Comparison of iceball diameter and temperature distribution achieved with 3-mm accuprobe cryoprobes in porcine and human liver tissue and human colorectal liver metastases in vitro

We aimed to assess the thermal profile and size of iceballs produced by Accuprobe cryoprobes in fresh porcine and human liver and human colorectal cancer liver metastases in vitro to allow better planning of cryosurgical treatment of liver metastases. Iceballs were produced by a 20-min single freeze cycle using 8-mm cryoprobes in pig liver in a waterbath at 37 degrees C (n = 8) and 3-mm cryoprobes in pig liver (n = 8), human liver (n = 3), and human colorectal cancer liver metastases (n = 8). The iceball diameters and the temperatures at different distances from the cryoprobe were measured. Mean iceball diameters produced by 8-mm cryoprobes in pig liver were 56.3 mm and varied from 38.7 to 39.6 mm for 3-mm cryoprobes in the different tissues used. There was no significant difference in iceball size in the different tissues. The diameter of the zone of -40 degrees C or less was approximately 44 mm using 8-mm cryoprobes in porcine liver and between 27 and 31 mm using 3-mm cryoprobes in the different tissues examined. The results may allow better preoperative planning of the cryosurgical treatment of liver metastases with Accuprobe cryoprobes.     

Imaging of interstitial cryotherapy--an in vitro comparison of ultrasound, computed tomography, and magnetic resonance imaging.

RATIONALE AND OBJECTIVES: To evaluate the imaging capabilities of ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI) in monitoring interstitial cryotherapy and to compare them with visual control. METHODS: An experimental MR-compatible, vacuum-insulated and liquid nitrogen-cooled cryoprobe was inserted under in vitro conditions into a porcine liver, which was kept at a temperature of 37 +/- 1 degrees C, in a water bath with continuous stirring. The freezing procedure was controlled macroscopically, by US (Toshiba Sonolayer, 7.5-MHz linear array transducer), by CT (Siemens Somatom Plus, slice thickness 2-8 mm, 165-210 mA at 120 kV), and by MRI (Philips Gyroscan ACS-NT, FFE TR/TE/FA = 15/5.4/25 degrees, T1-SE 550/20, T2-TSE 1800/100) after the iceball reached its maximum size. RESULTS: The maximum iceball diameter around the probe tip was 12.0 mm by visual control, 12.4 mm by US, 12.7 mm by CT, and within 12.8 mm by spin echo sequences and 11 mm by gradient echo sequence. Due to the nearly signal-free appearance of the frozen tissue on MR images, the ice/tissue contrast on T1-weighted and gradient echo images was superior to T2-weighted images and CT images. Sonographically, the ice formation appeared as a hyperechoic sickle with nearly complete acoustic shadowing. CONCLUSION: Due to the better ice/tissue contrast, T1-weighted or gradient echo MR images were superior to CT and US in monitoring interstitial cryotherapy. Gradient echo sequences generally underestimated the ice diameter by 15%.     

Controlled-rate freezing of human ES cells

A significant obstacle to using human embryonic stem cells (hESCs) arises from extremely poor survival associated with freezing, typically in the range of 1%. This report describes a slow controlled-rate freezing technique commonly used for mammalian embryo cryopreservation. Using a combination of surviving colony number and colony diameter; survival was determined relative to untreated hESCs. Using a dimethyl sulfoxide (DMSO) cryoprotectant and either a homemade controlled-rate freezing device or a commercial freezing device, survival rates of 20%-80% were obtained. To achieve the highest levels of survival, the critical factors were an ice crystal seed (at -7 degrees to -10 degrees C), a freeze rate between 0.3 degrees and 1.8 degrees C/min, and a rapid thaw rate using room temperature water. Slow controlled-rate cooling allows a rapid, simple, and reproducible means of cryopreserving hESCs.     

Experimental measurements and theoretical calculations on muscle and liver tissue during cryosurgery. I. Experiments on freezing of rabbit tissue.

Experiments on freezing of muscle and liver tissue of 113 rabbits were performed. The diameter on the frozen surface, and the thickness and the mass of the iceball were measured for the live and dead, body-temperature, animal. Four continuously cooled and six massive probes (2.5–20-mm diameter) were used with liquid nitrogen as the cooling agent. The following conclusions can be drawn: (1) with use of round probe-tips, the iceball has approximately spherical symmetry. However, the depth of the frozen tissue is about 15% smaller than the lateral extension on the visible surface. (2) For continuously cooled probes the diameter of the iceball in the steady state is about five times as large as the probe diameter. The maximal iceball diameter for massive probes is two times larger than the probe diameter. (3) The different blood circulation of liver and muscle tissue has an influence of only 10% on the size of the iceball. For clinical applications this difference is of little importance. (4) For live tissue the iceball is about 15% smaller than for body-temperature dead tissue. Thus, the main heat-transport process in tissue is heat condition.     

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)).     

Computerized tomographic imaging of cryosurgical iceball formation in brain

Computerized tomography (CT) was used to monitor the exact anatomical location and dimensions of the cryosurgical iceball within the brain. The gross and microscopic appearance of the tissue iceball was examined in both acute and chronic animals. Iceball formation was monitored in the brain of four dogs under a general anesthesia. The radiographic image of the iceball was that of a well-demarcated radiolucent sphere that disappeared upon thawing. The post-thaw contrast-enhanced CT scan revealed a zone of blood-brain barrier breakdown extending no more than 1 mm beyond the maximum diameter that the iceball had attained. Histological examination demonstrated a sharp transition from frankly necrotic brain within the iceball to the normal cytoarchitecture of the surrounding neuropil. The safety and efficacy of a neurosurgical ablative procedure depends on the precision with which it can be generated. The use of CT imaging to monitor the formation of the cryosurgical iceball offers the neurosurgeon a means to precisely control the size of the ablative lesion. Small deeply situated brain tumors can be incorporated into the iceball under direct CT observation, thereby ensuring the completeness of the cryoablation while minimizing damage to the surrounding brain.     

Mechanisms of inactivation of HSV-2 during storage in frozen and lyophilized forms

The structural integrity of herpes simplex virus 2 (HSV-2) during freezing, thawing, and lyophilization has been studied using scanning and transmission electron microscopy. Viral particles should be thawed quickly from -80 to 37 degrees C to avoid artifacts of thawing. To avoid freezing damage, the virus should be rapidly frozen (>20 K s(-1)) rather than slowly frozen as occurs on the shelf of a lyophilizer (<1 K s(-1)). Fast freezing and thawing allows six cycles of freeze thaw with no loss of viral titer TCID50. Viral particles were characterized using immunogold labeling methods. Freshly thawed virus had 19 +/- 4 polyclonal immunogold particles virus(-1); virus stored at -80 degrees C for at least 4 months had 17 +/- 3 particles virus(-1); virus stored for 1 week at 4 degrees C had 8 +/- 4 particles virus(-1). By bulk lyophilization the number of particles was 4 +/- 4, but by fast freezing and lyophilization the number of gold particles improved to 12 +/- 5. The loss of viral membrane was directly observed, and the in vitro loss was demonstrated to occur through three possible pathways, including (i) simultaneous release of tegument and membrane, (ii) sequential release of membrane and then tegument, and (iii) release like by in vivo infection. The capsids were not further degraded as indicated by the lack of free DNA, which was only released by boiling the viral samples with 1% SDS, followed by a dilution to 0.001% w/v SDS for the real-time PCR reaction.     

Experimental frostbite: freezing times, rewarming times, and lowest temperatures of pig skin exposed to chilled air

Frostbite was produced in the skin of five Hanford Miniature Swine by exposing local areas to chilled air (-75 degrees C) for 1, 3, 5, 10, or 20 min. A copper-constantan thermocouple was inserted into the dermis to measure the temperature. The mean freezing time (the time required to reach 0 degrees C) was approximately 1.9 min. The mean lowest temperatures were 8.8, -15.7, -20.9, -22.5, and -23.4 degrees C for the 1-, 3-, 5-, 10-, and 20-min freezes, respectively. The mean times to rewarm the skin to 25 degrees C were 3.1, 4.5, 5.5, 7.0, and 8.6 min for the 1-, 3-, 5-, 10-, and 20-min freezes, respectively. Significant linear correlations existed between duration of freeze and rewarming times, duration of freeze and lowest temperature, and lowest temperature and rewarming times.     

The relationship between xylem conduit diameter and cavitation caused by freezing

The centrifuge method for measuring the resistance of xylem to cavitation by water stress was modified to also account for any additional cavitation that might occur from a freeze-thaw cycle. A strong correlation was found between cavitation by freezing and mean conduit diameter. On the one extreme, a tracheid-bearing conifer and diffuse-porous angiosperms with small-diameter vessels (mean diameter <30 μm) showed no freezing-induced cavitation under modest water stress (xylem pressure = −0.5 MPa), whereas species with larger diameter vessels (mean >40 μm) were nearly completely cavitated under the same conditions. Species with intermediate mean diameters (30–40 μm) showed partial cavitation by freezing. These results are consistent with a critical diameter of 44 μm at or above which cavitation would occur by a freeze–thaw cycle at −0.5 MPa. As expected, vulnerability to cavitation by freezing was correlated with the hydraulic conductivity per stem transverse area. The results confirm and extend previous reports that small-diameter conduits are relatively resistant to cavitation by freezing. It appears that the centrifuge method, modified to include freeze–thaw cycles, may be useful in separating the interactive effects of xylem pressure and freezing on cavitation.     

Subzero water permeability parameters and optimal freezing rates for sperm cells of the southern platyfish, Xiphophorus maculatus

This study reports the subzero water transport characteristics (and empirically determined optimal rates for freezing) of sperm cells of live-bearing fishes of the genus Xiphophorus, specifically those of the southern platyfish Xiphophorus maculatus. These fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. Water transport during freezing of X. maculatus sperm cell suspensions was obtained using a shape-independent differential scanning calorimeter technique in the presence of extracellular ice at a cooling rate of 20 degrees C/min in three different media: (1) Hanks' balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol, and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder with a length of 52.35 microm and a diameter of 0.66 microm with an osmotically inactive cell volume (Vb) of 0.6 V0, where V0 is the isotonic or initial cell volume. This translates to a surface area, SA to initial water volume, WV ratio of 15.15 microm(-1). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best fit membrane permeability parameters (reference membrane permeability to water at 0 degrees C, Lpg or Lpg [cpa] and the activation energy, E(Lp) or E(Lp) [cpa]) were found to range from: Lpg or Lpg [cpa] = 0.0053-0.0093 microm/minatm; E(Lp) or E(Lp) [cpa] = 9.79-29.00 kcal/mol. By incorporating these membrane permeability parameters in a recently developed generic optimal cooling rate equation (optimal cooling rate, [Formula: see text] where the units of B(opt) are degrees C/min, E(Lp) or E(Lp) [cpa] are kcal/mol, L(pg) or L(pg) [cpa] are microm/minatm and SA/WV are microm(-1)), we determined the optimal rates of freezing X. maculatus sperm cells to be 28 degrees C/min (in HBSS), 47 degrees C/min (in HBSS+14% glycerol) and 36 degrees C/min (in HBSS+10% DMSO). Preliminary empirical experiments suggest that the optimal rate of freezing X. maculatus sperm in the presence of 14% glycerol to be approximately 25 degrees C/min. Possible reasons for the observed discrepancy between the theoretically predicted and experimentally determined optimal rates of freezing X. maculatus sperm cells are discussed.     

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

Comparison of dual- and triple-freeze protocols for pulmonary cryoablation in a Tibet pig model

The purpose of this study was to compare a dual-freeze protocol with a triple-freeze protocol for pulmonary cryoablation in a porcine lung model. Five dual- (10-5-10-5) and five triple-freeze (5-5-5-5-10-5) cryoablations were performed on an exposed operation field in normal porcine lung. Changes in the temperature of the cryoprobes and the diameter of the iceballs were measured during the ablation and pathologic changes in the cryozones (zones of tissue destruction) were reviewed 7 days after the procedure. The diameter of the iceball surface differed between the two protocols. Pathologically, the triple-freeze protocol was associated with a longer complete necrosis zone than the dual-freeze protocol, though the two protocols produced cryolesions and cryozones of similar length, and in both cases there were five areas of tissue destruction. With the same duration of freezing (20 min), the triple-freeze protocol may be better for pulmonary cryoablation than the dual-freeze protocol.     

Intracellular ice formation in yeast cells vs. cooling rate: Predictions from modeling vs. experimental observations by differential scanning calorimetry

To survive freezing, cells must not undergo internal ice formation during cooling. One vital factor is the cooling rate. The faster cells are cooled, the more their contents supercool, and at some subzero temperature that supercooled cytoplasm will freeze. The question is at what temperature? The relation between cooling rate and cell supercooling can be computed. Two important parameters are the water permeability (Lp) and its temperature dependence. To avoid intracellular ice formation (IIF), the supercooling must be eliminated by dehydration before the cell cools to its ice nucleation temperature. With an observed nucleation temperature of −25 °C, the modeling predicts that IIF should not occur in yeast cooled at <20 °C/min and it should occur with near certainty in cells cooled at ?30 °C/min. Experiments with differential scanning calorimetry (DSC) confirmed these predictions closely. The premise with the DSC is that if there is no IIF, one should see only a single exotherm representing the freezing of the external water. If IIF occurs, one should see a second, lower temperature exotherm. A further test of whether this second exotherm is IIF is whether it disappears on repeated freezing. IIF disrupts the plasma membrane; consequently, in a subsequent freeze cycle, the cell can no longer supercool and will not exhibit a second exotherm. This proved to be the case at cooling rates >20 °C/min.     

Assessment of in situ cellular glutathione labeling with naphthalene-2,3-dicarboxaldehyde using high-performance liquid chromatography

We have presently studied a dialdehydic reagent, i.e. naphthalene-2,3-dicarboxaldehyde (NDA), as a fluorogenic probe for the labeling of intracellular reduced glutathione (GSH), using a yeast strain Candida albicans as a cell model. Chemical reactivity of NDA with both amino and sulfhydryl groups of the GSH molecule leads to a highly selective detection. Moreover, fluorescence properties of the resulting adduct fit well with most of modern instruments adapted for in situ measurements, and equipped with an argon laser. After incubation of cells with 100 microM of NDA for 20 min, cells were harvested and corresponding lysates obtained after a freezing cycle, were suspended in 0.2M borate buffer pH 9.2 and analysed with HPLC (column: Spherisorb ODS-2 (125 mm x 4.6 mm i.d.) 5 microm; mobile phase: methanol-0.01 M phosphate buffer pH 6.5 (20:80, v/v) at a flow rate of 0.8 mL min(-1); spectrofluorimetric detection: lambda(exc)=430 nm and lambda(em)=530 nm). The GSH-NDA adduct was identified in the yeast strain extracts using the reported HPLC technique and quantified versus a calibration curve of NDA derivatized with an excess of GSH (linearity range: 9-230 nM). The cell loading step of the free probe NDA and the extraction efficiency of the resulting NDA-GSH adduct were optimized.     

Improvement of the cryopreservation of the fungal starter Geotrichum candidum by artificial nucleation and temperature downshift control

Food industry tends towards the use of controlled microorganisms in order to improve its technologies including frozen starter production. The fungus Geotrichum candidum, which is currently found in various environments, is widely used as ripening agent in some specific cheese making process. In order to optimize the cryopreservation of this microorganism, freezing experiments were carried out using a Peltier cooler-heater incubator, which permits to control the temperature downshift from +20 to -10 degrees C in time period ranges from 20 to 40min depending on the experiments. Concomitantly, study of the effect of an industrial ice nucleator protein derived from Pseudomonas syringae (SNOMAX) on the dynamic of freezing of G. candidum was carried out. Our results showed that the addition of this protein in the microbiological suspension has different complementary effects: (i) the synchronization of the different samples nucleation, leading to an homogeneous and earlier freezing, (ii) the increase of the freezing point temperature from -8.6 to -2.6 degrees C, (iii) a significant decrease of the lethality of G. candidum cells subjected to a freezing-thawing cycles challenge.     

The effects of starvation, environmental temperature and injury on the rate of disposal of glucose by the rat

1. The disposal rate of glucose, R, given by R=k(v)Q, where Q is the quantity of plasma glucose and k(v) is a rate coefficient, was determined from the disappearance of [U-(14)C]-glucose from blood after single intravenous injection. Values of R should be close to the carbohydrate oxidation rate in the states investigated. 2. Normal rats (i) experimental methodology was studied. The best (single sampling) method gave the following results. (ii) The plasma glucose concentration (C(p)) and R were temporarily increased by the stress of handling and injection. (iii) R was increased by decreasing the environmental temperature from 29 degrees C to 20 degrees C in line with previously published (Stoner & Marshall, 1971) changes in total body O(2) consumption. (iv) Starvation decreased R such that R=constantxC(p) (2). (v) The results suggested some central control of cell permeability to glucose. 3. Injured post-absorptive rats were studied in the ebb phase after three severe injuries: scalding at 20 degrees and 29 degrees C (non-lethal) and bilateral hind-limb ischaemia at 20 degrees C (85% mortality). (i) Handling and injection did not affect C(p). (ii) The rise in C(p) after injury was not closely correlated with its severity. (iii) The value of R was nearly independent of severity. (iv) Unlike in normal rats R varied little with ambient temperature (in line with O(2) consumption) or with C(p). Values of k(v) varied inversely as C(p). (v) The results were explained in terms of a centrally integrated response to injury involving the hypothalamus which over-rode the controls operating in normal rats. Hormonal factors are discussed.     

Freeze tolerance in Aporrectodea caliginosa and other earthworms from Finland

Earthworms that live in subarctic and cold temperate areas must deal with frost even though winter temperatures in the soil are often more moderate than air temperatures. Most lumbricid earthworms can survive temperatures down to the melting point of their body fluids but only few species are freeze tolerant, i.e. tolerate internal ice formation. In the present study, earthworms from Finland were tested for freeze tolerance, and the glycogen reserves and glucose mobilization (as a cryoprotectant) was investigated. Freeze tolerance was observed in Aporrectodea caliginosa, Dendrobaena octaedra, and Dendrodrilus rubidus, but not in Lumbricus rubellus. A. caliginosa tolerated freezing at -5 degrees C with about 40% survival. Some individuals of D. octaedra tolerated freezing even at -20 degrees C. Glycogen storage was largest in D. octaedra where up to 13% of dry weight consisted of this carbohydrate, whereas the other species had only 3-4% glycogen of tissue dry weight. Also glucose accumulation was largest in D. octaedra which was the most freeze-tolerant species, but occurred in all four species upon freezing. It is discussed that freeze tolerance may be a more common phenomenon in earthworms than previously thought.     

Cold hardiness abilities vary with the size of the land snail Cornu aspersum

The land snail Cornu aspersum (syn. Helix aspersa) living in Brittany (France) can be considered partially freezing tolerant as it possesses a low ability to supercool and a limited capacity to bear freezing of its body tissues. The absence of a marked cold hardiness strategy permits the emphasis of the role of parameters such as individual size or water mass (W(M)) contained by the organism. Adult snails (shell diameter 30-32 mm) had a supercooling ability, about 1-1.5 degrees C lower than that of immatures (shell diameter 12-20 mm) and survived longer to an exposure to -5 degrees C, with an Lt(50) comprised between 6.0 and 9.8 h against 2.6 to 4.2 h for immature snails. This better ability to bear freezing was explained by the faster dynamic of body ice formation observed in small individuals, which attained ice lethal quantity more rapidly. At the species level, large snails will then tend to be more tolerant to freezing and small ones to be freezing avoidant, a statement also observable at the phylum level.     

Effects of freezing rates and dimethyl sulfoxide concentrations on thermal expansion of rabbit aorta during freezing phase change as measured by thermo mechanical analysis

Thermal expansion data are essential for thermal stress analysis in order to predict the likelihood of fracture formation in tissues during cryopreservation as well as cryosurgery. The current study focuses on examining the thermal expansion behavior of rabbit aorta during freezing, especially phase change processes. Thermo mechanical analysis (TMA) was used to investigate the effects of different concentrations of dimethyl sulfoxide (DMSO) cryoprotective agent (CPA) (0%, 5%, 10%, and 15% (v/v) DMSO) and different freezing rates (3, 5 and 10 degrees C/min). The results showed that (1) the maximum of thermal strain for 10 degrees C/min was approximately four times greater than that for 3 degrees C/min, and 1.4 times greater than that for 5 degrees C/min, and the higher the freezing rate, the larger the corresponding thermal expansion coefficient; (2) the maximum thermal strain of sample permeated by 5% DMSO approached that of 0% DMSO (i.e., no DMSO was added); however, it showed very significant difference from that of 15% DMSO (only half of that with 5% DMSO), and the thermal expansion coefficient decreased when the concentration of DMSO solution increased; (3) comparison with data available from the literature and with theoretically calculated values illustrated that the thermal expansion change was not equal to the volume change from free water to ice during freezing, but was related to the freezing rate of samples and the DMSO concentration.     

Cryopreservation of cornea: a low cooling rate improves functional survival of endothelium after freezing and thawing

AIM: To investigate the influence of low cooling rates on endothelial function and morphology of corneas frozen with propane-1,2-diol (PROH). METHODS: Rabbit corneas, mounted on support rings, were exposed to 1.4mol/l (10% v/v) PROH, seeded to initiate freezing, and cooled at 0.2 or 1 degrees C/min to -80 degrees C. Corneas were frozen immersed in liquid or suspended in air. After being held overnight in liquid nitrogen, corneas were warmed at 1 or 20 degrees C/min. After stepwise removal of the cryoprotectant, the ability of the endothelium actively to control corneal hydration was monitored during normothermic perfusion. Morphology was assessed after staining with trypan blue and alizarin red S, and by specular microscopy during perfusion. RESULTS: Functional survival was achieved only after slow cooling (0.2 degrees C/min) with the cornea immersed in the cryoprotectant medium, and rapid warming (20 degrees C/min). These conditions also gave the best morphology after freezing and thawing. CONCLUSION: Cooling rates lower than those typically applied to cornea improved functional survival of the endothelium. This result is in accord with previous observations showing the benefit of low cooling rates for cell monolayers [CryoLetters 17 (1996) 213-218].