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J C Bischof

Publications and source records attributed to J C Bischof.

36 records · Page 2Linked to original sources
PubMed1999

Liver freezing response of the freeze-tolerant wood frog, Rana sylvatica, in the presence and absence of glucose. II. Mathematical modeling.

The "two-step" low-temperature microscopy (equilibrium and dynamic) freezing methods and a differential scanning calorimetry (DSC) technique were used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing, in Part I of this study. In this study, the experimentally determined dynamic water transport data are curve fit to a model of water transport using a standard Krogh cylinder geometry (Model 1) to predict the biophysical parameters of water transport: L(pg) = 1.76 microm/min-atm and E(L(p)) = 75.5 kcal/mol for control liver cells and L(pg)[cpa] = 1.18 microm/min-atm and E(L(p))[cpa] = 69.0 kcal/mol for liver cells equilibrated with 0.4 M glucose. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5 degrees C/min but do so when cooled at 2 degrees C/min. Cells also retained twice as much intracellular fluid in the presence of 0.4 M glucose than in control tissue when cooled at 5 degrees C/min. The ability of R. sylvatica liver cells to retain water during fast cooling (>/=5 degrees C/min) appears to be primarily due to its liver tissue architecture and not to a dramatically lower permeability to water, in comparison to mammalian (rat) liver cells which do dehydrate completely when cooled at 5 degrees C/min. A modified Krogh model (Model 2) was constructed to account for the cell-cell contact in frog liver architecture. Using the same biophysical permeability parameters obtained with Model 1, the modified Krogh model (Model 2) is used in this study to qualitatively explain the experimentally measured water retention in some cells during freezing on the basis of different volumetric responses by cells directly adjacent to vascular space versus cells at least one cell removed from the vascular space. However, at much slower cooling rates (1-2 degrees C/h) experienced by the frog in nature, the deciding factor in water retention is the presence of glucose and the maintenance of a sufficiently high subzero temperature (>/=-8 degrees C).

Animals↗
PubMed1999

A parametric study of freezing injury in AT-1 rat prostate tumor cells.

The connection between thermal history and cell injury in single AT-1 cells is studied systematically through a two-level, four-parameter (2(4)) experiment. The four parameters considered are cooling rate (CR), end temperature (ET), hold time (HT), and thawing rate (TR). Cryosurgically relevant high and low values of each parameter are chosen (CR, 5 to 50 degrees C/min; ET, -20 to -80 degrees C; HT, 0 to 15 min; TR, 20 to 200 degrees C/min) to maximize applicability of the results to cryosurgery; it is important to note that any conclusions drawn from the results are valid only for the range of parameter values studied. AT-1 cell suspensions are frozen in a controlled way on a directional solidification stage, and viability is assessed postthaw with a live/dead assay using the fluorescent dyes calcein-AM and propidium iodide to indicate live and dead cells, respectively. The parameters which most significantly affect short-term survival outcome are determined through calculation of the individual parameter effect values (E) according to the factorial experimental design guidelines. In addition, any synergy between two parameters in determining short-term survival outcome is revealed by calculation of the interaction value for those parameters (I). The results suggest that survival is most significantly affected by variation in end temperature and hold time, and the only significant parameter interaction found is between these two parameters. The analysis further suggests that survival depends nonlinearly on the thermal parameters, based on calculation of the survival curvature (C) in the parameter ranges studied. These results are discussed within the context of previously proposed mechanisms of cellular injury during freezing. Although coupling between several mechanisms is possible, single mechanisms which may explain the survival results include slow-cooling injury mechanisms such as solute effects injury, dehydration-induced membrane instabilities, and volume-catalyzed nucleation of intracellular ice.

Animals↗
PubMed1999

Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents.

Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp)[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp)[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions.

Animals↗
PubMed1998

Response of a liver tissue slab to a hyperosmotic sucrose boundary condition: microscale cellular and vascular level effects.

Transport of a non-permeating CPA in liver tissue was studied by experimental and theoretical techniques. The system consisted of a 20 mm x 15 mm x 500 microns (thick) slab of liver tissue which was exposed to culture media and hyperosmotic sucrose (0.3 or 0.6 M) at the boundary. The volumetric changes of cell and vascular spaces within the tissue slab at 125 microns from one of the symmetric boundaries was studied by slam freezing followed by freeze substitution microscopy. The experimental data was then theoretically investigated using two models; one based on an effective diffusion coefficient for sucrose, and another which incorporated the convective flux of water out of the cells (and the tissue) while sucrose diffuses in. We estimate the effective diffusion of sucrose as 16-33% of the actual diffusivity of sucrose in bulk water. The role of convection of water out of the tissue is against the flow of sucrose and appears to be important in reducing the effective diffusivity of the sucrose. The role of vascular compliance, porosity and tortuosity are also discussed with respect to our results.

Animals↗
PubMed1998

Biophysics of freezing in liver of the freeze-tolerant wood frog, R. sylvatica.

This study investigates the water transport characteristics during freezing in the liver tissue of the freeze-tolerant wood frog Rana sylvatica. Experiments were performed using both low temperature microscopy and a differential scanning calorimeter (DSC). Tissue samples were cooled at 2 and 5 degree C/min by a "two-step" freezing technique to end temperatures of -4, -6, -8, -10, and -20 degrees C, followed by a slam cooling (> 1000 degrees C/min) step. Stereological analysis of the low temperature microscopy results leads to the conclusions that 74% of the control tissue is cellular (26% vascular), Vb (osmotically inactive cell volume) is 0.4 Vo and the Krogh cylinder dimensions are: distance between adjacent sinusoid centers, delta X = 64 microns, original sinusoid (vascular) radius, rvo = 18.4 microns and length of the Krogh cylinder, L = 0.71 microns (assuming a single isolated hepatocyte cell diameter of 16 microns). A parallel study was also done using the DSC at 2 and 5 degrees C/min, and the measured heat releases from the tissue were used to calculate water transport data. Both techniques confirmed that tissue cooled at 5 degrees C/min does not dehydrate completely, but does so when cooled at 2 degrees C/min. By curve fitting a model to 5 degrees C/min water transport data from both techniques the biophysical parameters of water transport were obtained: Lpg = 1.76 microns/min-atm and ELp = 75.5 Kcal/mol. A modified Krogh model was used to account for the fact that approximately 24% of the hepatocytes were found not to be in direct contact with the vasculature. This model was then used to explain the experimentally measured water retention in some cells on the basis of different volumetric responses to dehydration of cells directly adjacent to vascular spaces and cells at least one cell removed from the vascular spaces.

Animals↗
PubMed1998

Measurement of water transport during freezing in cell suspensions using a differential scanning calorimeter.

A new technique using a differential scanning calorimeter (DSC) was developed to obtain dynamic and quantitative water transport data in cell suspensions during freezing. The model system investigated was a nonattached spherical lymphocyte (Epstein-Barr virus transformed, EBVT) human cell line. Data from the technique show that the initial heat release of a prenucleated sample containing osmotically active cells in media is greater than the final heat release of an identical sample of osmotically inactive or lysed cells in media. The total integrated magnitude of this difference, Deltaqdsc, was found to be proportional to the cytocrit and hence also to the supercooled water volume in the sample. Further, the normalized fractional integrated heat release difference as a function of temperature, Deltaq(T)dsc/Deltaqdsc, was shown to correlate with the amount of supercooled cellular water which had exosmosed from the cell as a function of subzero temperature at constant cooling rates of 5, 10, and 20 degrees C/min. Several important limitations of the technique are (1) that it requires a priori knowledge of geometric parameters such as the surface area, initial volume, and osmotically inactive cell volume and (2) that the technique alone cannot determine whether the heat released from supercooled cellular water is due to dehydration or intracellular ice formation. Cryomicroscopy was used to address these limitations. The initial cell volume and surface area were obtained directly whereas a Boyle-van't Hoff (BVH) plot was constructed to obtain the osmotically inactive cell volume Vb. Curve fitting the BVH data assuming linear osmometric behavior yielded Vb = 0.258V0; however, nonlinearity in the data suggests that the EBVT lymphocyte cells are not "ideal osmometers" at low subzero temperatures and created some uncertainty in the actual value of Vb. Cryomicroscopy further confirmed that dehydration was the predominant biophysical response of the cells over the range of cooling rates investigated. One notable exception occurred at a rate of 20 degrees C/min where evidence for intracellular ice formation due to a DSC measured heat release between -30 and -34 degrees C correlated with a higher end volume but no darkening of the cells during cryomicroscopy. For the cooling rate tested (5 degrees C/min) the cryomicroscopy data correlated statistically very well with the DSC water transport data. A model of water transport was fit to the DSC water transport data and the average (5, 10, and 20 degrees C/min) biophysical parameters for the EBVT lymphocytes were found to be Lpg = 0.10 micro m/min-atm, ELp = 15.5 kcal/mol. Finally, the decrease in heat release from osmotically active cells measured by the DSC during repetitive freezing and thawing was found to correlate strongly with the viability of the cells measured during identical freeze/thaw protocols with cryomicroscopy. This shows the additional ability of the technique to assess freeze/thaw injury. In summary, this DSC technique is a promising new approach for measuring water transport in cellular systems during freezing.

Biological Transport↗
PubMed1998

The effect of dimethylsulfoxide on the water transport response of rat hepatocytes during freezing.

Successful improvement of cryopreservation protocols for cells in suspension requires knowledge of how such cells respond to the biophysical stresses of freezing (intracellular ice formation, water transport) while in the presence of a cryoprotective agent (CPA). This work investigates the biophysical water transport response in a clinically important cell type--isolated hepatocytes--during freezing in the presence of dimethylsulfoxide (DMSO). Sprague-Dawley rat liver hepatocytes were frozen in Williams E media supplemented with 0, 1, and 2 M DMSO, at rates of 5, 10, and 50 degrees C/min. The water transport was measured by cell volumetric changes as assessed by cryomicroscopy and image analysis. Assuming that water is the only species transported under these conditions, a water transport model of the form dV/dT = f(Lpg([CPA]), ELp([CPA]), T(t)) was curve-fit to the experimental data to obtain the biophysical parameters of water transport--the reference hydraulic permeability (Lpg) and activation energy of water transport (ELp)--for each DMSO concentration. These parameters were estimated two ways: (1) by curve-fitting the model to the average volume of the pooled cell data, and (2) by curve-fitting individual cell volume data and averaging the resulting parameters. The experimental data showed that less dehydration occurs during freezing at a given rate in the presence of DMSO at temperatures between 0 and -10 degrees C. However, dehydration was able to continue at lower temperatures (< -10 degrees C) in the presence of DMSO. The values of Lpg and ELp obtained using the individual cell volume data both decreased from their non-CPA values--4.33 x 10(-13) m3/N-s (2.69 microns/min-atm) and 317 kJ/mol (75.9 kcal/mol), respectively--to 0.873 x 10(-13) m3/N-s (0.542 micron/min-atm) and 137 kJ/mol (32.8 kcal/mol), respectively, in 1 M DMSO and 0.715 x 10(-13) m3/N-s (0.444 micron/min-atm) and 107 kJ/mol (25.7 kcal/mol), respectively, in 2 M DMSO. The trends in the pooled volume values for Lpg and ELp were very similar, but the overall fit was considered worse than for the individual volume parameters. A unique way of presenting the curve-fitting results supports a clear trend of reduction of both biophysical parameters in the presence of DMSO, and no clear trend in cooling rate dependence of the biophysical parameters. In addition, these results suggest that close proximity of the experimental cell volume data to the equilibrium volume curve may significantly reduce the efficiency of the curve-fitting process.

Animals↗
PubMed1998

Measurement of water transport during freezing in mammalian liver tissue: Part II--The use of differential scanning calorimetry.

There is currently a need for experimental techniques to assay the biophysical response (water transport or intracellular ice formation, IIF) during freezing in the cells of whole tissue slices. These data are important in understanding and optimizing biomedical applications of freezing, particularly in cryosurgery. This study presents a new technique using a Differential Scanning Calorimeter (DSC) to obtain dynamic and quantitative water transport data in whole tissue slices during freezing. Sprague-Dawley rat liver tissue was chosen as our model system. The DSC was used to monitor quantitatively the heat released by water transported from the unfrozen cell cytoplasm to the partially frozen vascular/extracellular space at 5 degrees C/min. This technique was previously described for use in a single cell suspension system (Devireddy, et al. 1998). A model of water transport was fit to the DSC data using a nonlinear regression curve-fitting technique, which assumes that the rat liver tissue behaves as a two-compartment Krogh cylinder model. The biophysical parameters of water transport for rat liver tissue at 5 degrees C/min were obtained as Lpg = 3.16 x 10(-13) m3/Ns (1.9 microns/min-atm), ELp = 265 kJ/mole (63.4 kcal/mole), respectively. These results compare favorably to water transport parameters in whole liver tissue reported in the first part of this study obtained using a freeze substitution (FS) microscopy technique (Pazhayannur and Bischof, 1997). The DSC technique is shown to be a fast, quantitative, and reproducible technique to measure dynamic water transport in tissue systems. However, there are several limitations to the DSC technique: (a) a priori knowledge that the biophysical response is in fact water transport, (b) the technique cannot be used due to machine limitations at cooling rates greater than 40 degrees C/min, and (c) the tissue geometric dimensions (the Krogh model dimensions) and the osmotically inactive cell volumes Vb, must be determined by low-temperature microscopy techniques.

Animals↗
PubMed1997

Cryosurgery of dunning AT-1 rat prostate tumor: thermal, biophysical, and viability response at the cellular and tissue level.

This study investigates cryodestruction of the Dunning AT-1 rat prostate tumor at the single cell, tissue slice, and in vivo levels. The thermal history around a 3-mm-diameter cylindrical cryosurgical probe was predicted by solving the bioheat equation in a one-dimensional cylindrical geometry. At various radial positions in the iceball this thermal history was approximated by a constant cooling rate and a final, steady-state temperature (or end-temperature). The predicted cooling rates and end temperatures ranged from > or = 1000 degrees C/min to 5 degrees C/min and -196 degrees C to -20 degrees C, respectively. These cooling rates and end-temperatures were then imposed on single AT-1 cells, AT-1 tissue slices in vitro and AT-1 tumors in vivo. The single cells and tissue slices were frozen by LN2 immersion, copper block slam-freezing, or controlled cooling on a cryomicroscope or a directional solidification stage. LN2 immersion is lethal to AT-1 cells (presumably due to intracellular ice formation), while cooling at 5-100 degrees C/min leaves some viable cells (at end-temperatures ranging between -20 and -40 degrees C). AT-1 tumor slices show extensive intracellular ice formation due to slam cooling, extensive dehydration at 100 degrees C/min, and total dehydration at rates < or = 10 degrees C/min to end temperatures below -10 degrees C. Postfreeze culture and histology of the AT-1 tissue show that extensive intracellular ice formation is lethal, while cellular dehydration and vascular engorgement leave viable cells (at end-temperatures between -20 and -40 degrees C). Based solely on the single cell and in vitro tissue damage achieved by cooling rates and end-temperatures, a sizable portion of a cryosurgically frozen tumor would be expected to survive. However, in vivo cryosurgery performed on AT-1 tumors demonstrated that the tissue was damaged throughout the cryolesion, even at the periphery where the thermal history would be expected to allow single cells and tissue slices to survive in vitro. Taken together, these results suggest that damage mechanisms other than those due to cooling rate and end-temperature may be responsible for the increased cellular destruction at the periphery of the iceball in vivo and that cooling rate is less important than end-temperature in determining cryosurgical damage in AT-1 tumors. Experiments are ongoing to determine if the time held at an end temperature, thawing rate, vascular response, or other mechanisms are primarily responsible for the enhanced destructive capability in vivo.

Animals↗
PubMed1997

Rectal protection during prostate cryosurgery: design and characterization of an insulating probe.

This study presents the design and characterization of an insulating probe made of silicone that could be used for enhancing the safety and efficacy of prostate cryosurgery. The probe would be placed in Denonvilliers' fascia between the prostate and the rectum prior to freezing. During freezing, the iceball would be monitored by ultrasound through the silicone, and direct temperature monitoring of the rectal and prostatic tissue via thermocouples mounted on opposing sides of the device. Both theoretical and experimental studies were performed to verify the insulating and acoustic properties of the probe. The insulating effect of the silicone will enhance cell death within the prostate while minimizing tissue freezing injury and therefore fistula formation postfreeze in the rectum. Experiments were also performed with the insulator placed in gelatine which showed that the silicone material is transparent to ultrasound. In addition the silicone was itself visible under ultrasound imaging, a characteristic which may assist in the delivery of the device to the surgical site. One possible scenario for reconfiguration and delivery of the device is suggested prior to a cryosurgery. The success of this device in insulating and monitoring temperature during freezing suggests that it can also be useful in protecting sensitive tissues adjacent to a surgical site when extreme heat is applied (i.e., electron or hyperthermic surgery).

Cryosurgery↗
PubMed1997

Measurement and simulation of water transport during freezing in mammalian liver tissue.

Optimization of cryosurgical procedures on deep tissues such as liver requires an increased understanding of the fundamental mechanisms of ice formation and water transport in tissues during freezing. In order to further investigate and quantify the amount of water transport that occurs during freezing in tissue, this study reports quantitative and dynamic experimental data and theoretical modeling of rat liver freezing under controlled conditions. The rat liver was frozen by one of four methods of cooling: Method 1-ultrarapid "slam cooling" (> or = 1000 degrees C/min) for control samples; Method 2-equilibrium freezing achieved by equilibrating tissue at different subzero temperatures (-4, -6, -8, -10 degrees C); Method 3-two-step freezing, which involves cooling at 5 degrees C/min. to -4, -6, -8, -10 or -20 degrees C followed immediately by slam cooling; or Method 4-constant and controlled freezing at rates from 5-400 degrees C/min. on a directional cooling stage. After freezing, the tissue was freeze substituted, embedded in resin, sectioned, stained, and imaged under a light microscope fitted with a digitizing system. Image analysis techniques were then used to determine the relative cellular to extracellular volumes of the tissue. The osmotically inactive cell volume was determined to be 0.35 by constructing a Boyle van't Hoff plot using cellular volumes from Method 2. The dynamic volume of the rat liver cells during cooling was obtained using cellular volumes from Method 3 (two-step freezing at 5 degrees C/min). A nonlinear regression fit of a Krogh cylinder model to the volumetric shrinkage data in Method 3 yielded the biophysical parameters of water transport in rat liver tissue of: Lpg = 3.1 x 10(-13) m3/Ns (1.86 microns/min-atm) and ELp = 290 kJ/mole (69.3 kcal/mole), with chi-squared variance of 0.00124. These parameters were then incorporated into the Krogh cylinder model and used to simulate water transport in rat liver tissue during constant cooling at rates between 5-100 degrees C/min. Reasonable agreement between these simulations and the constant cooling rate freezing experiments in Method 4 were obtained. The model predicts that the water transport ceases at a relatively high subzero temperature (-10 degrees C), such that the amount of intracellular ice forming in the tissue cells rises from almost none (= extensive dehydration and vascular expansion) at < or = 5 degrees C/min to over 88 percent of the original cellular water at > or = 50 degrees C/min. The theoretical simulations based on these experimental methods may be of use in visualizing and predicting freezing response, and thus can assist in the planning and implementing of cryosurgical protocols.

Animals↗
PubMed1995

Dynamics of cell membrane permeability changes at supraphysiological temperatures.

A quantitative fluorescent microscopy system was developed to characterize, in real time, the effects of supraphysiological temperatures between 37 degrees and 70 degrees C on the plasma membrane of mouse 3T3 fibroblasts and isolated rat skeletal muscle cells. Membrane permeability was assessed by monitoring the leakage as a function of time of the fluorescent membrane integrity probe calcein. The kinetics of dye leakage increased with increasing temperature in both the 3T3 fibroblasts and the skeletal muscle cells. Analytical solutions derived from a two-compartment transport model showed that, for both cell types, a time-dependent permeability assumption provided a statistically better fit of the model predictions to the data than a constant permeability assumption. This finding suggests that the plasma membrane integrity is continuously being compromised while cells are subjected to supraphysiological temperatures.

3T3 Cells↗
PubMed1993

Large ice crystals in the nucleus of rapidly frozen liver cells.

Evidence in the literature shows that ice crystals that form in the nucleus of many rapidly cooled cells appear much larger than the ice crystals that form in the surrounding cytoplasm. We investigated the phenomenon in our laboratory using the techniques of freeze substitution and low temperature scanning electron microscopy on liver tissue frozen by liquid nitrogen plunge freezing. This method is estimated to cool the tissue at 1000 degrees C/min. The results from these techniques show that the ice crystal sizes were statistically significantly larger in the nucleus than in the cytoplasm. It is our belief that this finding is important to cryobiology considering its potential role in the process of freezing and the mechanisms of damage during freezing of cells and tissues.

Animals↗
PubMed1992

An analytical study of cryosurgery in the lung.

The process of freezing in healthy lung tissue and in tumors in the lung during cryosurgery was modeled using one-dimensional close form techniques and finite difference techniques to determine the temperature profiles and the propagation of the freezing interface in the tissue. A thermal phenomenon was observed during freezing of lung tumors embedded in healthy tissue, (a) the freezing interface suddenly accelerates at the transition between the tumor and the healthy lung, (b) the frozen tumor temperature drops to low values once the freezing interface moves into the healthy lung, and (c) the outer boundary temperature has a point of sharp inflection corresponding to the time at which the tumor is completely frozen.

Algorithms↗
PubMedDate not supplied

Evaluation of freezing effects on human microvascular-endothelial cells (HMEC).

There is mounting evidence that the endothelium may play an important role in traditional cryosurgical treatments by acting to locally foster thrombi in the microvasculature of various tissues after freezing. In addition, new catheter based cryosurgical probes are being designed for cardiovascular applications where endothelial and smooth muscle cell freezing is involved but not well understood. Therefore, this study was designed to investigate, at the cellular level in human microvascular endothelial cells (hMEC), the various biophysical changes that occur during freezing which can affect post-freeze viability. The hMECs were loaded on a cryomicroscope stage and freezing experiments at 5, 10, 15, 25, 100 and 130 degrees C/min were performed to experimentally evaluate dehydration (water transport) as well as intracellular ice formation (IIF) within this cell system. The dehydration kinetics at 5, 10 and 25 degrees C/min were found to be governed by a membrane permeability L(pg) and activation energy E(Lp) of 0.05 (microm/min.atm) and 14.8 (kcal/mole) respectively [R(2)=0.94]. These parameters were then tested for predictive ability against the experimentally measured behavior at 15 degrees C/min with a good agreement [R(2)=0.98]. Intracellular Ice Formation (IIF) was found to occur at lower temperatures than many cell types (i.e. TIIF 50% approximately -18 degrees C) and at cooling rates greater than or equal to 25 degrees C/min. At cooling rates above 50 degrees C/min, two types of IIF, cell darkening and twitching, were both observed and quantified and were assumed to be governed by Surface Catalyzed Nucleation (SCN). IIF parameters, omega(o) and kappa(o), which fit data from 50, 100 and 130 degrees C/min were found to be 6.8 x 10(-8) (m(2).s)(-1) and 8.3 x 10(-9) (K5) [R(2)=0.94] respectively. Preliminary results show that viability drops precipitously between -20 and -30 degrees C, however, further studies are warranted to address the role of cooling rate, end-temperature, hold time and thawing rate to establish the freeze sensitivity of this cell.

Biological Transport↗
PubMedDate not supplied

Ice formation in isolated human hepatocytes and human liver tissue.

Cryopreservation of isolated cells and tissue slices of human liver is required to furnish extracorporeal bioartificial liver devices with a ready supply of hepatocytes, and to create in vitro drug metabolism and toxicity models. Although both the bioartificial liver and many current biotoxicity models are based on reconstructing organ functions from single isolated hepatocytes, tissue slices offer an in vitro system that may more closely resemble the in vivo situation of the cells because of cell-cell and cell-extracellular matrix interactions. However, successful cryopreservation of both cellular and tissue level systems requires an increased understanding of the fundamental mechanisms involved in the response of the liver and its cells to freezing stress. This study investigates the biophysical mechanisms of water transport and intracellular ice formation during freezing in both isolated human hepatocytes and whole liver tissue. The effects of cooling rate on individual cells were measured using a cryomicroscope. Biophysical parameters governing water transport (Lpg = 2.8 microns/min-atm and ELp = 79 kcal/mole) and intracellular heterogeneous ice nucleation (omega het = 1.08 x 10(9) m-2s-1 and kappa het = 1.04 x 10(9) K5) were determined. These parameters were then incorporated into a theoretical Krogh cylinder model developed to simulate water transport and ice formation in intact liver tissue. Model simulations indicated that the cellular compartment of the Krogh model maintained more water than isolated cells under the same freezing conditions. As a result, intracellular ice nucleation occurred at lower cooling rates in the Krogh model than in isolated cells. Furthermore, very rapid cooling rates (1000 degrees C/min) showed a depression of heterogeneous nucleation and a shift toward homogeneous nucleation. The results of this study are in qualitative agreement with the findings of a previous experimental study of the response to freezing of intact human liver.

Cell Communication↗