The influence of gelatin polypeptides,potassium, calcium and osmolality on the hypothermic perfusion of rabbit hearts

After a period of perfusion at 37 °C with a standard perfusate, rabbit hearts were cooled to +10 °C and perfused at this temperature for 5 hr with a variety of solutions. The hearts were then rewarmed to 37 °C and perfused again with the standard perfusate to assess heart function. The effects on subsequent normothermic function of including gelatin polypeptides (Haemaccel) and of increasing the osmolality and the concentrations of K⁺ and Ca²⁺ in the solutions used for hypothermic perfusion were studied. The best results were achieved with a noncolloidal electrolyte solution containing 20 mm K⁺ and 4.8 mm Ca²⁺ which gave an average maximum percentage recovery of function of 57.9 ± 7.1%. The addition of sufficient mannitol to raise the osmolality from 300 mOsm/Kg to 420 mOsm/Kg improved (but not significantly) the maximum percentage recovery of function to 61.2 ± 8.5%     

The contribution of the cryoprotectant to total injury in rabbit hearts frozen with ethylene glycol.

Following the failure of hearts to recover function after freezing at ?20 ° in the presence of 3 m ethylene glycol, a variety of experimental treatments was devised to determine the relative harmfulness of ice, high concentrations of electrolytes and high ethylene glycol concentration. Neither cooling to ?20 °C without freezing in a Ca²⁺-free solution containing twice the normal salt concentration and 6 m ethylene glycol (freezing 3 m ethylene glycol at ?20 °C doubles the solute concentration in the liquid phase), nor perfusion at ?1 °C with this solution were conducive to the recovery of hearts. However, perfusion with Ca²⁺-free 3 m ethylene glycol solution with twice the normal concentration of salts did allow full recovery of function, whereas perfusion with Ca²⁺-free 6 m ethylene glycol solution with normal salt concentrations did not. Therefore, the high ethylene glycol concentration encountered during freezing was the main cause of damage.     

Isolated sheep heart hypothermic (5–13 °C) perfusion with fresh blood: Successful preservation for 24–72 hours with continuous strong ventricular activity

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.     

Viability and Morphology of rat heart cells after freezing and thawing of the whole heart

Intact adult rat hearts were cooled in the presence of 10% DMSO according to an external cooling program which approximated the optimal external three-step cooling program for the isolated adult heart cells: 20 min at ?20 °C, 0.2 °C/min from ?20 to ?25, ?30, or ?50 °C, and rapid cooling to ?196 °C. Following rapid thawing, cells were isolated after perfusion with a 0.1% collagenase solution. Only cells which originated from the free wall of the right ventricle could be isolated, even after cooling to ?20 °C. Most cells from hearts cooled to ?196 °C did not survive. When the third cooling step was omitted and the end temperature of the second cooling step was ?30 °C, 38% of the cells excluded trypan blue, 29% were morphologically intact, and 30% showed spontaneous contractions after thawing, expressed as percentages of the control, A much lower survival was found after cooling to ?50 °C.Histological and electron microscopical study of the heart immediately after thawing revealed no differences between hearts cooled to ?20, ?30, or ?196 °C. Also no marked differences were observed between the morphological integrity after freezing and thawing of the atrium, the left and right ventricle walls, and the ventricular septum. The survival data suggest the presence of nonmorphologically detectable alterations in cells frozen to ?196 °C, compared to cells frozen to ?30 °C. The morphological investigations indicate no essential differences in resistance of atrial and ventricular cells to the freezing process.Experiments involving neonatal rat hearts cooled to ?196 °C, according to the method which gave optimal preservation of the isolated cells, revealed that after thawing cells are present from which growing and contracting cultures can be derived. It appears that cells in the neonatal rat heart are more resistant to freezing to ?196 °C than cells in the adult rat heart.     

Winter survival of an adult bark beetle Ips acuminatus Gyll

Freezing-susceptible adult Ips acuminatus hibernate underneath bark of Scots pine. The beetles lower their supercooling points from ?20 to ?34°C due to accumulation of low molecular weight antifreezes. The capability of specimens to supercool to about ?20°C in the absence of cryoprotective solutes during winter, seemed to be at least partially attributable to the presence of a thermal hysteresis factor at 3–4°C.Using a GC-MS-COM technique, a unique combination of accumulated solutes present only in specimens demonstrating supercooling points below ?20°C was identified as ethylene glycol, mannitol, sorbitol and dulcitol. Not previously found in nature, ethylene glycol was the major solute (90%) synthesized at sub-zero temperatures. Exposure to ?10°C was an effective cue to accumulation of ethylene glycol and nearly 5 times as effective in promoting sorbitol synthesis than was ?5°C. When low molecular weight substances were lost at high temperatures, they were not re-synthesized in beetles re-exposed to sub-zero temperature. The supercooling point was closely related to both the concentration of ethylene glycol and to the haemolymph melting point. Attempts to correlate changes in sorbitol concentrations to changes in supercooling points were not conclusive.Proliferation of thermal hysteresis was observed in the beginning of November. A melting-hysteresis freezing point differential of about 3.6°C was demonstrated in the haemolymph of beetles during December. No thermal hysteresis was demonstrated in the haemolymph of positive phototactic beetles or in the outdoor beetles in May. The combination of high temperature and long photoperiod appeared to be a more effective cue to the final loss of thermal hysteresis than was high temperature alone.     

Silica-free silicone rubber in isolated heart perfusion with blood at 13 °C

Isolated lamb hearts were perfused at 13 °C for 24 hr with whole fresh blood using a silicone rubber circuit and a membrane lung (N = 7); there was formation of thrombi, deposition of fibrin, and an increase in resistance to blood flow in the membrane lung. The perfused hearts fibrillated at hypothermia and showed unequal recovery of function upon final rewarming.There was less rise in membrane lung resistance when the perfusion circuit was primed with blood at 38 instead of 13 °C and then cooled progressively to 13 °C. Some hearts perfused in these circuits were well preserved but others became edematous with loss of ventricular contractility (N = 6).Coating the perfusion circuit with a hypothrombogenic material, silica-free silicone rubber and priming the circuit at 38 °C prevented any rise in membrane lung resistance during blood perfusion. All the hearts perfused in these circuits (N = 6) had the same left ventricular function before and after cold perfusion.Thus isolated hearts can be perfused in vitro with whole blood at hypothermic temperature without loss in function when attention is paid to thrombogenicity of materials used to construct the perfusion circuit.     

Ultrastructure-function correlative studies for cardiac cryopreservation. II. Hearts frozen to various temperatures without a cryoprotectant

Isolated rat hearts were perfused with balanced salt solution (BSS) for 20 min, sealed in a metal cannister, and cooled in a −20 °C acetone bath at a rate of 1 °C/min to one of four subzero core temperatures (−10, −12, −17, or −20 °C). Upon attainment of the desired temperature the hearts were rapidly thawed (40–50 ° C/min) and reperfused with BSS for an additional 20 min. Approximately half of the hearts cooled to −10 or −12 °C resumed spontaneous contractile activity after thawing. One of 16 hearts survived cooling to −17 °C, while no heart survived cooling to −20 °C. Nonfrozen controls gave a positive inotropic response to a standard test dose of ouabain; none of the thawed survivors did.     

Temperature dependence of the microwave properties of aqueous solutions of ethylene glycol between +15 degrees C and -70 degrees C.

The values of the dielectric constant and of the loss tangent for samples of 0 m (distilled H₂O), 1 m, 2 m, 3 m, 4 m, 5 m, 10 m, and 100% ethylene glycol were determined over the temperature range of +15 °C to ?70 °C. An operating frequency of 1.40 GHz to 1.55 GHz was used, allowing the results to be applied directly to both 0.915 GHz and 2.450 GHz studies. Strong temperature and concentration dependencies were found; low concentration solutions tended to behave similarly to water, while higher concentration solutions were more independent. Peak values and discontinuities occurred at different temperatures, depending on concentration. Five molar ethylene glycol was indicated as a near optimum concentration based on toxicity, cryoprotection, and microwave power absorption considerations. A study of blends of cryoprotectants is suggested.     

Visualization of freezing damage. II. Structural alterations during warming

There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from ?20 ° to ?50 °C and then rapidly cooled to ?196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above ?40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as ?45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as ?20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.     

Optimal temperature ranges for control of cooling rate.

Survival of hamster fibroblasts following cooling at 1 °C/min to various subzero temperatures in the presence of penetrating or nonpenetrating cryoprotective agents was examined. In the presence of nonpenetrating agents maximum recovery was obtained when the cooling rate was controlled between ?5 and ?20 °C followed by rapid cooling to ?196 °C. For penetrating agents recovery was maximal in samples cooled at 1 °C/min to ?30 °C or lower. These different temperature ranges for maximum recovery indicate different modes of actions of penetrating and nonpenetrating cryoprotective agents. The action of penetrating agents appear to be based on their colligative properties. Nonpenetrating agents may promote electrolyte leaks out of the cell and a corresponding osmotic efflux of cell water during slow cooling, thereby reducing the amount of intracellular ice present at ?196 °C.     

Membrane energization at subzero temperatures: calcium uptake and oxonol-V responses

The use of aprotic solvents for preserving the electron transport properties of mitochondria at subzero temperatures is based upon the use of binary water and ethylene glycol mixtures or upon ternary and quaternary mixtures that include dimethyl sulfoxide and the lower aliphatic alcohols. In order to better preserve the respiratory control properties of mitochondria at subzero temperatures, detailed studies have been made of the effects of these mixtures on the respiratory control and electron transport from NADH or succinate of mitochondrial preparations. It is found that ADP is not metabolized at a measurable rate below 0 °C, but that Ca²⁺ is rapidly taken up and can thus be used to assay respiratory control ratios down to ?8 °C. In the region below ?8 °C the charge-sensitive probe oxonol-V has been used to evaluate energy coupling. By using Ca²⁺ to stimulate respiration at 0 °C good results are obtained with ethylene glycol/water alone and optimal results are obtained with a quaternary mixture. A mixture that freezes at ?21 °C gives about 50% inhibition of the respiratory control ratio for electron transport at 0 °C with NADH or succinate as substrates. The mixtures permit low-temperature studies of mitochondrial functions under conditions of minimal respiratory rate, including the kinetics of electron transfer reactions, the formation of intermediate compounds, and the rapid freeze-trapping of mitochondrial reactions for analytical chemistry or ³¹P NMR.     

Interaction of rate of latent heat removal during freezing and rates of subsequent cooling and warming on survival of the blood protozoan, Babesia rodhaini

Babesia rodhaini parasites in murine blood containing 1.5 m DMSO were frozen at two rates, as judged by the duration of the “freezing plateau”, then cooled to ?196 °C and rewarmed at two rates to detect interactions between the duration of the plateau and rates of subsequent cooling and rewarming. Infectivity tests showed that fast and slow freezing (plateau times of about 1 sec and 30 sec, respectively) had similar effects on parasite survival when cooling was at 130 °C/min and warming was at 800 °C/min. However, when either the cooling rate was increased to 3500 °C/min or the warming rate was decreased to 2.3 °C/min, fast freezing decreased parasite survival more than did slow freezing. It is suggested that fast freezing accentuated the damaging effects of fast cooling and slow warming by increasing intracellular ice formation.     

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

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

Storage of Porcine Articular Cartilage at High Subzero Temperatures

Objective: Transplantation of osteochondral allograft tissue can treat large joint defects but is limited by tissue availability, surgical timing, and infectious disease transmission. Fresh allografts perform the best but requirements for infectious disease testing delay the procedure with subsequent decrease in cell viability and function. Hypothermic storage at lower temperatures can extend tissue banking time without loss of cell viability and, therefore, increase the supply of allograft tissue. This study investigated the effects of different cryoprotectant solutions on intact AC at various subzero temperatures. Design: 10 mm porcine osteochondral dowels were immersed for 30 minutes in various combinations of solutions [(XVIVO, propylene glycol (51% w/w), sucrose (46% w/w)] cooled to various subzero temperatures (−10, −15, and −20 °C), and held for 30 min. After warming, 70 μm slices were stained with membrane integrity dyes, viewed under fluorescence microscopy and cell recovery calculated relative to fresh controls. Results: Results demonstrated excellent cell recovery (>75%) at −10°C provided ice did not form. Excellent cell recovery (>70%) occurred at −15°C in solutions containing 51% propylene glycol but formation of extra-matrix ice in other solutions resulted in significant cell loss. All groups had <6% cell recovery at −20°C and propylene glycol did not provide a protective effect even though extra-matrix ice did not form Conclusions: These results suggest that extra-matrix ice plays an important role in cell damage during cryopreservation. Excellent cell recovery can be obtained after storage at subzero temperatures if ice does not form. Hypothermic preservation at high subzero temperatures may extend AC storage time in tissue banks compared to current techniques.     

An Efficient Method for Separating Ascospores from Sporulating Cultures in Saccharomyces cerevisiae by Hydrostatic Pressure

A new oxidative reaction of ethylene glycol was found with two alcohol oxidases from methanol yeast, Candida sp. and Pichia pastoris. Both alcohol oxidases oxidized ethylene glycol to glyoxal via glycolaldehyde. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Candida alcohol oxidase were around 8.5 and 5.5, respectively, and their apparent K_ms were 2.96 m and 28.6 mm, respectively. The optimum temperature was 40°C at pH 7.0. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Pichia alcohol oxidase were around 8.0 and 6.0, respectively, and their optimum temperatures were 50 and 45°C, respectively, at pH 7.0. The apparent K_m for glycolaldehyde was found to be 83.3 mm. For the accumulation of glyoxal, addition of catalase was effective, and a higher amount of glyoxal was obtained at a much lower temperature than the optimum for the alcohol oxidase. When 0.1 m ethylene glycol and glycolaldehyde were incubated with 80 units of the Pichia enzyme at 10°C, both substrates were almost completely converted to glyoxal after 10 and 3h of incubation, respectively.     

An evaluation of colloidal solutions for normothermic perfusion of rabbit hearts: An improved perfusate containing Haemaccel

Rabbit hearts were perfused at 37 °C with the aim of establishing a relatively simple and preferably synthetic perfusate which would give reliable and consistent perfusion performances for periods of several hours. Of the perfusates devised and tested, a 1.75% solution of Haemaccel most fully satisfied the above criteria and maintained myocardial function for 30.0 ± 4.0 hr.     

The influence of temperature on the function of renal cortical slices frozen in various cryoprotectants

Renal cortical slices were frozen to various subzero temperatures after treatment with 2.1 M of one of three cryoprotectants, dimethyl sulfoxide (Me2SO), ethylene glycol, or glycerol. The effects on tissue [K+]/[Na+] of cooling to these temperatures were tested (using identical procedure times, cooling rates, and warming rates) by holding the slices at each experimental temperature for appropriate periods of time prior to rewarming. The effects of the holding time were assessed by comparison with slices which were cooled and rewarmed with no intermediate holding time. Slices treated with ethylene glycol or glycerol were found to exhibit a continuous decrease in [K+]/[Na+] with lowered temperatures, in contrast to those treated with Me2SO. Slices treated with Me2SO actually experienced a continuous increase in [K+]/[Na+] with lowered temperature (-12 to -33 degrees C). Me2SO does exhibit toxic effects at subzero temperatures. Adverse effects of holding time on viability are seen for Me2SO-treated slices at higher subzero temperatures. These effects were alleviated as the temperature is reduced, suggesting that temperature has a greater effect on survival of renal cortical tissue than Me2SO concentration. However, the toxicity observed at higher subzero temperatures is expected to be of importance, particularly for slowly cooled tissues which are exposed to these temperatures for relatively long periods of time.     

Effect of dimethylsulphoxide on the functional characteristics of isolated perfused rat liver

Exposure of rat liver, perfused with 7% BSA in Krebs-Ringer bicarbonate buffer, to 1.4 m Me₂SO at 35 °C had no effect on the release of potassium from the livers, but the rate of urea synthesis fell from 0.6 to 0.1 μmol/min. Bile production also decreased and the total amount collected during perfusion was only half that produced by controls. After perfusion for 4 hr at 35 °C control livers and those exposed to Me₂SO started to release GOT into the perfusate but livers exposed to the cryoprotective compound released the enzyme at a faster rate.Exposure of livers to Me₂SO at 5 °C resulted in potassium being released at a slower rate (0.98 μmol/min) than from cooled controls (1.19 μmol/min) and urea synthesis was decreased from 0.8 to 0.2 μmol/min. Bile production also declined but, because bile flow normally ceases during hypothermia, the effect on this aspect of liver function was probably less than was found at 35 °C. Release of GOT from livers exposed to Me₂SO at 5 °C was quite different from that observed at 35 °C; the enzyme appeared in the perfusate after about 8 hr and it was present in much lower concentration than was found with appropriately cooled controls which started to release the enzyme after 6 hr.Thus, exposure of rat liver to Me₂SO at 5 °C appears to be slightly less damaging than exposure at 35 °C and it may even have a beneficial effect on some aspects of liver function in vitro.     

Ethylene Glycol Utilization,Cold and Ethylene Glycol Shockand Acclimation Proteins in a Psychrotrophic Bacterium

A psychrotrophic Pseudomonas fluorescens was isolated that utilizes ethylene glycol as a sole carbon source, with removal efficiencies of 98% and 96% in 20 and 55 days at 25° and 5°C, respectively. The response of the psychrotroph to environmental shifts was investigated using two-dimensional SDS-PAGE and computing scanning laser densitometry. During a 25°C to 5°C cold shock, the microorganism induced ten cold shock proteins. Under conditions of constant growth at 5°C, five cold acclimation proteins were synthesized. Ethylene glycol shock induced 14 ethylene glycol shock proteins. Ten ethylene glycol acclimation proteins were found. Similarities between the shock proteins and acclimation proteins for cold shock and acclimation and the ethylene glycol shock and acclimation may suggest that these proteins are of significance to both shock recovery as well as constant growth in a new environment.     

The contuation of the phase-boundary between ice I and liquid into the region of ice III and II and its relation to freeze-pressing of biological material.

The trajectory of the phase-boundary between ice I and liquid has been continuously followed by compression of deionized water, 0.10 m KCl, 0.10 m NaCl, and deionized water with suspended yeast cells (Saccharomyces cerevisiae, 180 mg/g) in a close-ended pressure chamber at temperatures below 0 °. Upon increasing pressure on deionized H₂O at ?8.6 °C the temperature first increases, until the transition line between ice I and liquid is reached. Then the sample cools on further compression, which is concomitant with an increase in electrical conductivity, indicating the gradual formation of liquid. At ?34.8 °C the pressure drops spontaneously from 3 × 10⁸ to 2.4 × 10⁸ Pa, the conductivity decreases, and the volume of the samples becomes further reduced to ?3.1 cm³/mole of H₂O, making the formation of ice III probable. On increase of pressure on 0.10 m KCl and 0.10 m NaCl the sample is gradually cooled, as the fusion line of the respective eutectic solid is reached. 0.10 m KCl is then super-cooled into the region of ice III and II, whereas 0.10 m NaCl is desalinated with a final conductivity of the suspension of 3–10 nmho/cm. In the sample with S. cerevisiae 180 mg/g the ice I-liquid phase-boundary was followed to ?36.0 °C into the region and ice III and II.These results are of great importance to the understanding of the freeze-pressing process, since they indicate that a transition from ice I to liquid may occur even at temperatures between ?22 °C and ?35 °C, thus facilitating flow of material through the press. This way they shed light on the pressures needed to initiate flow at different temperatures and compositions of the sample to be freeze-pressed.