E.G. Cravalho

Thermodynamics and kinetics of intracellular ice formation during freezing of biological cells

A physicochemical model based on the modified classical heterogeneous nucleation theory was proposed to analyze the ice formation inside biological cells during freezing. According to this model, intracellular ice formation (IIF) may be catalyzed either by the plasma membrane via the effects of the external ice on the plasma membrane, called surface‐catalyzed nucleation (SCN), or by the intracellular particles, called volume‐catalyzed nucleation (VCN), depending on the freezing conditions. The model for IIF was coupled with the model describing the kinetics of water transport to predict the thermodynamic state of the cytoplasm.A method to estimate the nucleation frequency from the observed probability of nucleation was suggested. This method was based on the assumption that each cell has the same heteronucleating particles with identical properties to alter the nucleation kinetics in an identical way. This allowed the correlation of the experimental probability of IIF with the nucleation rate. It was sugg...

Microscopic observation of intracellular ice formation in unfertilized mouse ova as a function of cooling rate

Abstract A physical-chemical analysis of water loss from cells at subzero temperatures had shown that the likelihood of intracellular ice formation increased with increasing cooling rate (22). We have now used a modified version of a unique conductioncooled cryomicroscope stage (8) to observe the freezing of unfertilized mouse ova suspended in dimethyl sulfoxide. Survival measurements showed that the respective survivals of ova were about 65, 56, and 0% when they were cooled at rates of 0.2 to 1.5, 2.5, and 5.4 °C/min. Direct microscopic observation of mouse ova during freezing showed that the respective fractions of cells that froze intracellularly were 13, 72, and 100% when they were cooled at rates of 1.3, 2.9, and 4.8 °C/min or faster. These values agree with those predicted from the physical-chemical analysis for cells the size of mouse ova. The microscopic observations have also shown that intracellular freezing generally occurred at about −40 to −45 °C. We had previously observed that mouse embryos must be cooled slowly to −50 °C or below if they are to survive subsequent rapid cooling to −196 °C. The observation of intracellular ice formation at −45 °C supports the interpretation that at temperatures above −50 °C the embryos still contain water capable of freezing intracellularly.

Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures.

A MODEL OF DIFFUSION-LIMITED ICE GROWTH INSIDE BIOLOGICAL CELLS DURING FREEZING

A theoretical model for predicting the kinetics of ice crystallization inside cells during cryopreservation was developed, and applied to mouse oocytes, by coupling separate models of (1) water transport across the cell membrane, (2) ice nucleation, and (3) crystal growth. The instantaneous cell volume and cytosol composition during continuous cooling in the presence of glycerol were predicted using the water transport model. Classical nucleation theory was used to predict ice nucleation rates, and a nonisothermal diffusion‐limited crystal‐growth model was used to compute the resulting crystallization kinetics. The model requires knowledge of the nucleation rate parameters Ω and κ, as well as the viscosity η of a water‐NaCl‐glycerol solution as a function of both the composition and temperature of the solution. These dependences were estimated from data available in the literature. Cell‐specific biophysical parameters were obtained from previous studies on mouse oocytes. A sensitivity analysis showed that...

A membrane model describing the effect of temperature on the water conductivity of erythrocyte membranes at subzero temperatures.

Thermodynamic models show that the loss of intracellular water from human erythrocytes during freezing depends heavily upon the water conductivity of the erythrocyte membrane. These calculations, which are based on the simple extrapolation of ambient conductivity data to subzero temperatures, show that more than 95% of cell water is transferable during freezing, whereas experiments show that at least 20% of cell water is retained. A study of the effects of different published values for the membrane water conductivity on cell water retained during freezing shows that this discrepancy may be a consequence of the simple extrapolation procedure. For a homogeneous membrane system, absolute reaction rate theory was used to develop a surface-limited permeation model that includes the resistance to the flow of water not only through the interior region of the membrane but also across possible rate-limiting barriers at the solution-membrane interfaces. The model shows that it is unlikely that a single rate-limiting process dominates water transport in the red cell as it is being cooled from ambient to subzero temperatures. The effective membrane conductivity at subzero temperatures could possible be much lower than a simple extrapolation of existing data would predict. With the aid of this model analytical predictions of intracellular water during freezing are more consistent with experimental observations.

Correlation of American Urological Association Symptom Index With Obstructive and Nonobstructive Prostatism

The precise role of the American Urological Association (AUA) symptom index in the management of benign prostatic hyperplasia (BPH) is not well established. The AUA symptom index has been recommended only for quantifying the symptoms of BPH but not for its diagnosis. However, to our knowledge the ability to discriminate obstructive from nonobstructive BPH using the AUA symptom index has never been investigated. To establish the relationship between the AUA symptom index and prostatic obstruction 125 men (mean age 67.7 +/- 8.4 years) with voiding dysfunction presumably related to BPH were analyzed. Patients were given the AUA symptom questionnaire, following which video urodynamic studies were done, including micturitional urethral pressure profilometry for specifically diagnosing outlet obstruction. The patients were divided into 2 groups: group 1-78 with primary BPH dysfunction and group 2-47 with prostatism of ambiguous etiology. The mean AUA symptom index in group 1 (15.5 +/- 7.1) was not statistically different from that in group 2 (14.8 +/- 7.9). In both groups the mean AUA symptom index in the patients with obstruction (15.3 +/- 7.2 for group 1 and 13.9 +/- 7.9 for group 2) was not statistically different from that in the nonobstructed group (17.0 +/- 5.4 and 16.1 +/- 7.9, respectively). Of the severely symptomatic patients 22% did not have obstruction whereas all mildly symptomatic patients did. No significant correlations were found between the severity of obstruction and the AUA symptom index in either group. These observations indicate that the AUA symptom index cannot discriminate obstructed from nonobstructed BPH cases, not all severely symptomatic BPH patients will have outlet obstruction, a significant proportion of mildly symptomatic BPH patients can have outlet obstruction and voiding dysfunctions in elderly men, regardless of the etiology, produce similar symptoms.

A cryomicroscope for the study of freezing and thawing processes in biological cells

Abstract A cryomicroscope capable of effecting controlled freezing and thawing in biological cell suspensions has been developed. A dual capability for both heating and cooling is employed in conjunction with an analog control system to provide for precise regulation of the specimen temperature between 77 °K and 310 °K at time rates of temperature change between zero and several thousand degrees centigrade per minute. The microscope can be fitted with a high speed motion picture to record the dynamics of the freezing and thawing processes in slow motion. A miniature freezing and thawing system adapted to the stage of a light microscope enables a broad spectrum of cooling and warming rates to be achieved by cooling the specimen at a constant rate with a steady flow of refrigerant fluid through the system and by simultaneously dissipating electrical energy at a variable rate in a resistance heater immersed in the fluid stream and in thermal communication with the specimen. The control system is of the analog type in which a signal generator, inverter, and integrator are used to generate a voltage representative of the desired linear temperature profile, be it for cooling or warming. This profile is continuously compared in a summing unit with the amplified output of a thermocouple monitoring the specimen temperature. The output from the summing unit, which is proportional to the difference between the generated reference temperature and the specimen temperature, is amplified in a power amplifier having the resistance heater as its load. Energy is dissipated in the heater only when the specimen temperature is lower than the reference temperature. The maximal temperature for warming processes is set by a limiting diode in the integrator, whereas the minimal temperature for cooling processes is determined by the refrigerant temperature.

An experimental comparison of intracellular ice formation and freeze-thaw survival of hela S-3 cells

Abstract A small-volume fluorescent dye viability assay has been successfully applied to a conduction cryomicroscope freezing-thawing stage as a means of determining post-thaw survival of the nucleated mammalian cell HeLa S-3. The survival signature for HeLa S-3 cells has been determined, revealing an optimum cooling rate of −30 °C/min where the maximum survival is 30%. No cells survive for cooling rates greater than −128 °C/min and the decreased survival at supraoptimal cooling rates coincides with a linear increase in the percentage of cells containing intracellular ice from 0% at −16 °C/min to 100% at −128 °C/min. Although no data were taken to identify increased salt concentration as the mechanism responsible for cell injury at suboptimal cooling rates, the post-thaw leakage of intracellular fluorescent dye at these rates takes approximately 4–10 min as opposed to instantaneous release of dye for cells which contain ice at the high cooling rates. This indicates two modes of damage. Cell number density has been identified as an important parameter in freezing studies since survival can be enhanced at slow rates by packing cells together in groups. Packing also causes a greater fraction of the cells in a sample to have intracellular ice present, thus decreasing survival at the faster rates. These responses can be explained by assuming that the outer cells in a group protect the inner ones from solution damage at slow rates, yet restrict water flux from the inner cells at faster rates, causing an increased likelihood of intracellular ice formation. Both of these results are consistent with the dual-mechanism freezing damage theory proposed by Mazur.

A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration

Current methods of cryopreservation of hepatocytes in single cell suspensions result in low overall yields of hepatocytes, demonstrating long-term preservation of hepatocellular functions. A novel culture method has recently been developed to culture liver cells in a sandwich configuration of collagen layers in order to stabilize the phenotypic expression of these cells in vitro (J. C. Y. Dunn, M. L. Yarmush, H. G. Koebe, and R. G. Tompkins, FASEB J. 3, 174, 1989). Using this culture system, rat hepatocytes were frozen with 15% (v/v) Me2SO to -70 degrees C, and stored at approximately -100 degrees C. Following rapid thawing, long-term function was assessed by measuring albumin secretion in culture for 7-14 days postfreezing. Comparison was made with cryopreservation of liver cells in single cell suspensions. Cryopreservation of liver cells in suspension resulted in only a 2% yield of cells which could be successfully cultured; albumin secretion rates in these cultured cells over 48 hr were 26-30% of secretion rates for nonfrozen hepatocytes. Freezing cultured liver cells in the sandwich configuration after 3, 7, and 11 days in culture maintained 0, 26, and 19% of the secretion rates of nonfrozen hepatocytes, respectively. Morphology of the cryopreserved cells appeared grossly similar to cells without freezing; however, this morphological result was patchy and represented approximately 30% of the cells in culture. These results represent the first demonstration of any quantitative long-term preservation of hepatocellular function by cryopreservation, suggesting that cultured hepatocytes can survive freezing and maintain function.

Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential

A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.