-alpha-tubulin labeling followed by immunogold-silver staining. Cooling and exposure to 1 M sucrose or to 1.5 M PROH did not induce major abnormalities in the microtubular or chromosomal organization. On the other hand, a significant percentage of deformities such as spindle size reduction and loss of bipolarity were observed after exposure to 3 M PROH. The results of the present study demonstrate that the use of PROH as a single cryoprotectant for the freezing of mature unfertilized oocytes cannot be recommended in procedures involving ambient temperature or concentrations exceeding 1.5 M PROH. On the other hand, the potential beneficial effect of low temperatures may outweigh the effect of concentration at subzero temperatures and could be explored further in the tailoring of conditions for slow controlled freezing.
 
Red blood cells are cooled in buffered solutions containing 10, 15, 20, 30, or 35% (w/w) 1,2-propanediol or glycerol. Cell survival is measured after cooling to -196 degrees C at rates between 1 and 3500 degrees C/min, followed by rewarming rapidly, except in a few cases. At low cooling rates, where the injuries are due to solution effects, for the same (w/w) concentrations of 15 or 20% (w/w), 1,2-propanediol protects erythrocytes better than glycerol. Differences are still observed when the two cryoprotectants are compared on a mole-fraction basis. At high cooling rates the survival passes through a minimum and then increases again. For the same concentrations, the minimum occurs at much lower cooling rates with 1,2-propanediol than with glycerol, in agreement with the better glass-forming tendency of 1,2-propanediol solutions. These cooling rates almost coincide with those at which the quantity of ice crystallized begins to decrease in the corresponding solutions. Thus, survival seems to be closely related to the glass-forming tendency at the survival minimum, and at higher cooling rates. After the fastest cooling rates, the warming rates necessary to avoid damage on warming are much smaller than those necessary to avoid devitrification. Therefore, in the present experiments the survivals are not related to the stability of the wholly amorphous state. However, injury follows the presumed transition from cubic to hexagonal ice, in erythrocytes as well as in other kinds of cells.
 
In this paper, we report on the suitability of solutions containing propane-1,2-diol (propylene glycol, PD), sugars, and salts for the vitrification of the human cell line, ECV304. Cooling (at 10 degrees C/min) and rewarming (at 80 degrees C/min) were at rates that are practicable for the tissues to be studied later. Under these conditions, 45% PD in phosphate-buffered saline (PBS) sometimes froze during cooling and always devitrified during rewarming but both events were avoided if the PBS salts were replaced by an osmotically equivalent concentration of sucrose or trehalose. The effect of such solutions on cells was evaluated using a cell culture assay in which the number of cells recovered after 3 days of culture was divided by the number cells plated, giving a cell multiplication factor or CMF. In the absence of PD the cells tolerated a low-salt concentration in solutions that were made isotonic with sugars, but they recovered poorly when 45% PD was also present. Trehalose gave significantly better recovery than sucrose. When 39% PD and 15% trehalose were included in a low-salt vehicle solution (LSV) that contained approximately 5% of the total salt concentration of PBS (this solution was designated LSV/39/15), the cells exhibited approximately 40% of untreated control CMF following exposure for 9min. LSV/39/15 vitrifies with a glass transition temperature of -102 degrees C, does not devitrify when warmed at 80 degrees C/min, and has suitable dielectric properties for uniform and rapid dielectric heating. An improved method for adding and removing LSV/39/15 gave a CMF of approximately 55% of untreated controls. Using this method, 1.0ml suspensions of ECV304 cells was cooled to, and stored briefly at, -120 degrees C and then rewarmed by immersion in a 37 degrees C water bath ( approximately 75 degrees C/min). The CMF of the cooled samples was similar to that of the exposure-only controls, approximately 50% of the untreated control CMF in both cases.
 
The tolerance and cryoprotective effect of propane-1,2-diol on human skin was evaluated using tetrazolium reductase enzyme activity to assess tissue viability. The tolerance of human skin was tested by exposing the skin to increasing concentrations of propane-1,2-diol (10, 20, and 40% v/v), at two permeation temperatures (4 degrees C or room temperature), in a stepwise manner, to reduce osmotically induced changes in cell volume. There was no evidence of specific toxicity attributable to the cryoprotectant during the permeation period, even at a concentration of 40%; however a significant decrease in human skin viability was observed after freezing and thawing with high concentrations of cryoprotectant. The only useful protocol was that employing a low cooling rate (-1 degree C min-1) with 10 or 20% (v/v) propane-1,2-diol at a low permeation temperature. The data suggest that propane-1,2-diol does not produced improved results when compared with other cryoprotectants used for skin cryopreservation.
 
For the same water contents, the stability of the wholly amorphous state of the aqueous solutions of 1,2-propanediol is much greater than that for all the solutions previously studied by us with glycerol, dimethylsulfoxide, ethanol, and ethylene glycol. To the degree that cyroprotection is related to that stability, 1,2-propanediol should be a better cryoprotectant than all these other compounds. The aqueous solutions of 1,2-propanediol have a simple behavior. No hydrate cyrstallizes on cooling, and for intermediate concentrations, on warming, after fast cooling, only ice crystallizes from the wholly amorphous state—first cubic, then hexagonal. The great stability of the amorphous state is shown by the critical warming rates above which no crystallization occurs, as well as by the difficulty in crystallizing on cooling.
 
The Kedem-Katchalsky equations and permeability data previously reported (F. G. Arnaud and D. E. Pegg. Permeation of glycerol and propane-1,2-diol into human platelets. Cryobiology 27, 130-136, 1990) have been used to design methods for adding and removing propane-1,2-diol (propylene glycol, PG) with human platelets. Mean platelet volume was kept within the tolerated range of 60 to 120% of normal. PG concentrations of 0.5, 1.0, 2.0, 2.5, and 3.0 M were studied at 2, 21, and 37 degrees C. PG was removed only at 21 degrees C. The effects of concentration of PG, temperature, and duration of exposure on the hypotonic stress response and ADP-induced aggregation were measured. It was found that platelets would tolerate exposure to PG concentrations up to 2 M at 21 or 2 degrees C for up to 15 min. The extent of damage increased considerably at higher temperatures and concentrations. These data provide the necessary basis for experiments to cryopreserve platelets with PG.
 
The effect of sugars or reduced saccharides trehalose, sucrose, sorbitol, or mannitol on the glass-forming tendency during cooling and the stability of the wholly amorphous state during warming has been studied with 2,3-butanediol, 1,2-propanediol, or 1,3-butanediol in three different carrier solutions. The 2,3-butanediol contained 96.7% (w/w) racemic mixture of the levo and dextro isomers and 3.1% (w/w) of the meso isomer (called 2,3-butanediol 97% dl). The carrier solutions were water, a phosphate-buffered saline, and two organ preservation solutions (Euro-Collins and Saint Thomas). The latter two were chosen because they are often used for kidney and heart preservation, respectively. The concentrations of 2,3-butanediol, 1,2-propanediol, and 1,3-butanediol varied respectively from 25 to 34, 30 to 35, and 30% (w/w). The concentrations of saccharides were 4 or 5% (w/w). In the absence of saccharides, for a given 2,3-butanediol concentration, the glass-forming tendency increased in the following order: water, Saint Thomas, the phosphate buffer, Euro-Collins. Addition of 4 or 5% (w/w) saccharide resulted in a large increase in the glass-forming ability of the solution during cooling and increased the stability of the glass during warming; but replacement of 4 or 5% diol by an equivalent weight (percentage) of a saccharide decreased, though to a lesser extent, these properties.
 
Two experiments were conducted to investigate the relationship between survival rates and ultrastructural appearance, using the freeze-replica technique, of bovine blastocysts after vitrification. In experiment 1, blastocysts obtained from in vitro-matured and in vitro-fertilized (IVM-IVF) bovine oocytes were either equilibrated in a stepwise manner in a vitrification solution (VS; 22.5% glycerol + 22.5% 1,2-propanediol) using 16 steps for 18 min in total (the 16-step method) or equilibrated with 10% glycerol + 20% 1,2-propanediol for 10 min and then exposed to VS (the 2-step method). The blastocysts were then vitrified by plunging them into liquid nitrogen. All samples were subsequently thawed in a water bath at 37 degrees C and cultured in vitro with a monolayer of cumulus cells. The survival rate obtained for blastocysts equilibrated by the 16-step method was 83.3% (25/30). In contrast, no blastocysts survived by the 2-step method (0/30). In experiment 2, freeze-replica observations were carried out on blastocysts vitrified by the 16-step method and the 2-step method. In all the blastocysts, no ice crystals were observed in the cytoplasm, blastocoelic cavity, or extracellular areas, which confirmed the occurrence of complete vitrification. Little ultrastructural change was observed in the plasma membrane of the blastocysts equilibrated by the 16-step method. In contrast, small vesicles and distinct intramembrane particle (IMP) aggregation were frequently observed in the plasma membranes of blastocysts equilibrated by the 2-step method. These results indicate that the successful cryopreservation of blastocysts following the 16-step equilibration requires not only complete vitrification, but also minimization of ultrastructural damage to the plasma membrane.
 
The permeability of human platelets to glycerol (GLY) and propane-1,2-diol (propylene glycol, PG) has been determined by measuring the time course of their change in volume following abrupt immersion in solutions of these solutes. A simple light-scattering method, and its calibration to measure mean platelet volume is described. The data are analyzed by means of the Kedem-Katchalsky (K-K) equations, modified to take into account the nonideal behavior of both intracellular and extracellular solutes. The values of the K-K parameters at 2, 21, and 37 degrees C, respectively, were as follows: the hydraulic conductivities (Lp) were 1 x 10(-7), 7 x 10(-7) and 3 x 10(-6) cm.sec-1.atm-1; the solute permeabilities for PG (omega RTPG) were 1.9 x 10(-6), 2.8 x 10(-5), and 1.3 x 10(-4) cm.sec-1; the solute permeabilities for GLY (omega RTGLY), at 21 and 37 degrees C only, were 2.6 x 10(-7) and 1.4 x 10(-6) cm.sec-1. The reflection coefficient (sigma) was 1 throughout. The relevant activation energies were -Lp, 16.5 kcal.mol-1; omega RTPG, 20.5 kcal.mol-1; and omega RTGLY, 17.9 kcal.mol-1. The use of these data is illustrated by computing schedules for the addition and removal of GLY and PG so that the amplitudes of changes in platelet volume are held within predetermined limits.
 
We investigated the slow freezing of one-cell mouse embryos with either dimethyl sulfoxide (Me2SO) or 1,2-propanediol (PROH) as the cryoprotectant. One-cell embryos, collected from superovulated C57BL/6J x CBA/Ca females were exposed to 1.5 M solutions of either Me2SO or PROH. The embryos were cooled at 0.3 degrees C/min to temperatures between -10 degrees and -80 degrees C before being plunged into LN2 and then warmed at either 20 degrees C/min or 450 degrees C/min. Survival was expressed as the percentage of hatching or hatched blastocysts per frozen-thawed embryo. When the slow cooling was in 1.5 M PROH, the temperature at which survival rates after slow thawing began to increase was -35 degrees C (52.6 +/- 5.2% survival). For slow cooling in 1.5 M Me2SO this temperature was -50 degrees C (45.0 +/- 2.9% survival). The addition of sucrose to the 1.5 M PROH solution raised the temperature at which survival rates after slow thawing began to increase to -30 degrees C (54.8 +/- 3.7% survival). If slow cooling was stopped at high subzero temperatures, embryos survived better after rapid thawing than slow thawing. If slow cooling was stopped at low subzero temperatures, the survival rate was not dependent on the thawing rate if freezing was done in 1.5 M PROH. When freezing was in Me2SO solutions and to subzero temperatures of -60 degrees and -80 degrees C, slow thawing gave better survival than rapid thawing. The addition of sucrose to the Me2SO freezing solution restored the survival rates at -60 degrees and -80 degrees C. These results indicate that high rates of survival may be obtained from one-cell mouse embryos by a rapid or a slow thawing procedure, as has been found for other developmental stages. The results also indicate that PROH provides superior protection compared to Me2SO against freezing-thawing damage and that the addition of sucrose to the freezing solutions prior to freezing improves the overall survival rates. Embryos that survived freezing and developed in culture implanted and formed normal fetuses at rates similar to those of nonfrozen control embryos (60% vs 68% and 53% vs 58%, respectively.
 
Propane-1,2-diol (propylene glycol, PG) permeates more rapidly than glycerol, has a strong glass-forming tendency, and appears to have a low toxicity. It is therefore attractive as a potential cryoprotectant for renal preservation. In this paper we compared the effect on subsequent function, of exposing rabbit renal cortical slices to 1 M PG or glycerol in a range of vehicle solutions and we demonstrated a remarkably low toxicity of PG at this concentration. Rabbit kidneys were then perfused with solutions containing PG up to a maximum concentration of 3 M, after which the cryoprotectant was removed and the function of cortical slices prepared from the perfused kidneys was assessed. Marked differences in perfusion characteristics were found between PG and glycerol and between different vehicle solutions for PG, but the two most suitable perfusates, both containing about 100 mM mannitol, permitted normal function in slices prepared after removal of PG. These results indicate that, with an appropriate vehicle perfusate, exposure to PG up to a concentration of 3 mol/liter has remarkably little effect upon vascular resistance and the renal cortical functions measured.
 
Corneas must first be equilibrated with multimolar concentrations of cryoprotectants if the formation of ice during cryopreservation is to be avoided by vitrification at practicable cooling rates. Rabbit corneas were exposed to equimolar mixtures of the cryoprotectants propane-1,2-diol and glycerol in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, and 6% w/v bovine serum albumin. Endothelial function was assessed by monitoring its ability to control stromal hydration during perfusion of the endothelial surface at 34 degrees C for 6 h. Endothelial morphology was observed by specular microscopy during perfusion and by scanning electron microscopy after perfusion. Endothelial pump activity and structural integrity of the endothelial layer were demonstrated after 20 min exposure at 4 degrees C to a total concentration of 1.4 mol/liter cryoprotectant (i.e., 0.7 mol/liter propane-1,2-diol + 0.7 mol/liter glycerol). Exposure to 2.0 and 3.4 mol/liter cryoprotectant for 20 min at 4 degrees and -5 degrees C, respectively, resulted in initial endothelial damage; but this repaired and a functioning endothelial pump was subsequently demonstrated. Although exposure to 4.1 mol/liter cryoprotectant for 10 min at -10 degrees C caused irreparable damage to 2/4 corneas, reduced dilution temperatures together with increased dilution time allowed exposure to 4.8 and 5.5 mol/liter cryoprotectant with retention of endothelial pump activity. Exposure to 6.1 mol/liter cryoprotectant for 10 min at -15 degrees C caused endothelial damage which was not mitigated by the presence of 2.5% w/v chondroitin sulfate. Endothelial function may be improved by further modification of addition and dilution protocols or by exposure to the cryoprotectants at lower temperatures.
 
Devitrification is a major problem which must be overcome for successful organ cryopreservation. Devitrification can be initiated on fracture planes and on bubbles, but the focus of attention here is on devitrification by ordinary heterogeneous and homogeneous mechanisms, which are the most relevant for organ preservation by vitrification. The purpose of the present studies was to define the devitrification process: to determine nucleation rates, ice-crystal growth rates, and the distribution of ice-crystal size and to evaluate the applicability of existing quantitative models of these processes which have successfully approximated the behavior of other aqueous systems. The present work was done using differential scanning calorimetry and cryomicroscopy. The amount of ice formed has been estimated for highly concentrated solutions. Kinetic parameters are presented here for isothermal conditions and continuous heating rate experiments. The classical theory based on the Johnson-Avrami equation has been evaluated and the results are compared with the theory of Boutron. The agreement is good for the continuous heating rate conditions, but results differ for the isothermal conditions.
 
1,3-Butanediol and 2,3-butanediol have been used in buffered solutions with 20, 30, or 35% (w/w) alcohol to cool erythrocytes to -196 degrees C at different cooling rates between 1 to 3500 degrees C/min, followed by slow or rapid rewarming. 1,3-butanediol shows the same shapes of red blood cell survival curves as 1,2-propanediol. Having nearly the same physical properties, they have comparable effects on cell survival. The classical maximum of survival for intermediate cooling rates and an increase for the highest cooling rates are observed. This increase seems to be correlated with the glass-forming tendency of the solution. After the fastest cooling rates, a warming rate of 5000 degrees C/min is sufficient to avoid cell damage, but a warming rate of 100-200 degrees C/min is not. Yet both of these rates would be insufficient to avoid the intracellular ice crystallization on warming. The damage on warming after fast cooling seems once again to be correlated with the transition from cubic to hexagonal ice. For all our results, 1,3-butanediol is like a "second" 1,2-propanediol and could be useful as a cryoprotectant for preservation by total vitrification. 2,3-Butanediol always gives extremely low survival rates, though it presents good physical properties. The crystallization of its hydrate seems to be lethal on cooling or on rewarming.
 
Experiments were carried out on the cryopreservation of common carp (Cyprinus carpio) sperm in order to test the suitability of using 1.2 and 5 ml straws and to investigate the ploidy of malformed larvae found among the hatched progeny. In the first set of experiments, the effect of freezing time was investigated on the hatch rate of embryos. The highest hatch rate for 1.2 ml straws was 69+/-16% at the freezing time of 4 min, and 39+/-27% for 5 ml straws at 5 min. In the second set, the effect different egg volumes fertilized with one straw of sperm on the hatch rate and the rate of malformed larvae was investigated. The highest hatch rate with 1.2 ml straws (86+/-12%) was observed when 10 g of eggs were fertilized with one straw, whereas with 5 ml straws the hatch rate was highest (65+/-18%) when 40 g of eggs were fertilized. The highest rate of malformed larvae (15+/-9%) was found in the control, whereas the highest rate of malformed larvae among the groups fertilized with cryopreserved sperm (13+/-7%) was found in the 1x dose group fertilized with 5 ml straw. The chromosome numbers of malformed larvae were investigated and haploids were found among those hatched from eggs fertilized with cryopreserved sperm whereas only diploids were found in the controls.
 
This study was conducted to investigate the ability of cryoprotective chemicals to induce phenotypic cryoadaptation in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T. Tolerance to negative temperature stress (freezing at -20 degrees C and thawing at 37 degrees C) was induced by pretreatment with Me(2)SO, glycerol, lactose, sucrose, and trehalose. Interestingly, Me(2)SO has a significantly greater cryoprotective effect than glycerol. Furthermore, lactose, sucrose, and trehalose, often referred to as osmotica, were shown to have greater cryoadaptive than cryoprotective properties. These results suggest that bacteria such as L. delbrueckii ssp. bulgaricus could be phenotypically adapted to freezing and thawing by an osmotic stress applied prior to freeze-thaw stress.
 
Microbial communities occur throughout the cryosphere in a diverse range of ice-dominated habitats including snow, sea ice, glaciers, permafrost, and ice clouds. In each of these environments, organisms must be capable of surviving freeze-thaw cycles, persistent low temperatures for growth, extremes of solar radiation, and prolonged dormancy. These constraints may have been especially important during global cooling events in the past, including the Precambrian glaciations. One analogue of these early Earth conditions is the thick, landfast sea ice that occurs today at certain locations in the Arctic and Antarctic. These ice shelves contain liquid water for a brief period each summer, and support luxuriant microbial mat communities. Our recent studies of these mats on the Markham Ice Shelf (Canadian high Arctic) by high performance liquid chromatography (HPLC) showed that they contain high concentrations of chlorophylls a and b, and several carotenoids notably lutein, echinenone and beta-carotene. The largest peaks in the HPLC chromatograms were two UV-screening compounds known to be produced by cyanobacteria, scytonemin, and its decomposition product scytonemin-red. Microscopic analyses of the mats showed that they were dominated by the chlorophyte genera cf. Chlorosarcinopsis, Pleurastrum, Palmellopsis, and Bracteococcus, and cyanobacteria of the genera Nostoc, Phormidium, Leptolyngbya, and Gloeocapsa. From point transects and localized sampling we estimated a total standing stock on this ice shelf of up to 11,200 tonnes of organic matter. These observations underscore the ability of microbial communities to flourish despite the severe constraints imposed by the cryo-ecosystem environment.
 
The objective was to cryopreserve sperm recovered from the canine epididymal cauda immediately after an orchiectomy. The sperm was stored for 12 h at 4°C using ACP-106c and TRIS as extenders. Sixty adult male dogs were used. The testis-epididymis complex (TEC) was removed, immersed in 0.9% saline and transported to the laboratory. The 60 TEC were divided into groups according to the 4°C cooling time (0 h or 12 h) and according to the extender used for sperm recovery (ACP-106c or TRIS), forming 4 experimental groups: G0h-ACP, G12h-ACP, G0h-TRIS and G12h-TRIS. The sperm were recovered from the epididymal cauda using the retrograde flow technique. Next, 1.0 mL of ACP-106c or 1.0 mL of TRIS (preheated to 37°C for 5 min) was added to the sperm of each epididymis. One week later, the sperm was thawed at 37°C for 1 min, and its morphology, functionality and total and progressive sperm motilities were analyzed. Other parameters were obtained by Computer Assisted Semen Analysis (CASA). The data were submitted to multivariate analysis of variance (MANOVA) (P < 0.05). The total motility values were 52.17 ± 1.78 and 49.8 ± 1.93 for groups G0h-ACP and G12h-ACP and 50.7 ± 2.06 and 43.90 ± 2.51 for groups G0h-TRIS and G12h-TRIS, respectively. A decrease in total sperm motility was observed after 12 h of cooling for both extenders (P < 0.05). ACP-106c can be used as an extender for freezing canine epididymal sperm, and the freezing procedure must be performed immediately after sperm recovery.
 
Plasma membranes of sperm subjected to low temperatures undergo changes in their structure and permeability. The addition of fatty acids in semen cryopreservation media may influence the sperm motility after thawing, possibly by maintaining the membrane fluidity due to their incorporation in lipid bilayers. In this work, different concentrations of the isomers cis-9,trans-11 and trans-10,cis-12 of conjugated linoleic acid (CLA) were added in the cryopreservation medium of bovine sperm. Four Jersey bulls were used, and the ejaculates were processed as a pool. The Tris-based extender (Dilutris®) was supplemented with 20% egg yolk (MB). The treatments with CLA (Luta-CLA®), which had oily presentation, were prepared from MB with addition of 1% sodium lauryl sulfate, and denominated MBL. The concentrations of CLA tested were 50, 100 and 150 μM. The motility characteristics of the post-thaw semen were analyzed by computerized analysis system (CASA), and plasma membrane integrity and acrosomal and mitochondrial function assessed by the association of the fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), JC-1 and Hoechst 33342. No significant differences were observed among treatments, excepting for a decreased mitochondrial potential of cells treated with 150 μM CLA. The addition of CLA, at the concentrations used, showed no advantages on the integrity and functionality of bovine sperm submitted to cryopreservation.
 
There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to -196°C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250°C/min to -196°C and for each cooling rate, subjecting them to five warming rates back above 0°C at rates ranging from 610 to 118,000°C/min. In samples warmed at the highest rate (118,000°C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610°C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000°C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.
 
Urea cycle (UC) is the main pathway of ammonium removal. A deficiency in any of the five classical enzymes of the pathway causes a urea cycle disorder. Hepatocellular transplantation is one of the techniques applicable to treat this disorder. In the present work, we investigated the activities and the relative expression levels of two of the UC enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), in isolated hepatocytes preserved up to 120 h in University of Wisconsin (UW) solution at 0 degrees C, and during the rewarming of these suspensions. During preservation, CPSI showed differences in mRNA levels respect to time 0, while ornithine transcarbamylase remained unchanged. At the end of the rewarming, CPSI showed values of enzymatic activity and relative mRNA level comparable with the control, meanwhile, there was an increment in OTC activity. In line with these results, we found that hepatocytes cold preserved up to 120h in UW solution maintained their ability to remove an ammonium load comparable to freshly isolated hepatocytes. These data indicated that our preservation conditions up to 120h in UW solution followed by rewarming, preserves UC enzymes at levels similar to freshly isolated hepatocytes, allowing the use of these cells in bioartificial liver devices or hepatocellular transplantation.
 
Thymomas are the most common tumors of the mediastinum. These tumors often compress vital mediastinal organs and severely impact the quality of life of thymoma patients. To avoid the side effects of chemoradiotherapy, some patients with unresectable malignant thymomas have opted to undergo cryotherapy in our hospital. We reviewed the cryosurgery, nursing and follow-up records of our hospital for the past 8 years, and evaluated the safety and efficiency of cryotherapy in 19 patients with unresectable malignant thymomas. No severe complications involving the vital organs surrounding the tumor occurred during or after cryosurgery. The most common side effect was pleural effusion, which occurred in 11 patients and healed after drainage within 1 week. Cough, mediastinal and pericardial effusions, pneumothorax, mild fever and chest tightness also occurred and resolved 1 week after symptomatic treatment. Since our patients had high KPS scores and mild myasthenia gravis symptoms before the treatment, myasthenia gravis did not occur after the treatment. The progression-free survival of the patients was 14-29 months (median, 18 months), and did not differ between patients with large tumors and those with small tumors (P = 0.6753). In conclusion, cryotherapy is a safe and efficient method for the treatment of unresectable malignant thymoma.
 
An 125IUDR incorporation assay is described which has been used to assess cell viability after freeze-thawing. A −10 °C/ min program and a −1 °C/min program, to be used on a Cryoson BV4 biological freezer, were designed to minimize the latent heat plateaus. These programs and a Knight and Farrant two-step program were compared with respect to survival of cells obtained from patients with malignant ascites and the established Chang cell line, originally from normal human liver. All the programs tested gave survival rates above 44% and the −5 °C/min program gave the best overall rates.
 
Quantitative measurements of edema following standardized freezing traumas of depilated rat skin have been made by means of intravenously injected 125I human albumin. It is shown that the edema following a vigorous, cryosurgerytype freezing develops fully in about 30 min, after which it remains on a constant level for the duration of the experimental period.Little or no edema results if the tissues are treated with epinephrine before they are frozen.The rapid extravasation of a macromolecular substance such as human albumin is taken as an indication of severe vascular damage.It is believed that this vascular damage can to some extent be prevented by application of vasoconstrictors to the tissues prior to freezing.
 
A blood-borne trigger for natural mammalian hibernation is described and details of the conditions necessary for its successful utilization are enumerated: 1.1. It is present in serum and cells of hibernation blood both in ground squirrels (Citellus tridecemlineatus) and woodchicks (Marmota monax).2.2. It acts intra and interspecifically insofar as appropriate hibernation serum of 13-lined ground squirrel when transfused can trigger hibernation in other ground squirrels of the same species, and appropriate hibernation serum of woodchucks can similarly trigger hibernation in both woodchucks and in 13-lined ground squirrels. Withdrawal, preparation, and transfusions follow a careful technique outlined in References 1 and 2.3.3. The whole blood, serum, and cells retain the hibernation-inducing power for at least 6 months whether kept at freezer temperature (−15 °C) or liquid nitrogen temperature (−196 °C).4.4. The trigger is effective whether used as serum or as dialyzate of that serum (prepared as described). The nondialyzed residue was not effective, and evidence has been found which indicates that an antitrigger may be present in that residue.5.5. Cold adaptation may be contraindicated for trigger effectiveness.6.6. The titer of trigger in a donor's blood evidently increases as a bout of hibernation lengthens. This is noted by the fact that trigger taken from a donor long in hibernation induces hibernation more rapidly in recipients than does trigger from a donor in a short bout of hibernation.7.7. Trigger manifests its effect in winter whether a recipient animal is placed in a colddark or a warm window-lit room. (Trigger effect in a warm window-lit room in a nonhibernating season has not as yet been studied.)8.8. Trigger has not been found either in the blood of active or of aroused animals. Thus, effective material is not circumscribed by the expression “winter blood,” but rather by the expression “hibernation blood.”9.9. Female animals in all cases are more effective recipients than males, whereas neither sex is a better donor.10.10. Recipient animals receiving a trigger and maintained in cold-dark rooms will not show seasonal reversal, rather, have been observed to show bouts of hibernation intermittently for as long as 2 years. They seemingly do not return to the active state.A theoretical approach to hibernation induction is advanced which holds that the trigger is a small molecular material (SM) found in blood taken in hibernation. It is excreted, complexed, and/or metabolized in the arousing process. It may complex with another blood-borne trigger-inhibitor (LM) to form a complex (CM). The entire circannual cycle of hibernation can be related to relative concentrations of SM, LM, and CM. Speculation is further set forth as to hibernation of tissue as contrasted to hibernation of animals. This notion was presaged by others, notably by Lyman, Kayser, and Suomalainen. As Lyman stated (7) “… entrance into hibernation is more than the abandonment of the warm-blooded state.”
 
Even though the existence of the blood-borne "hibernation induction trigger" has been reported in the 13-lined ground squirrel, transfusion of plasma from hibernating rodents with other hibernating species as the recipients failed to induce the occurrence of summer hibernation. In order to verify whether the response to the "trigger" substance is species specific, the present study was carried out to compare the effect of plasma from hibernating Richardson's ground squirrels on the incidence of summer hibernation in both juvenile Richardson's and adult 13-lined ground squirrels. In two series of experiments, 13-lined ground squirrels entered hibernation quite readily independent of the treatment. The rate of occurrence of hibernation ranged from 78% after sham injection to 86% after warm saline, fresh summer active plasma, and fresh hibernating plasma, respectively. There were no differences in the number of hibernation bouts and the number of days in hibernation after each treatment. In contrast, none of the juvenile Richardson's ground squirrels entered hibernation after any of the treatments up to the end of the 8-week observation period. These results not only argue against the existence of blood-borne "trigger" substance, at least in the Richardson's ground squirrel, but also caution against the use of the 13-lined ground squirrel as a standard test animal for the bioassay of the "trigger" substance.
 
Among several mammalian hibernators, an endogenous circannual sequence of physiological events is believed to mediate the timing of torpor. Dawe and Spurrier (3) reported that a bloodborne substance (hibernation induction trigger) is important in initiating the torpor phase of those events in the 13-lined ground squirrel. We have reported the induction of summer hibernation among 13-lined ground squirrels using dialysates of serum from hibernating golden hamsters (a nonseasonal hibernator). While those animals receiving saline injections hibernated in 36.3 ± 2.9 days, an earlier induction (22 ± 8.8 days) occurred among those receiving the hibernation serum dialysate (P = 0.05). It was also observed that naive animals departed from a strict circannual rhythm and displayed a high incidence of hibernation, although not significant when compared to the experimental saline controls. The spontaneity of torpor in summer among the naive sample may in part be a characteristic of wild-caught animals employed in the bioassay. Nevertheless, the induction of hibernation among those animals receiving the hibernation serum preparations is supportive of the studies of Dawe and Spurrier (2, 3). That a “trigger” material apparently is present in the hamster, a phylogenetically distinct nonseasonal hibernator, suggests that a characteristic of rodent hibernators is the presence of a material which is associated with the initiation of torpor.
 
In an endeavor to more clearly define the physiological action of hibernation trigger (previously identified by the authors) an experiment was devised using infant ground squirrels. In one case, pregnant ground squirrel mothers were transfused intravenously with cold dialysate of serum of hibernating woodchuck, and in another case, infant (1 week to 3 week) squirrels were transfused intraperitoneally with the same material. When isolated and placed in a cold room (15 °C) at 6 weeks post partum, both the infant born of the transfused mothers, as well as the infant which has been transfused directly, hibernated in the summer. This was in contrast to controls of several kind, i.e., animals not injected, or receiving active or arousing woodchuck serum or heat-treated hibernation serum. Some details for a theory, which had been advanced in a previous article (4), were essentially substantiated by this experiment. It is our belief that this represents the first time mammalian hibernation has been observed in infant hibernators.
 
The function of isolated strips of smooth muscle from the guinea pig were assessed by isometric contractile responses to histamine before and after storage at −13 °C in high potassium, Pipes-buffered solutions containing 30% (w/v) Me2SO and having different pH values. The structural integrity of the tissue during and after cooling was also assessed by electron microscopy.The pH-dependence of tissue recovery in these muscles was clearly demonstrated after about 17-hr storage at −13 °C. Despite the wide difference in functional recoveries between groups of muscles cooled in either “high” (7.7) or “low pH” (6.4) media, however, there were no apparent ultrastructural differences between the two differently treated groups of muscles. Muscles from both pH groups, fixed after rewarming to 37 °C, showed the morphological characteristics typically seen in noncooled muscles. An examination of muscle cells which were fixed at −13 °C (w/v) Me2SO revealed that the contractile myofibrils of specimens in both experimental groups had undergone a noticeable reorganization and aggregation.
 
Because cholecystokinin octapeptide and angiotensin II are directly involved in intestinal food and water absorption, the effect of these two compounds on intestinal motor responses of hibernating and alert 13-lined ground squirrels was investigated. Both cholecystokinin octapeptide and angiotensin II caused a greater increase in the composite motility of intestinal segments of normothermic in contrast to hypothermic hibernating ground squirrels. Additionally, cholecystokinin caused an increase in the contraction frequency of the intestine from normothermic as compared to hypothermic squirrels. This differential response may provide an adaptive advantage by decreasing food and water consumption; depressing motility and inhibiting digestive enzyme release; reducing the release of bile which also may have an irritant effect on the intestinal mucosa under prolonged exposure. As a result, the GI tract is devoid of food and digestive enzyme irritants during torporous periods.
 
Diabetes is a global epidemic that affects about 285 million people worldwide. For severely-ill patients with type I diabetes, whole pancreas or islet transplantation is the only therapeutic option. Islet transplantation is hindered by the scarce supply of fresh functional islets and limitations in cryopreservation procedures. Thus, improved cryopreservation procedures are needed to increase the availability of functional islets for clinical applications. Towards this goal, this work developed a cryopreservation protocol for pancreatic cells using proteins that accumulate naturally in freezing-tolerant plants. A preincubation of cells with 1% lecithin-1% glycerol-1% N-methylpyrrolidone followed by cryopreservation with partially purified proteins from wheat improved the viability and insulin-secreting properties of INS832/13 cells, compared to cryopreservation with 10% dimethyl sulfoxide (Me(2)SO). The major factor that enhanced the cryoprotective effect of the wheat protein formulation was preincubation with the lipid lecithin. Expression profiles of genes involved in metabolic and signaling functions of pancreatic cells (Ins, Glut1/2/3, Pdx1, Reg1α) were similar between fresh cells and those cryopreserved with the plant protein formulation. This novel plant-based technology, which is non-toxic and contains no animal material, is a promising alternative to Me(2)SO for cryopreservation of insulin-secreting pancreatic cells.
 
Isolated lamb hearts perfused with fresh whole blood at 10 and 13 °C in an ex vivo perfusion circuit continuously contracted at a rate of 15 to 20 times/min with a peak left ventricular systolic pressure (LVPSP) up to 70 mm Hg. These contractions persisted for the duration of the hypothermic study, up to three days with no change in vascular resistance. On rewarming to 38 °C, the hearts resumed regular and efficient contractions. Hearts perfused at 5 °C, however, exhibited no electrical or mechanical activity during hypothermic preservation and were uniformly poorly preserved.Quality of heart preservation was improved if, prior to final cooling, hearts were first rewarmed to 38 °C, followed by cooling. Change of the support animal, or interruption of flow of fresh blood into the perfusion circuit, resulted in cessation of ventricular contractions, ventricular fibrillation, and poor organ preservation.
 
Previously, transplantation of the leukemia L2C/NB of strain 2 guinea pigs (100% successful in strain 2 guinea pigs or their F1 hybrids) was only partially successful when transplanted homologously into newborn Hartley and strain 13 animals (foreign strains) and unsuccessful into such adults. In recent studies, increased takes (60%) occurred in adults of these foreign strains. In experiments utilizing 400 inbred strain 13 and Hartley guinea pigs, dimethyl sulfoxide treatment (intraperitoneal injection of 1.0 ml as a 50% solution in saline) was associated with a significant shortening of the out of strain latent period of this leukemia, permitting deaths to occur closer in time to those seen in inbred strain 2 controls. In some experiments such takes increased to as high as 93%. On the other hand, in other experiments similarly conducted, this effect of DMSO was not observed, suggesting to us that an optimal infectious dose range may be critical for the pertinent DMSO effect to come into play. In a related but smaller series (32 animals), ACTH and Dexamethasone also appeared to hasten the speed of lethality of this leukemia in homoiotransplantation. A combination of possible factors, corticosteroids, alterations in chromosomal number, an increased infectiousness attributable perhaps to a recently discovered associated virus and an enhanced penetrability of the virus afforded by combinations of drugs with DMSO, are offered as explanations of the results.
 
(1) Tyrosine and tryptophan metabolism in brain and peripheral tissues were studied in hypothermic hibernating and normothermic nonhibernating 13-lined ground squirrels (Spermophilus tridecemlineatus). (2) In the hypothermic hibernating state, there were significant elevations of brain stem tyrosine, norepinephrine, and dopamine levels; forebrain norepinephrine and dopamine levels; and cerebellum norepinephrine and tyrosine levels. (3) On the other hand, plasma norepinephrine levels were significantly decreased in hypothermic hibernating squirrels while plasma tyrosine levels were increased. Kidney norepinephrine levels were significantly increased in hypothermic hibernating squirrels, while kidney tyrosine levels were decreased. Total plasma tryptophan and free plasma tryptophan were significantly reduced in hypothermic hibernating squirrels. Hepatic tyrosine aminotransferase Km and Vmax were decreased in hypothermic hibernating squirrels, while tryptophan 2,3-dioxygenase activity was not altered. Plasma and liver albumin were increased in hypothermic hibernating squirrels, while plasma and liver total protein were not altered. (4) These results demonstrate that significant changes in tyrosine and tryptophan metabolism occur in both central and peripheral tissues with concomitant alterations in metabolites during hypothermic hibernation in 13-lined ground squirrels.
 
A “trigger” substance was again indicated to be present in sera of hibernating animals. Sera from the hibernating 13-lined ground squirrel, hibernating woodchuck, hibernating Arctic ground squirrel, and hibernating Arctic marmot were all capable of inducing the 13-lined ground squirrel to hibernate in the summer, a season when that species would normally be active. The hibernation trigger is thus not species specific. It is effective whether drawn from these two Arctic species of hibernators or drawn from these two species of hibernators from the midwestern states. The normothermic Arctic marmot appears to have an “anti-trigger” substance in its serum in the summer, which impedes fall hibernation in the transfused 13-lined ground squirrel. This is similar to the anti-trigger observed in the summer serum of active 13-lined ground squirrels and active woodchucks. With respect to hypothermia, it was induced in Artic marmots and in Arctic foxes at Point Barrow, Alaska, in summer. Though in such cases body temperatures fell significantly (as in hibernation), no trigger was recovered from their hypothermic sera that could be shown to be capable of inducing summer hibernation in the ground squirrel. Neither was anti-trigger found in the serum of hypothermic experimentals. These latter experiments thus suggest that the release of trigger into the blood during hibernation is dependent on a mechanism more complex than simply lowering body temperature.
 
The induction of summer hibernation in the 13-lined ground squirrel (Citellus tridecemlineatus) by intravenous injection of plasma obtained from winter hibernating ground squirrels was confirmed. Hibernation was also induced by injection of urine from arousing winter ground squirrels. Results support the “trigger” theory of hibernation proposed by Dawe and Spurrier (3) and also suggest that tissues are set free from “trigger” influence during winter arousal by the excretion of “trigger.”
 
By means of xenon-133 injections in the skin, local circulation has been examined before, during, and after rapid local freezing.Monitoring of the xenon clearance curves has shown that circulation continues unimpaired up to the time the tissue freezes. Blood flow is completely arrested while the tissue is frozen. Immediately upon thawing of the tissue, blood flow is resumed.
 
Time-tissue concentrations of Cs137 in rats were measured for two exposure times and using two methods of sacrifice, with the intention of showing whether any important amount of chemical mobility exists in the span of time between the initiation of sacrifice and the isolation of organs.It is concluded that the differences in the specific activity ratio between the 1-min exposure and the 15-min exposure cases are due to metabolic processes that were going on before and at the time of sacrifice, and that there is little effect on these ratios by the choice of method of sacrifice.
 
Harvesting of stem cells during the early phases of treatment with no immediate intention to perform a stem cell transplant is becoming an increasingly common practice. Such "insurance" harvests are often stored for many years before being needed for transplant in a subsequent relapse. The effect of long-term cryostorage (5-14 years) on the viability and functional capacity of haematopoietic stem cells (HSCs) was investigated in 40 bone marrow and peripheral blood harvests using standard in vitro methods, the colony forming unit-granulocyte/macrophage (CFU-GM) assay and a single platform viable CD34(+) cell absolute count by flow cytometry. Forty percent of harvests had CD34(+) HSC counts of at least 0.7 x 10(6)/kg bodyweight and 85% had CFU-GM counts of at least 1.0 x 10(5)/kg bodyweight, these values representing our institutional minimum requirements for safe transplantation. Based on these results, it appears that HSC collections can remain adequate for safe transplantation after up to 14 years of cryostorage. However, as deterioration of HSC quality and viability may occur, some precautions may be warranted, namely harvesting higher than normal numbers of HSCs in collections intended for long-term storage and repeating in vitro assays on harvests after long-term storage prior to transplantation.
 
Low temperatures can assure the long-term or even indefinite preservation of important biological specimens. Nematode cryopreservation allows for the availability of large numbers of living nematodes at any one time, especially for experimental purposes. New isolates of Bursaphelenchus have recently been collected, including Bursaphelenchus eremus (Rühm) Goodey. This species was identified in north-central Italy on dying oak trees and from the bark beetle Scolytus intricatus Ratzeburg as dauer larvae. We therefore, sought to develop a cryopreservation technique for the long-term storage of all available Bursaphelenchus spp. The technique consists of a rapid-cooling protocol involving immersion in a liquid nitrogen bath before storage of the frozen samples in a mechanical freezer at -140 degrees C. The survival of nematodes subjected to this rapid-cooling protocol was higher than previously reported using slow-cooling methods and is suitable for several species of Bursaphelenchus and other phytoparasitic nematodes.
 
Top-cited authors
Mustafa Numan Bucak
  • Selcuk University Veterinary Faculty, Konya, Turkey
Pürhan Barbaros Tuncer
  • Mersin University
Peter Mazur
  • University of Tennessee
Shinsuke Seki
  • Akita University
Barry J Fuller
  • University College London