Supplemented phase diagrams for vitrification CPA cocktails: DP6, VS55 and M22.

DP6, VS55 and M22 are the most commonly used cryoprotective agent (CPA) cocktails for vitrification experiments in tissues and organs. However, complete phase diagrams for the three CPAs are often unavailable or incomplete (only available for full strength CPAs) thereby hampering optimization of vitrification and rewarming procedures. In this paper, we used differential scanning calorimetry (DSC) to measure the transition temperatures including heterogeneous nucleation temperatures (Thet), glass transition temperatures (Tg), rewarming phase crystallization (devitrification and/or recrystallization) temperatures (Td) and melting temperatures (Tm) while cooling or warming the CPA sample at 5 °C/min and plotted the obtained transition temperatures for different concentrations of CPAs into the phase diagrams. We also used cryomicroscopy cooling or warming the sample at the same rate to record the ice crystallization during the whole process, and we presented the cryomicroscopic images at the transition temperatures, which agreed with the DSC presented phenomena.

A guide to successful mL to L scale vitrification and rewarming.

Cryopreservation by vitrification to achieve an "ice free" glassy state is an effective technique for preserving biomaterials including cells, tissues, and potentially even whole organs. The major challenges in cooling to and rewarming from a vitrified state remain ice crystallization and cracking/fracture. Ice crystallization can be inhibited by the use of cryoprotective agents (CPAs), though the inhibition further depends upon the rates achieved during cooling and rewarming. The minimal rate required to prevent any ice crystallization or recrystallization/devitrification in a given CPA is called the critical cooling rate (CCR) or critical warming rate (CWR), respectively. On the other hand, physical cracking is mainly related to thermomechanical stresses, which can be avoided by maintaining temperature differences below a critical threshold. In this simplified analysis, we calculate deltaT as the largest temperature difference occurring in a system during cooling or rewarming in the brittle/glassy phase. This deltaT is then used in a simple "thermal shock equation" to estimate thermal stress within the material to decide if the material is above the yield strength and to evaluate the potential for fracture failure. In this review we aimed to understand the limits of success and failure at different length scales for cryopreservation by vitrification, due to both ice crystallization and cracking. Here we use thermal modeling to help us understand the magnitude and trajectory of these challenges as we scale the biomaterial volume for a given CPA from the milliliter to liter scale. First, we solved the governing heat transfer equations in a cylindrical geometry for three common vitrification cocktails (i.e., VS55, DP6, and M22) to estimate the cooling and warming rates during convective cooling and warming and nanowarming (volumetric heating). Second, we estimated the temperature difference deltaT and compared it to a tolerable threshold (deltaTmax) based on a simplified "thermal shock" equation for the same cooling and rewarming conditions. We found, not surprisingly, that M22 achieves vitrification more easily during convective cooling and rewarming for all volumes compared to VS55 or DP6 due to its considerably lower CCR and CWR. Further, convective rewarming (boundary rewarming) leads to larger temperature differences and smaller rates compared to nanowarming (volumetric rewarming) for all CPAs with increasing failure at larger volumes. We conclude that as more and larger systems are vitrified and rewarmed with standard CPA cocktails, this work can serve as a practical guide to successful implementation based on the characteristic length (volume/surface area) of the system and the specific conditions of cooling and warming. doi.org/10.54680/fr22610110112.

Cryoinjury of MCF-7 human breast cancer cells and inhibition of post-thaw recovery using TNF-alpha.

Cryoinjury of MCF-7 human breast cancer cells and its enhancement using tumor necrosis factor-alpha (TNF-alpha) as an adjuvant, were investigated. Through a series of experiments in a two level factorial design critical parameters affecting cryotherapy responses were identified. The cryoinjury was investigated by quantifying the effects of four freeze/thaw (F/T) parameters, selected to be within the expected range for a cryosurgical iceball. Thermal parameters considered were cooling rate (5 and 50 degrees C/min), end temperature (-20 and -80 degrees C), hold time (0 and 10 min), and thawing rate (20 and 100 degrees C/min). After exposing the cells to the selected F/T conditions, survival was assessed and statistically analyzed to determine the effect of each parameter and their interactions. A statistical analysis shows that the end temperature and hold time were the two most significant parameters in the range studied. This suggests that proper control of these two parameters is important to achieve desired cryodestruction of MCF-7 cells. Enhancement of cryoinjury by TNF-alpha was also investigated in a tissue equivalent cryoinjury model in which a cryosurgical iceball is formed. MCF-7 cells cultured in a collagen matrix underwent a controlled F/T with or without TNF-alpha pre-treatment at 100 ng/ml for 24 hours. Post-thaw viability of MCF-7 cells was assessed at three hours, and at one and three days after freezing. Although the TNF-alpha treatment alone induced neither apoptotic nor necrotic cell death, the combination of TNF-alpha pre-treatment and freezing enhanced the immediate cryoinjury of MCF-7 cells, and significantly impaired the post-thaw recovery. Without TNF-alpha treatment, MCF-7 cell cultures were repopulated, reaching approximately 80% survival at day 3 even after severe cryoinjury (< or = 20% survival) at three hours. In contrast, this repopulation was significantly inhibited by TNF-alpha pre-treatment, in which case the viability of the frozen region remained below 40% at day 3. The effects of TNF-alpha on the cryoinjury of MCF-7 cells suggest that TNF-alpha may serve as a potent adjuvant to cryosurgery of breast cancer.

Use of a fluorescently labeled poly-caspase inhibitor for in vivo detection of apoptosis related to vascular-targeting agent arsenic trioxide for cancer therapy.

Arsenic trioxide (ATO, Trisenox) is a potent anti-vascular agent and significantly enhances hyperthermia and radiation response. To understand the mechanism of the anti-tumor effect in vivo we imaged the binding of a fluorescently-labeled poly-caspase inhibitor (FLIVO) in real time before and 3 h or 24 h after injection of 8 mg/kg ATO. FSaII tumors were grown in dorsal skin-fold window chambers or on the rear limb and we observed substantial poly-caspase binding associated with vascular damage induced by ATO treatment at 3 and 24 h after ATO injection. Flow cytometric analysis of cells dissociated from the imaged tumor confirmed cellular uptake and binding of the FLIVO probe. Apoptosis appears to be a major mode of cell death induced by ATO in the tumor and the use of fluorescently tagged caspase inhibitors to assess cell death in live animals appears feasible to monitor and/or confirm anti-tumor effects of therapy.

Re-purposing cryoablation: a combinatorial 'therapy' for the destruction of tissue.

It is now recognized that the tumor microenvironment creates a protective neo-tissue that isolates the tumor from the various defense strategies of the body. Evidence demonstrates that, with successive therapeutic attempts, cancer cells acquire resistance to individual treatment modalities. For example, exposure to cytotoxic drugs results in the survival of approximately 20-30% of the cancer cells as only dividing cells succumb to each toxic exposure. With follow-up treatments, each additional dose results in tumor-associated fibroblasts secreting surface-protective proteins, which enhance cancer cell resistance. Similar outcomes are reported following radiotherapy. These defensive strategies are indicative of evolved capabilities of cancer to assure successful tumor growth through well-established anti-tumor-protective adaptations. As such, successful cancer management requires the activation of multiple cellular 'kill switches' to prevent initiation of diverse protective adaptations. Thermal therapies are unique treatment modalities typically applied as monotherapies (without repetition) thereby denying cancer cells the opportunity to express defensive mutations. Further, the destructive mechanisms of action involved with cryoablation (CA) include both physical and molecular insults resulting in the disruption of multiple defensive strategies that are not cell cycle dependent and adds a damaging structural (physical) element. This review discusses the application and clinical outcomes of CA with an emphasis on the mechanisms of cell death induced by structural, metabolic, vascular and immune processes. The induction of diverse cell death cascades, resulting in the activation of apoptosis and necrosis, allows CA to be characterized as a combinatorial treatment modality. Our understanding of these mechanisms now supports adjunctive therapies that can augment cell death pathways.

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.

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.

Evaluation of thermal therapy in a prostate cancer model using a wet electrode radiofrequency probe.

PURPOSE: To determine the temperature-time threshold of local cell death in vivo for thermal therapy in a prostate cancer animal model and to use this value as a benchmark to quantify global tissue injury. MATERIALS AND METHODS: Two studies were designed in the Dunning AT-1 rat prostate tumor hind limb model. For both studies, a wet electrode radiofrequency (RF) probe was used to deliver 40 W of energy for 18 to 62 seconds after a 30-second infusion of hypertonic saline/Hypaque through the RF antenna. Thermal history measurements were obtained in tumors from at least two Fluoroptic probes placed radially 5 mm from the axis of a RF probe and 10 mm below the surface of the tissue. In study 1, the thermal history required for irreversible cell injury was experimentally determined by comparing the predicted injury accumulation (omega) with cell viability at the fluoroptic probe locations using an in vivo-in vitro assay. The omega value was calculated from the measured thermal histories using an Arrhenius damage model. In study 2, RF energy was applied for 40 seconds in all cases. At 1, 3, and 7 days after thermal therapy, triphenyltetrazolium chloride dye (TTC) and histologic analyses were performed to assess global tissue injury within a 5-mm radius from the axis of the RF probe. RESULTS: Study 1 showed that cell survival dropped to 0 for 0.42 < omega < 0.7. This result was the basis for selection of 40 seconds of RF thermal therapy in study 2, which yielded omegaave = 0.5 in the tissue 5 mm from the probe axis. Both TTC and histology analysis showed that sham-treated tissue was not irreversibly injured. However, there was an inherent heterogeneity present in the tumor that accounted for as much as 15% necrosis in control or sham-treated tissue. In contrast, at 1, 3, and 7 days after therapy, significantly less enzyme activity was observed by TCC in thermally treated tissue compared with sham-treated tissue (35 v 85%; P < 0.001). Histologic analysis of thermally treated tissues revealed a gradual increase in the percent of coagulative necrosis (47%-70%) with a concomitant decrease in the percentage of shocked cells (53%-28%). At day 7, <3% viability was observed in treated tumors compared with 90% viability in sham-treated tissue. CONCLUSION: The threshold of cellular injury in vivo corresponded to omega > 0.7 (> or =48 degrees C for 40 seconds). Global tissue injury could be conservatively predicted on the basis of local thermal histories during therapy.

Measurement and prediction of thermal behavior and acute assessment of injury in a pig model of renal cryosurgery.

PURPOSE: To analyze in vivo end temperatures and histologic injury in a standardized cryo-iceball using a porcine kidney model in order to establish the threshold temperature for tissue ablation. To evaluate the ability to predict end temperatures using a thermal finite element model. MATERIALS AND METHODS: A single freeze/thaw cryolesion was created in five pig kidneys and the temperature history recorded. End temperature was calculated using a thermal finite element model. The threshold temperature for tissue injury was established by directly correlating end temperature and histologic injury. RESULTS: Reproducible geometry and temperature profiles of the cryo-iceball were found. End temperature could be accurately predicted through thermal modeling, and correlation with histologic injury revealed a threshold temperature of -16.1 degrees C for complete tissue ablation. CONCLUSION: Thermal modeling may accurately predict end temperature within a cryo-iceball. Provided threshold temperatures for tissue destruction are known, modeling may become a powerful tool in cryosurgery, improving the assessment of damage in normal and malignant tissue.

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.

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.

Optimizing magnetic nanoparticle based thermal therapies within the physical limits of heating.

Magnetic nanoparticle (mNP) based thermal therapies have demonstrated relevance in the clinic, but effective application requires an understanding of both its strengths and limitations. This study explores two critical limitations for clinical use: (1) maximizing localized mNP heating, while avoiding bulk heating due to inductive coupling of the applied field with the body and (2) the limits of treatable volumes, related to basic heat transfer. Two commercially available mNPs are investigated, one superparamagnetic and one ferromagnetic, thereby allowing a comparison between the two fundamental types of mNPs (both of which are being evaluated for clinical use). Important results indicate that in dispersed solutions, the superparamagnetic mNPs outperform on a per mass basis (2× better), but the ferromagnetic mNPs outperform on a per nanoparticle basis (170× better), at the fields of highest clinical relevance (approximately 100 kHz and 20 kA/m). We also demonstrate a new method of observing heating in microliter droplets of mNP solution, leading to scaling analyses that suggest treatable tumor volumes should be ≥2 mm in diameter (for mNP loading of ≥10 mg Fe/g tumor), to achieve therapeutic temperatures ≥43 °C. This technique also provides a novel platform for quantifying heating from microgram quantities of mNPs.

Use of X-ray tomography to map crystalline and amorphous phases in frozen biomaterials.

The outcome of both cryopreservation and cryosurgical freezing applications is influenced by the concentration and type of the cryoprotective agent (CPA) or the cryodestructive agent (i.e., the chemical adjuvants referred to here as CDA) added prior to freezing. It also depends on the amount and type of crystalline, amorphous and/or eutectic phases formed during freezing which can differentially affect viability. This work describes the use of X-ray computer tomography (CT) for non-invasive, indirect determination of the phase, solute concentration and temperature within biomaterials (CPA, CDA loaded solutions and tissues) by X-ray attenuation before and after freezing. Specifically, this work focuses on establishing the feasibility of CT (100-420 kV acceleration voltage) to accurately measure the concentration of glycerol or salt as model CPA and CDAs in unfrozen solutions and tissues at 20 degrees C, or the phase in frozen solutions and tissue systems at -78.5 and -196 degrees C. The solutions are composed of water with physiological concentrations of NaCl (0.88% wt/wt) and DMEM (Dulbecco's Modified Eagle's Medium) with added glycerol (0-8 M). The tissue system is chosen as 3 mm thick porcine liver slices as well as 2 cm diameter cores which were either imaged fresh (3-4 h cold ischemia) or after loading with DMEM based glycerol solutions (0-8 M) for times ranging from hours to 7 days at 4 degrees C. The X-ray attenuation is reported in Hounsfield units (HU), a clinical measurement which normalizes X-ray attenuation values by the difference between those of water and air. NaCl solutions from 0 to 23.3% wt/wt (i.e. water to eutectic concentration) were found to linearly correspond to HU in a range from 0 to 155. At -196 degrees C the variation was from -80 to 95 HU while at -78.5 degrees C all readings were roughly 10 HU lower. At 20 degrees C NaCl and DMEM solutions with 0-8 M glycerol loading show a linear variation from 0 to 145 HU. After freezing to -78.5 degrees C the variation of the NaCl and DMEM solutions is more than twice as large between -90 and +190 HU and was distinctly non-linear above 6 M. After freezing to -196 degrees C the variation of the NaCl and DMEM solutions increased even further to -80 to +225 HU and was distinctly non-linear above 4 M, which after modeling the phase change and crystallization process is shown to correlate with an amorphous phase. In all tissue systems the HU readings were similar to solutions but higher by roughly 30 HU, as well as showing some deviations at 0 M after storage, probably due to tissue swelling. The standard deviations in all measurements were roughly 5 HU or below in all samples. In addition, two practical examples for CT use were demonstrated including: (1) glycerol loading and freezing of tissue cores and, (2) a mock cryosurgical procedure. In the loading experiment CT was able to measure the permeation of the glycerol into the sample at 20 degrees C, as well as the evolution of distinct amorphous vs. crystalline phases after freezing to -196 degrees C. In the mock cryosurgery example, the iceball edge was clearly visualized, and attempts to determine the temperature within the iceball are discussed. An added benefit of this work is that the density of these frozen samples, an essential property in measurement and modeling of thermal processes, was obtained in comparison to ice.

Quantitative measurement and prediction of biophysical response during freezing in tissues.

Cryopreservation and cryosurgery are important biomedical applications used to selectively preserve or destroy cellular systems through freezing. Studies using cryomicroscopy techniques, which allow the visualization of the freezing process in single cells, have shown that a drop in viability correlates with the extent of two biophysical events during the freezing process: (a) intracellular ice formation and (b) cellular dehydration. These same biophysical events operate in tissue systems; however, the inability to visualize and quantify the dynamics of the freezing process in tissues has hampered direct correlation of these events with freezing-induced changes in viability. This review highlights two new techniques that use freeze substitution and differential scanning calorimetry to provide dynamic freezing data in tissue. Characteristic dimensions and parameters extracted from these new data are then used in a predictive model of biophysical freezing response in several tissues, including liver and tumor. This approach promises to help guide improved design of both cryopreservation and cryosurgical applications of tissue freezing.

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.

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.

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.

Cryosurgery of normal and tumor tissue in the dorsal skin flap chamber: Part I--thermal response.

Current research in cryosurgery is concerned with finding a thermal history that will definitively destroy tissue. In this study, we measured and predicted the thermal history obtained during freezing and thawing in a cryosurgical model. This thermal history was then compared to the injury observed in the tissue of the same cryosurgical model (reported in companion paper (Hoffmann and Bischof, 2001)). The dorsal skin flap chamber, implanted in the Copenhagen rat, was chosen as the cryosurgical model. Cryosurgery was performed in the chamber on either normal skin or tumor tissue propagatedfrom an AT-1 Dunning rat prostate tumor. The freezing was performed by placing a approximately 1 mm diameter liquid-nitrogen-cooled cryoprobe in the center of the chamber and activating it for approximately 1 minute, followed by a passive thaw. This created a 4.2 mm radius iceball. Thermocouples were placed in the tissue around the probe at three locations (r = 2, 3, and 3.8 mm from the center of the window) in order to monitor the thermal history produced in the tissue. The conduction error introduced by the presence of the thermocouples was investigated using an in vitro simulation of the in vivo case and found to be <10 degrees C for all cases. The corrected temperature measurements were used to investigate the validity of two models of freezing behavior within the iceball. The first model used to approximate the freezing and thawing behavior within the DSFC was a two-dimensional transient axisymmetric numerical solution using an enthalpy method and incorporating heating due to blood flow. The second model was a one-dimensional radial steady state analytical solution without blood flow. The models used constant thermal properties for the unfrozen region, and temperature-dependent thermal properties for the frozen region. The two-dimensional transient model presented here is one of the first attempts to model both the freezing and thawing of cryosurgery. The ability of the model to calculate freezing appeared to be superior to the ability to calculate thawing. After demonstrating that the two-dimensional model sufficiently captured the freezing and thawing parameters recorded by the thermocouples, it was used to estimate the thermal history throughout the iceball. This model was used as a basis to compare thermal history to injury assessment (reported in companion paper (Hoffmann and Bischof, 2001)).