Freezing preservation of adult mammalian heart at high subzero temperatures

The present study adapted the overwintering strategy employed by freeze-tolerant amphibians and reptiles to freeze-preserve the isolated rat heart. The heart was flushed with a cardioplegic solution and supercooled to -1.2 and -3 degrees C. Then freezing was induced by inoculation of ice crystal. The viability of the heart explant was assessed after reanimation by the isolated working heart perfusion. There was no recovery of function in hearts flushed with solution containing 0.28 mM CaCl2. Lowering the concentration of CaCl2 to 0.15 mM, however, rendered good functional return. Furthermore, inclusion of 50 mM glycerol in the flush solution dramatically improved functional preservation. Under the best conditions defined here, the recoveries of aortic flow, coronary flow, cardiac output, systolic pressure, and work in hearts stored at -1.2 degrees C for 3 h were 72.8 +/- 6.8, 87.2 +/- 4.2, 77.6 +/- 5.4, 83.4 +/- 2.8, and 66.6 +/- 5.9% (mean +/- SEM, n = 8) of the unstored control levels, respectively. The myocardial ice content was 18.6 +/- 5.4% (n = 5) of tissue water. Prolonging the storage time to 5 h increased the ice content to 45.3 +/- 8.1% and reduced the recovery of cardiac output to 23 +/- 11% of the control value (mean +/- SEM, n = 5). Hearts frozen at -3 degrees C for 1.5 h showed 29.4 +/- 8.7% (n = 3) of control cardiac output during reperfusion. This novel approach may provide an opportunity to advance our knowledge about freezing preservation of not only the heart but other solid organs as well.     

Water transport in a cluster of closely packed erythrocytes at subzero temperatures

A one-dimensional model has been developed to describe the kinetics of water transport in a cluster of closely packed cells. For the case of human red blood cells, the intracellular medium has been treated as an ideal, hydrated, nondilute multicomponent electrolyte solution. Results show that the volume flux of water out of the interior cells of the cluster lags behind that of the exterior cells. At any given temperature (or time), the amount of water retained within a cluster of closely packed cells of a given type exceeds (on an overall percentage basis) the amount of water retained within a single isolated cell of the same type. For a given cooling rate the probability of intracellular ice nucleation at any given temperature will therefore be greater for cells in the interior of a cluster, and the survival signature for a cell cluster should peak at a cooling rate which is less than the corresponding optimal value for a single, isolated cell. These results are consistent with experimental observations.     

A quantitative analysis of the thermal properties of porcine liver with glycerol at subzero and cryogenic temperatures

There is a lack of information on the effect of cryoprotective agents (CPAs) on the thermal properties of biomaterials at cryobiologically relevant temperatures (i.e. <233.15 K, −40 °C). Thermal properties that are of most interest include: thermal conductivity, density, specific heat, and latent heat resulting from phase change in tissue systems. Availability of such information would be beneficial for accurate mathematical modeling of cryobiological applications. Recently, we reported these thermal properties in phosphate buffered saline (PBS) with varying concentrations of glycerol, a widely used cryoprotective agent. In this study we extend these results by assessing the effects of glycerol on the thermal properties of porcine liver at subzero temperatures. Differential scanning calorimeter (DSC) was used to measure the specific heat and the latent heat release of porcine liver immersed in PBS and varying concentrations of glycerol. The specific heat data obtained from the DSC experiments were also used to predict the bulk thermal conductivity. This was done using a transient heat transfer model with a thermistor probe technique. Results show that the introduction of glycerol significantly alters thermal properties from known values for H₂O and non-treated liver. Therefore, inaccuracies in thermal predictions can be expected due to the application of measured vs. predicted thermal properties such as from weight averaging. This supports the need for these and other measurements of biomaterial thermal properties, with and without CPA addition, in the cryogenic regime.     

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.     

Kinetics of water loss from cells at subzero centigrade temperatures

A mathematical model is developed for the calculation of the kinetics of water loss from cells at subzero centigrade temperatures. In this model it is assumed that the cell surface membrane is permeable to water only, the protoplasm is a nonideal solution, the cells are spherical, and during the cooling process the cell temperature is not uniform inside the cell. It is also assumed that because of water loss due to cooling process the cell volume and the cell surface area reduce and the reductions in surface area and volume of the cell are functions of the amount of water loss from the cell. Based on this model, and for different conditions, the fractions of supercooled intracellular water remaining in the cells at various temperatures are calculated.It is shown that for cooling cells at subzero centigrade temperatures. (1) the consideration of Clausius-Clapeyron equation for vapor pressures of water and ice, instead of the exact vapor pressure relations, may produce errors in the prediction of the amount of water loss from the cells at high cooling rates only, (2) the assumption of intact cells will produce considerable deviation in the prediction of water loss from the cells as compared to the more realistic assumption of shrinkable cells, (3) the nonideality of protoplasm solution is very effective on the prediction of the amount of water loss from the cells, and (4) the assumption of uniform-temperature cells during the cooling process may be erroneous only for cells with small fractions of water in their protoplasms.     

Deformability and stability of erythrocytes in high-frequency electric fields down to subzero temperatures.

High-frequency electric fields can be used to induce deformation of red blood cells. In the temperature domain T = 0 degrees to -15 degrees C (supercooled suspension) and for 25 degrees C this paper examines for human erythrocytes (discocytes, young cell population suspended in a low ionic strength solution with conductivity sigma(25 degrees) = 154 microS/cm) in a sinusoidal electric field (nu = 1 MHz, E0 = 0-18 kV/cm) the following properties and effects as a function of field strength and temperature: 1) viscoelastic response, 2) (shear) deformation (steady-state value obtained from the viscoelastic response time), 3) stability (by experimentally observed breakdown of cell polarization and hemolysis), 4) electrical membrane breakdown and field-induced hemolysis (theoretical calculations for ellipsoidal particles), and 5) mechanical hemolysis. The items 2-4 were also examined for the frequency nu = 100 kHz and for a nonionic solution of very low conductivity (sigma(25 degrees) = 10 microS/cm) to support our interpretations of the results for 1 MHz. Below 0 degrees C with decreasing temperature the viscoelastic response time tau(res)(T) for the cells to reach steady-state deformation values d(infinity,E) increases and the deformation d(infinity,E)(T) decreases strongly. Both effects are especially high for low field strengths. The longest response time of approximately 30 s was obtained for -15 degrees C and small deformations. For 1 MHz the cells can be highly elongated up to 2.3 times their initial diameter a0 for 25 degrees and 0 degrees C, 2.1a0 for -10 degrees C and still 1.95a0 for -15 degrees C. For T > or = 0 degrees C the deformation is limited by hemolysis of the cells, which sets in for E0(lysis)(25 degrees) approximately 8 kV/cm and E0(lysis)(0 degrees) approximately 14 kV/cm. These values are approximately three times higher than the corresponding calculated critical field strengths for electrically induced pore formation. Nevertheless, the observed depolarization and hemolysis of the cells is provoked by electrical membrane breakdown rather than by mechanical forces due to the high deformation. For the nonionic solution, where no electrical breakdown is expected in the whole range for E0, the cells can indeed be deformed to even higher values with a low hemolytic rate. Below 0 degrees C we observe no hemolysis at all, not even for the frequency 100 kHz, where the cells hemolyze at 25 degrees C for the much lower field strength E0(lysis) approximately 2.5 kV/cm. Obviously, pore formation and growth are weak for subzero temperatures.     

Motility of Colwellia psychrerythraea strain 34H at subzero temperatures

We examined the Arctic bacterium Colwellia psychrerythraea strain 34H for motility at temperatures from -1 to -15 degrees C by using transmitted-light microscopy in a temperature-controlled laboratory. The results, showing motility to -10 degrees C, indicate much lower temperatures to be permissive of motility than previously reported (5 degrees C), with implications for microbial activity in frozen environments.     

Partition of synaptic membranes in aqueous two-phase systems at subzero temperatures by using anti-freeze solvent

The freezing point of aqueous two-phase (liquid-liquid) systems containing water, dextran and poly(ethylene glycol) has been lowered by including glycerol. Biological membranes, obtained by fragmentation of a crude synaptosomal preparation from calf brain cortex, have been included in the two-phase systems. The effects of temperature and the concentration of glycerol on the partition of the membranes within the systems have been investigated. Considerable stabilisation of the membranes was noticed when they were partitioned at -10 degrees C compared with 0 degrees C. The influences of glycerol, ethylene glycol, N,N-dimethylformamide and tetrahydrofuran on the phase-forming properties of the systems and on enzyme activities are also presented. Possible use of the above systems for studies and separation of biological membranes are discussed.     

High-pressure inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum at subzero temperatures

High hydrostatic pressure is a new technology in the food processing industry, and is used for cold pasteurization of food products. However, the pressure inactivation of food-borne microorganisms requires very high pressures (generally more than 400 MPa) and long pressure holding times (5 min or more). Carrying out pressure processing at low temperatures without freezing can reduce these parameters, which presently limit the application of this technology, in keeping the quality of fresh raw product. The yeast, Saccharomyces cerevisiae and the bacterium, Lactobacillus plantarum were pressurized for 10 min at temperatures between -20 and 25 degrees C and pressure between 100 and 350 MPa. Pressurization at subzero temperatures without freezing significantly enhanced the effect of pressure. For example, at a pressure of 150 MPa, the decrease in temperature from ambient to -20 degrees C allowed an increase in the pressure-induced inactivation from less than 1 log up to 7-8 log for each microorganism studied. However, for comparable inactivation levels, the kinetics of microorganism inactivation did not differ, which suggests identical inactivation mechanisms. Implications of water thermodynamical properties like compression, protein denaturation, as well as membrane phase transitions, are discussed.