The flow regimes and the pressure-flow relationship in the canine urethra.

Classical fluid dynamics predicts that the pressure difference Deltap between any two points along a fully developed, viscous flow stream is linearly proportional to the flow rate Q (the Poiseuille relation). However, the passive urethral resistance relationship (PURR) widely used in modern urodynamics describes the pressure difference Deltap between two points along the urethra as linearly proportional to the flow rate squared (Q(2)). It is our hypothesis that this functional dependence may have its origins in the developing flow field within the urethra. That is, rather than being fully developed hydrodynamically, urethral flow is more likely representative of flow within the entry length of a rigid conduit. In our study, we used a canine model of the lower urinary tract to investigate the possibility of entrance effects. Although the most rigorous model of urethral fluid mechanics would include the elastic properties of the urethra into its configuration, the solutions from such a model would be unnecessarily complex and not readily lend themselves to the analysis of clinical data. Therefore, we chose to model the canine urethra at each instant in time as a rigid tube, and characterized its instantaneous flow using viscous flow theory for a rigid tube. All urodynamic analyses were performed on a surgically exposed urinary tract. Solid state pressure transducers were used to measure the intravesical and distal urethral pressures, whereas an ultrasonic flowmeter was used to obtain a simultaneous measure of the urinary flow rate. Detrusor contractions were induced using bilateral electrical stimulation of the pelvic nerves. Varying degrees of outlet obstruction were created using an inflatable sphincter cuff secured around the bladder outlet. The experimental data were evaluated using the well-known laminar entry length model of Atkinson and Goldstein. The peak Reynolds numbers under nonobstructed R(p)(e non-obs) and obstructed R(p)(e obs) outlet conditions ranged between 500 < R(p)(e non-obs) < 1,500 and 300 < R(p)(e obs) < 1,700, respectively. Under non-obstructed outlet conditions, the urethral diameters D and total lengths l(T) ranged between 1.5 mm < D < 2.5 mm and 75 mm < l(T) < 95 mm, respectively, whereas the peak entrance lengths L(p)(e non-obs) ranged between 55 mm < L(p)(e non-obs) < 215 mm. These data suggest that flow in the canine urethra under both non-obstructed and obstructed outlet conditions is typically laminar. The data further support the hypothesis that non-obstructed flows are predominantly entry length in nature. Entry length flows are fluid dynamically described by a quadratic pressure-flow relationship, thus suggesting a physiological basis for Schäfer's quadratic pressure-flow relationship, and therefore, for the PURR. Neurourol. Urodynam. 18:521-541, 1999.

The maximum watts factor as a measure of detrusor contractility independent of outlet resistance.

The maximum watts factor (WFmax) is often used to characterize detrusor contractility. It was recently shown that the WFmax may increase in some patients with chronic outlet obstruction. It is, however, unclear whether this increase reflects a dependence of the WFmax on the degree of outlet obstruction or whether it represents a true increase in detrusor contractility secondary to chronic outlet obstruction. Therefore, this study was performed to investigate this issue using a canine model of acute outlet obstruction. Urodynamic studies were performed on adult canines with surgically exposed lower urinary tracts. Pressure transducers were used to measure the intravesical and the distal urethral pressures, whereas an ultrasonic flow meter was used to obtain a simultaneous measure of the urinary flow rate. Detrusor contractions were induced by electrically stimulating the pelvic nerves bilaterally. Varying degrees of outlet obstruction were created using an inflatable sphincter cuff secured around the bladder outlet. The WFmax, the detrusor pressure at voiding terminus (Pdet.clos), and the passive urethral resistance (R) were computed from measured pressure-flow rate data at each degree of outlet obstruction. The WFmax was not significantly correlated to either the sphincter cuff volume (r = 0.025, p = 0.871), the Pdet.clos (r = 0.286, p = 0.073) or the R (r = 0.110, p = 0.509). The WFmax was not significantly different among mild, moderate, and severe degrees of outlet obstruction (p = 0.176). Our results suggest that the WFmax is independent of the degree of acute outlet obstruction (defined in terms of the sphincter cuff volume, Pdet.clos and R). This validates the current practice of using the WFmax to evaluate detrusor function in patients with voiding dysfunction regardless of outlet resistance. Further, since the WFmax is independent of outlet obstruction acutely, it is reasonable that it would also be independent of outlet obstruction under chronic conditions. Our results, therefore, also imply that the increase in the WFmax with chronic outlet obstruction may represent a true increase in detrusor contractility and not a WFmax dependence on outlet resistance.

Fertilization and development of mouse oocytes cryopreserved using a theoretically optimized protocol.

Rational design of a cryopreservation protocol was demonstrated by using theoretical models of the cryopreservation process to develop an optimal freezing protocol for mouse oocytes. A coupled mechanistic model of the processes of freeze-induced cell dehydration and intracellular ice formation was developed, and cryomicroscopical measurements of intracellular ice formation kinetics were used to determine biophysical parameters required by the model, and to test model predictions of the freezing behaviour of mouse oocytes. A simple phenomenological model for oocyte damage resulting from exposure to concentrated electrolyte and cryoprotectant solutions during cryopreservation was obtained by defining a cost function equal to the duration of the freezing protocol. A two-step freezing protocol was theoretically optimized by using a sequential simplex algorithm to minimize the cost function, subject to the constraint that the predicted probability of intracellular ice formation remain below 5%, yielding a putative optimum at the cooling rate B = 0.59 degrees C/min, and plunge temperature Tp = -67 degrees C. By systematically varying B and Tp about these values in experiments with mouse oocytes cryopreserved in 1.5 M dimethyl sulphoxide, the maximal recovery of intact oocytes with a normal morphology (82%) was obtained for B = 0.5 degrees C/min and Tp = -80 degrees C. Further evaluation of the fertilizability and developmental capacity of oocytes cryopreserved using the optimized protocol yielded cleavage to the 2-cell stage in 65% of oocytes inseminated, and blastocyst formation in 50% of these 2-cell embryos.

Detrusor internal and external work in relation to passive urethral resistance in a canine model of the lower urinary tract.

This study was conducted to evaluate whether passive urethral resistance, detrusor internal work, and detrusor external work are independent measures of the voiding process. Passive urethral resistance, detrusor internal work, detrusor external work, and detrusor total work of 5 canines were determined under nonobstructive and obstructive outlet conditions. All urodynamic analyses were performed on a surgically exposed urinary tract. Solid-state pressure transducers were used to measure the intravesical and distal urethral pressures, while an ultrasonic flow meter was used to obtain a simultaneous measure of urinary flow rate. Detrusor contractions were induced using bilateral electrical stimulation of the pelvic nerves. Varying degrees of outlet obstruction were created using an inflatable sphincter cuff secured around the proximal urethra. Urethral resistance, internal work, and total work increased with increasing obstruction, while external work decreased with increasing obstruction. Internal work was linearly and negatively correlated to external work. At low degrees of obstruction, internal and external work changed more rapidly than passive urethral resistance per unit change in obstruction. As obstruction was increased, the change in work parameters per unit change in obstruction decreased, while the change in passive urethral resistance per unit change in obstruction increased. Our results indicate that at low degrees of outlet obstruction (cuff volume < 60% of isometric cuff volume), detrusor internal and external work are more sensitive to changes in obstruction than passive urethral resistance. It therefore appears prudent to use these work parameters with a passive urethral resistance relation (PURR) when evaluating early BPH and also when monitoring its progression. The data also show that a detrusor will acutely respond to a change in outlet obstruction by modulating its total work output. The mechanisms responsible for this response are presently under investigation.

Continuous occlusion test to determine detrusor contractile performance.

PURPOSE: A study was conducted to determine the merits of the continuous occlusion test as a method of detrusor contractility assessment by comparing it with other stop tests and with contractility derived from pressure-flow analysis. MATERIALS AND METHODS: The continuous occlusion test was performed in elderly men by occluding the bladder outlet before the onset of a detrusor contraction and it was repeated to assess reproducibility. The magnitude of the isovolumetric contraction, maximum slope of the detrusor contraction, and duration of detrusor activation were determined. Voluntary and mechanical stop tests were performed during the mid voiding phase. Continuous occlusion test parameters were compared with the pressure-flow contractility parameters. RESULTS: The continuous occlusion test was evaluated in 159 patients. The maximum isovolumetric contraction pressure of the continuous occlusion test was significantly higher than that of the voluntary stop test (49 patients). Continuous occlusion test contractility parameters were reproducible. The maximum isovolumetric contraction pressure and the maximum slope of the detrusor contraction of the continuous occlusion test significantly correlated with the estimated maximum isovolumetric contraction pressure and estimated velocity of shortening (derived from pressure-flow), respectively (r = 0.79, p < 0.0001 and r = 0.385, p = 0.016, 39 patients). The watts factor was well correlated with maximum isovolumetric contraction pressure (r = 0.75, 39 patients). CONCLUSIONS: Our study suggests that the continuous occlusion test can be used as an effective alternative method of assessing detrusor contractility.

Correlation between micturitional urethral pressure profile and pressure-flow criteria in bladder outlet obstruction.

PURPOSE: We correlate micturitional urethral pressure profilometry with pressure-flow diagnoses of outlet obstruction. MATERIALS AND METHODS: Urodynamic evaluation was done of 86 consecutive men with voiding symptoms. Obstruction criteria were a micturitional urethral pressure profile (MUPP) gradient greater than 5 cm. water, Schäfer's linear passive urethral resistance relation (PURR) greater than grade 1 and Abrams-Griffiths nomogram. RESULTS: Interpretable results were completed in 99% of the patients undergoing MUPP and 60% undergoing pressure-flow studies (p < 0.00001). MUPP diagnosis agreed with PURR (p = 0.0015) and Abrams-Griffiths nomogram results (p = 0.00004). MUPP gradients correlated well with PURR (r = 0.70, p < 0.00001). Using optimum cutoff values (11 cm. water), the sensitivity of MUPP was 83%, specificity 82% and positive predictive value 94%. CONCLUSIONS: MUPP correlates well with and yields interpretable results more often than pressure-flow studies.

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.

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.

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.

Cellular response of mouse oocytes to freezing stress: prediction of intracellular ice formation.

Successful protocols for cryopreservation of living cells can be designed if the physicochemical conditions to preclude intracellular ice formation (IIF) can be defined. Unfortunately, all attempts to predict the probability of IIF have met with very limited success. In this study, an analytical model is developed to predict ice formation inside mouse oocytes subjected to a freezing stress. According to the model, IIF is catalyzed heterogeneously by the plasma membrane (i.e., surface catalyzed nucleation, SCN). A local site on the plasma membrane is assumed to become an ice nucleator in the presence of the extracellular ice via its effects on the membrane. This interaction is characterized by the contact angle between the plasma membrane and the ice cluster. In addition, IIF is assumed to be catalyzed at temperatures below -30 degrees C by intracellular particles distributed throughout the cell volume (i.e., volume catalyzed nucleation, VCN). In the present study, these two distinctly coupled modes of IIF, especially SCN, are applied to various experimental protocols from mouse oocytes. Excellent agreement between predictions and observations suggests that the proposed model of IIF is adequate.

Thermal model for the local microwave hyperthermia treatment of benign prostatic hyperplasia.

A system for the noninvasive localized, hyperthermia treatment of benign prostatic hyperplasia was investigated. The system uses a microwave transrectal antenna with a water cooled jacket to achieve localized hyperthermia. The purpose of this study is to model the temperature rise in the prostate and in the surrounding tissue during treatment. The SAR distribution for the transrectal probe is measured in a muscle tissue equivalent phantom. The SAR information is used with a finite element solution of the bioheat transfer equation to give the temperature rise during the treatment. Also the finite element solution is further used to determine the effect of the microwave power, the cooling fluid temperature and the blood perfusion on the tissue temperature rise. The results of the solution are compared to temperature measurements in a canine protocol. It was found that the maximum temperature rise in the tissue during treatment is 44 degrees C at a depth of 2 cm from the rectal mucosa.

Survival of directionally solidified B-lymphoblasts under various crystal growth conditions.

Reduction of temperature during freezing brings about two complex and interrelated phenomena: (1) crystal nucleation and subsequent growth processes and (2) change in biophysical properties of a biological system. The purpose of this investigation is to relate the morphology of the solid phase with the survival of a cell. To this end, B-lymphoblasts were exposed to directional solidification in phosphate-buffered saline + 0.05 M dimethyl sulfoxide. Directional solidification is a freezing technique which allows the morphology of the interface to be varied without varying the chemical history that a cell would experience during a constant cooling rate protocol. Results indicated that, for the range of experimental conditions tested, a maximum survival of approximately 78% could be achieved using a temperature gradient of 25(10)3 K/m and an interface velocity of 23(10)-6 m/s (cooling rate: 35 K/min). Survival dropped off sharply for freezing at faster cooling rates with little or no variation in survival for different crystal growth conditions. Survival at slower cooling rates decreased with decreasing cooling rate. It was observed, however, that the presence of secondary branches in the ice phase correlated with lower survival for a given cooling rate. These results indicated that not only is the redistribution of solute during freezing a potential source of damage during freezing but ice/cell interactions are also. Thus, the cooling rate alone may not be adequate to describe the freezing process.

Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel.

During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me2SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B50) was 70 degrees C/min for cells frozen after 1 day in culture and decreased to 15 degrees C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me2SO + DMEM solution, the value of B50 decreased from 70 to 50 degrees C/min for cells in culture for 1 day and from 15 to 10 degrees C/min for cells in culture for 7 days. The value of the average temperature for IIF (TIIF) for cultured cells was only slightly depressed by the addition of Me2SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me2SO on IIF characteristics implies that lower concentrations of Me2SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B50 measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.

Cryopreservation of isolated hepatocytes: intracellular ice formation under various chemical and physical conditions.

Kinetics of intracellular ice formation (IIF) for isolated rat hepatocytes was studied using a cryomicroscopy system. The effect of the cooling rate on IIF was investigated between 20 and 400 degrees C/min in isotonic solution. At 50 degrees C/min and below, none of the hepatocytes underwent IIF; whereas at 150 degrees C/min and above, IIF was observed throughout the entire hepatocyte population. The temperature at which 50% of hepatocytes showed IIF (50TIIF) was almost constant with an average value of -7.7 degrees C. Different behavior was seen in isothermal subzero holding temperatures in the presence of extracellular ice. 50TIIF from isothermal temperature experiments was approximately -5 degrees C as opposed to -7.7 degrees C for constant cooling rate experiments. These experiments clearly demonstrated both the time and temperature dependence of IIF. On the other hand, in cooling experiments in the absence of extracellular ice, IIF was not observed until approximately -20 degrees C (at which temperature the whole suspension was frozen spontaneously) suggesting the involvement of the external ice in the initiation of IIF. The effect of dimethyl sulfoxide (Me2SO) on IIF was also quantified. 50TIIF decreased from -7.7 degrees C in the absence of Me2SO to -16.8 degrees C in 2.0 M Me2SO for a cooling rate of 400 degrees C/min. However, the cooling rate (between 75 and 400 degrees C/min) did not significantly affect 50TIIF (-8.7 degrees C) in 0.5 M Me2SO. These results suggest that multistep protocols will be required for the cryopreservation of hepatocytes.

Viscoelastic properties of the contracting detrusor. II. Experimental approach.

Mechanical properties of detrusor muscle were studied with small-amplitude oscillatory volume perturbations in isometrically contracting bladders of anesthetized dogs. Contractions were studied at oscillatory frequencies (f) of 2 and 4 Hz and at bladder volumes (Vbl) ranging from 30 to 110 ml. The magnitude of bladder hydrodynamic stiffness (magnitude of G) increased linearly with mean detrusor pressure (Pdet) while the phase angle remained relatively constant during contraction. The slope (mG) of magnitude of G-Pdet relations had a positive dependence on f and a negative dependence on Vbl. Analysis of oscillatory data, described in the companion paper, was performed using incremental lumped-parameter models consisting of a spring with incremental constant (S = dF/dL), a viscous element with incremental viscosity (b = dF/du), and a mass (m). Only the model where elastic and viscous elements were placed in series with each other and in parallel with mass was compatible with the experimental data. Both S and b increased linearly with effective force (F), defined as Pdet times the cross-sectional area of the intravesical cavity. Slopes of the S-F and b-F relationships (ms and mb) were independent of Vbl and varied only slightly with f. The importance of this finding stems from recognizing that ms and mb correspond to the exponential coefficients of nonlinear series elastic and internal viscosity elements. These parameters, when normalized by resting muscle length, represent fundamental muscle properties independent of muscle cross-sectional area, stretch, or level of activation and compare well with parameters derived from other muscle systems using techniques such as quick releases and isotonic contractions.

Cryomicroscopic analysis of intracellular ice formation during freezing of mouse oocytes without cryoadditives.

Kinetics of intracellular ice formation (IIF) under various freezing conditions was investigated for mouse oocytes at metaphase II obtained from B6D2F1 mice. A new cryostage with improved optical performance and "isothermal" temperature field was used for nucleation experiments. The maximum thermal gradient across the window was less than 0.1 degrees C/10 mm at sample temperatures near 0 degrees C. The dependence of IIF on the initial concentration of the suspending medium was found to be pronounced. The mean IIF temperatures were found to be -9.56, -12.49, -17.63, -22.20 degrees C for freezing at 120 degrees C/min in 200, 285, 510, and 735 mosm phosphate-buffered saline, respectively. For concentrations higher than 735 mosm, the kinetics of IIF showed a break point at approximately -31 degrees C. Below -31 degrees C, all the remaining unfrozen oocytes underwent IIF almost immediately over a temperature range of less than 3 degrees C. This dramatic shift in the kinetics of IIF suggests that there were two distinct mechanisms responsible for IIF during freezing. The effect of the cooling rate on the kinetics of IIF was also investigated in isotonic PBS. At 1 degrees C/min none of the oocytes contained ice, whereas, at 5 degrees C/min all the oocytes contained ice. The mean IIF temperatures for cooling rates between 1 and 120 degrees C/min were almost constant with an average of -12.82 +/- 0.6 degrees C (SEM). In addition, constant temperature experiments were conducted in isotonic PBS. The percentages of oocytes with IIF were 0, 50, 60, and 95% for -3.8, -6.4, -7.72, and -8.85 degrees C. In undercooling experiments, IIF was not observed until approximately -20 degrees C (at which temperature the whole suspension was frozen spontaneously), suggesting the involvement of the external ice in the initiation of IIF between approximately -5 and -31 degrees C during freezing of oocytes.

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.

Redefining cooling rate in terms of ice front velocity and thermal gradient: first evidence of relevance to freezing injury of lymphocytes.

A freezing process and the resulting injury or survival of biological cells is commonly characterized in terms of the cooling rate, B. Under certain circumstances, the cooling rate can be expressed as B = G.v, where G denotes the thermal gradient at the ice-liquid interface and v its velocity, respectively. To determine the influence of G and v on the morphology of the ice-liquid interface and on cell survival, a gradient freezing stage was designed. Flat capillaries could be pushed with constant velocity from a warm to a cold heat reservoir. With this setup both parameters, G and v, are independently adjustable and the resulting process of directional solidification can be observed dynamically in a light microscope. Human lymphocytes in phosphate-buffered saline with 10 vol% of dimethyl sulfoxide were used as biological test material. Viability was assessed by a membrane integrity test with fluorescein diacetate and ethidium bromide. All cells were cooled down to a final temperature of -196 degrees C and then rapidly thawed. The results obtained with this technique show that the viability determined after freezing and thawing with a certain cooling rate, B = G.v, may vary considerably depending on the imposed values of the thermal gradient, G, and the ice front velocity, v. In addition, the data seem to suggest that, first, the maximum viability which can be reached is governed by the cooling rate, and, second, this maximum for a given cooling rate could be achieved by establishing small temperature gradients and high interface velocities (about 30 degrees K/cm and 500 microns/sec, respectively, for the range of values of G and v tested).

Water transport and estimated transmembrane potential during freezing of mouse oocytes.

The kinetics of water transport and the changes in transmembrane potential during freezing of mouse oocytes in isotonic phosphate buffered saline (PBS) were simulated using thermodynamic models. The permeability to water at 0 degree C, Lpg, and the activation energy, ELp, of metaphase II mouse oocytes from B6D2F1 mice were determined to be 0.044 +/- 0.008 micron/min-atm and 13.3 +/- 2.5 kcal/mol during freezing at 2 degrees C/min. The inactive cell volume was determined to be 0.214 with a correlation coefficient of 0.995, indicating that the oocytes closely follow the ideal Boyle-van't Hoff relation. The mean value of the oocyte diameter was 79.41 +/- 4.62 microns. These results were used to predict the behavior of mouse oocytes under various freezing conditions. The effect of the cooling rate on the cell volume and cytoplasm undercooling was investigated. The changes in transmembrane potential were also investigated during freezing of mouse oocytes. The computer simulations showed that at the beginning of the freezing process (-1 degrees C), the fast growth of ice in the extracellular solution causes a sharp increase of the membrane potential. It is predicted that the change in membrane potential is substantial for almost all cooling rates. Estimations show that values as high as -90 mV may be reached during freezing. The hyperpolarization of the membrane may cause orientation of the dipoles within the membrane. For membrane proteins with 300 debye dipole moment, the theoretical prediction suggests that the percentage of dipoles aligned with the membrane potential increases from 16% at 0 degrees C prior to freezing to 58% at -8 degrees C after seeding of the external ice followed with a cooling at 120 degrees C/min.