Effects of cooling and warming rate to and from -70 degrees C, and effect of further cooling from -70 to -196 degrees C on the motility of mouse spermatozoa

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.     

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

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

Modifying effect of concanavalin A and diamide on erythrocyte sensitivity to cold shock]

The temperature (0 degrees C and 37 degrees C) and the medium tonicity (0.15-1.20 M NaCl) were shown to affect erythrocyte agglutination by concanavalin A. Treatment of cells with lectin caused no significant decrease in the erythrocyte hemolysis upon cooling. Diamide, unlike concanavalin A used at concentrations above 2.0 M decreases the cell sensitivity to the cold shock. The changes in the erythrocyte susceptibility to cooling within the temperature range of 37-0 degrees C correlate with changes in the electrophoretic spectrum of membrane proteins. The progressive decrease in the spectrin bands intensity with a simultaneous formation of high molecular weight protein aggregates not included in the gel composition was observed after diamide treatment. The diamide effect depends on the medium tonicity, at which the treatment was performed, being especially well pronounced in hypertonic media with 0.8-1.2 M NaCl concentrations, the maximal spectrin aggregation being observed under these conditions. It is suggested that the main factor of the mechanism underlying the erythrocyte hypertonic cold shock is the increase in the association of peripheral cytoskeleton proteins with plasma membrane in osmotically dehydrated cells which limits the ability of lipids to adapt during cooling and results in the stabilization of defects in the membrane structure at low temperatures. Diamide eliminates these unfavourable changes eventually resulting in the dissociation of peripheral proteins from the cytoplasmic surface of the membrane on the protein aggregation.     

Effects of linear cooling rate on motion characteristics of stallion spermatozoa

Three experiments were designed to analyze the effects of cooling rate on survival of stallion spermatozoa in a milk-based extender, at 0 to 96 hours after reaching the desired temperature. The samples were warmed to 37 degrees C and were evaluated by computer-assisted analysis of sperm motility. In Experiment 1, rate of cooling between 37 and 20 degrees C was evaluated. Sperm motion was not affected by cooling at plunge, -0.42 or -0.28 degrees C/minute. However, storage of spermatozoa at 5 degrees C after slow cooling below 20 degrees C was superior to storage at 20 degrees C. In Experiment 2, 3 cooling rates from 37 degrees to 5 degrees C were evaluated. Cooling at either -0.05 or -0.7 degrees C/minute was superior (P<0.05) to plunging spermatozoa to 5 degrees C. Cooling at -0.05 degrees C/minute rather than -0.7 degrees C/minute maximized the percentage of motile spermatozoa and their curvilinear velocity. In Experiment 3, cooling rates from 20 to 5 degrees C were evaluated, with all samples cooled at -0.7 degrees C/minute from 37 to 20 degrees C. Sperm motion was similar (P>0.05) after cooling below 20 degrees C at -0.012, -0.05 or -0.10 degrees C/minute, and the 2 slower rates were superior (P<0.05) to cooling at -0.3 degrees C/minute. It was concluded that stallion spermatozoa can be cooled rapidly from 37 to 20 degrees C, but should be cooled at 相似文献   

The effect of cooling rate and warming rate on the packing effect in human erythrocytes frozen and thawed in the presence of 2 M glycerol

The effect of hematocrit (2 versus 75%) has been studied on human red blood cells frozen and thawed in 2 M glycerol at a range of cooling rates (0.8-850 degrees C/min) and warming rates (0.1-200 degrees C/min). The data obtained at a hematocrit of 2% agree well with the data of R. H. Miller and P. Mazur (Cryobiology 13, 404-414, 1976). The results at a hematocrit of 75% show a decrease in recovery with increased cell packing, primarily dependent on warming rate at cooling rates less than 100 degrees C/min and on cooling rate at higher cooling rates. Rapid warming reduced the packing effect, whereas cooling faster than 100 degrees C/min accentuated it. It has been argued that these effects are unlikely to be due to modulation of the generally accepted mechanisms of freezing injury, that is, solution effects and intracellular freezing. It has been suggested that they may be explained by effects of cooling and warming rates on the dimensions of the liquid channels in which the cells are accommodated during freezing and thawing.     

Effect of the cryopreservation conditions on the viability of the rumen ciliate Diploplastron (Metadinium) affine

AIMS: To study the viability of Diploplastron (Metadinium) affine after its cryopreservation at two cooling rates, and the effect of procedure conditions on viability. METHODS AND RESULTS: There were differences in viability between cooling rates (1 and 4 degrees C min(-1)) at 15 or 5 degrees C, but not after thawing. When the equilibrium temperature (25 or 5 degrees C), the cryopreservant (glycerol or dimethyl sulfoxide [DMSO]) and the use of membrane protector were tested, there were no differences caused by the cryopreservant or the membrane protector. However, the equilibrium at 25 degrees C increased the viability (P = 0.005) compared with 5 degrees C. CONCLUSIONS: Viability after thawing was 0.10-0.19. Adding the cryopreservant (either glycerol or DMSO) at 25 degrees C instead of 5 degrees C improves viability of D. affine after thawing. SIGNIFICANCE AND IMPACT OF THE STUDY: Conditions of cryopreservation are largely dependent on the species of rumen protozoa. Number of viable cells after thawing would indicate the possibility of culture recovery for D. affine.     

急性低温/再复温对大鼠心室肌膜电位和钾电流的影响

本文旨在研究急性低温/再复温对大鼠心室肌膜电位和钾电流的影响.膜电位和膜电流分别在全细胞膜片钳的电压钳和电流钳模式下记录.当细胞外灌流液从25℃降低到4℃后,一过性外向电流(transient outward current, Ito)完全消失,膜电位为 60mV时的稳态外向K 电流(sustained outward K current, Iss)和膜电位为-120mV时的内向整流K 电流(inward rectifier K current, IK1)分别降低(48.5±14.1)%和(35.7±18.2)%,同时,膜电位绝对值降低.当细胞外灌流液从4℃再升高到36℃后,膜电位出现一过性超级化,然后恢复到静息电位水平;在58个细胞中,有36个细胞伴随复温出现ATP-敏感性K (ATP-sensitive K , KATP)通道的激活.再复温引起的上述变化可以被Na /K -ATP酶抑制剂哇巴因(100μmol/L)所抑制.再复温引起的KATP通道激活也能被蛋白激酶A抑制剂H-89(100μmol/L)所抑制.在细胞膜电位被钳制在0mV时,当细胞外灌流液温度从25℃降低到4℃后,细胞的体积没有发生明显改变,但当再复温引起KATP通道激活后,细胞很快发生皱缩,同时细胞内部出现许多折光较强的斑点.上述结果表明急性低温/再复温对大鼠心室肌膜电位和K 电流有明显影响,并提示KATP通道激活可能与心肌低温/再复温损伤有关.     

Influence of cooling rate on Saccharomyces cerevisiae destruction during freezing: unexpected viability at ultra-rapid cooling rates

The purpose of this work was to study cell viability as a function of cooling rate during freezing. Cooling rate strongly influences the viability of cells during cold thermal stress. One of the particularities of this study was to investigate a large range of cooling rates and particularly very rapid cooling rates (i.e., faster than 20000 degrees C min (-1)). Four distinct ranges of cooling rates were identified. The first range (A(')) corresponds to very slow cooling rates (less than 5 degrees C min (-1)), and results in high cell mortality. The second range (A) corresponds to low cooling rates (5-100 degrees C min (-1)), at which cell water outflow occurs slowly and does not damage the cells. The third range (B) corresponds to rapid cooling rates (100-2000 degrees C min (-1)), at which there is competition between heat flow and water flow. In this case, massive water outflow, which is related to the increase in extracellular osmotic pressure and the membrane-lipid phase transition, can cause cell death. The fourth range (C) corresponds to very high cooling rates (more than 5000 degrees C min (-1)), at which the heat flow is very rapid and partially prevents water exit, which seems to preserve cell viability.     

Factors affecting the cryosurvival of mouse two-cell embryos

A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Freeze-etching study on membrane ultrastructural changes caused by freezing: cooling rate-dependent intramembrane particle aggregation in erythrocyte stripped ghosts

The changes of membrane ultrastructures by freezing stresses were examined on stripped ghosts which were made by removing almost all peripheral membrane proteins from human erythrocyte membranes. By freezing these stripped ghost membranes showed cooling rate-dependent intramembrane particle (IMP) aggregation. With the cooling rates at and faster than 30,000 degrees C/min, their IMPs were evenly distributed on the fracture faces. However, cooling rates at and slower than 8000 degrees C/min resulted in IMP aggregation. The degree of IMP aggregation increased in parallel with decreasing cooling rates. Without freezing, the IMP aggregation in stripped ghosts could be induced by exposing these ghosts to hypertonic salt solutions, but lowering the temperature did not affect IMP aggregation. The cooling rate-dependent IMP aggregation during freezing was suppressed by adding cryoprotective agents which were known to reduce the salt concentration of the medium during freezing. It is suggested that the IMP aggregation in stripped ghosts by freezing occurs by exposure to concentrated salt solutions during freezing. This result indicates the possibility that IMP aggregation may arise during slow freezing of some biomembranes as a result of an increase in salt concentration rather than as a result of reduction in temperature.     

A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing

The changes in morphology of Penicillium expansum Link and Phytophthora nicotianae Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage. Viability of 0.5-1.0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 degrees C in the presence or absence of glycerol. In P. expansum shrinkage occurred in all hyphae at rates of cooling of less than 15 degrees C min-1; at faster rates intracellular ice nucleation occurred. The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18 degrees C min-1 to 55 degrees C min-1. This species was particularly resistant to freezing injury and recovery was greater than 60% at all rates of cooling examined. At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated. In contrast, during the cooling of Ph. nicotianae in the growth medium, shrinkage occurred and no samples survived on thawing from -196 degrees C. However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 degrees C min-1 the loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.     

Influence of cooling rates and plunging temperatures in an interrupted slow-freezing procedure for semen of the African catfish, Clarias gariepinus

The objective of this study was to optimize interrupted slow-freezing protocols for African catfish semen. Semen diluted with methanol and extender was frozen in 1-ml vials in a programmable freezer. The temperatures of the freezer (T(chamber)) and of the semen (T(semen)) were measured simultaneously. We first tested two-step freezing protocols with different cooling rates (-2, -5, and -10 degrees C/min) and different temperatures at plunging into liquid N2. The difference between T(semen) and T(chamber) increased with faster cooling rates. In all programs, survival of spermatozoa, expressed as hatching rates, increased from near zero when T(semen) at plunging was higher than -30 degrees C to values equal to those of control when T(semen) at plunging was equal to or lower than -38 degrees C. The inclusion of an isothermal holding period before plunging into liquid N2 (three-step freezing protocols) resulted in an equilibration between T(semen) and T(chamber) and improved semen survival. Semen could be plunged at temperatures as high as -36 degrees C when cooled at -5 or -10 degrees C/min, without compromising postthaw semen survival. Cooling at -2 degrees C/min in combination with a 5-min holding period reduced postthaw survival. We conclude that with slow cooling rates of -2 to -5 degrees C/min, hatching rates can be maximized by plunging as soon as T(semen) reaches -38 degrees C. The isothermal holding period is beneficial when faster rates are used. A simple and efficient protocol for freezing African catfish semen can be obtained by cooling at a rate of -5 to -10 degrees C/min combined with a 5-min holding period in the freezer, at -40 degrees C.     

The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing

Dendritic cells (DCs) are now regarded as specialized leucocytes with distinctive morphological and functional characteristics as accessory or stimulator cells for many lymphocyte responses. While knowledge of the response of other leucocytes (e.g., lymphocytes, macrophages, and granulocytes) to freezing and thawing has been established for some years, an understanding of the cryobiological properties of DCs has not, hitherto, been determined specifically. Such information is important both for establishing procedures for the long-term storage of these cells for use in immunological procedures and for defining freezing conditions that might selectively kill DCs in attempts to modulate the immunogenicity of transplantable tissues during cryopreservation. Preparations of rat and human spleen cells enriched for DCs were frozen to -60 degrees C at one of six cooling rates (0.3, 1.5, 10, 20, 70, or 150 degrees C/min) using a procedure that was established for pancreatic islets with 2 M dimethyl sulfoxide (Me2SO) as the cryoprotectant. Following storage at -196 degrees C the survival of thawed cells was assessed by evaluating both the numbers of cells recovered after the complete process and the membrane integrity of the recovered cells using a supravital fluorescent probe assay. Survival profiles for DCs showed a dependence upon cooling rate similar to other lymphoid cells but DCs were more sensitive to freezing injury than either lymphocytes or macrophages: Optimum survival (75% recovery of numbers and 57% membrane integrity) of rat DCs was achieved by slow cooling (0.3 degrees C/min). Optimal recovery of human DCs was significantly higher (83% recovery of numbers and 72% membrane integrity) after cooling at either 0.3 or 1.5 degrees C/min. The viable yield of DCs from both species declined abruptly as cooling rate was increased, with less than 10% survival after cooling at 20 degrees C/min and negligible survival after cooling at 70 degrees C/min or greater. Analysis of variance of the survival data showed that the response of DCs to freezing and thawing was significantly different (P less than 0.005) from that of either lymphocytes or macrophages, thus providing additional evidence that DCs are distinct from other leucocytes, especially macrophages. This study defines conditions that either will provide effective cryopreservation of DCs for immunological purposes or are most likely to bring about their inactivation in cryobiological approaches to modulating tissue immunogenicity.     

Effect of cooling rates on the cold hardiness and cryoprotectant profiles of locust eggs

To examine the relationship between cooling rate and cold hardiness in eggs of the migratory locust, Locusta migratoria, the survival rates and cryoprotectant levels of three embryonic developmental stages were measured at different cooling rates (from 0.05 to 0.8 degrees C min(-1)) in acclimated and non-acclimated eggs. Egg survival rate increased with decreasing cooling rate. The concentration of cryoprotectants (myo-inositol, trehalose, mannitol, glycerol, and sorbitol) increased in non-acclimated eggs, but varied significantly in response to different cooling rates in acclimated eggs. The acclimation process (5 degrees C for 3 days) did not increase eggs resistance to quick cooling ("plunge" cooling and 0.8 degrees C min(-1)). Earlier stage embryos were much more sensitive than later stage embryos to the same cooling rates. Time spent at subzero temperatures also had a strong influence on egg survival.     

Protective effect of intracellular ice during freezing?

Injury results during freezing when cells are exposed to increasing concentrations of solutes or by the formation of intracellular ice. Methods to protect cells from the damaging effects of freezing have focused on the addition of cryoprotective chemicals and the determination of optimal cooling rates. Based on other studies of innocuous intracellular ice formation, this study investigates the potential for this ice to protect cells from injury during subsequent slow cooling. V-79W Chinese hamster fibroblasts and Madin-Darby Canine Kidney (MDCK) cells were cultured as single attached cells or confluent monolayers. The incidence of intracellular ice formation (IIF) in the cultures at the start of cooling was pre-determined using one of two different extracellular ice nucleation temperatures (-5 or -10 degrees C). Samples were then cooled at 1 degrees C/min to the experimental temperature (-5 to -40 degrees C) where samples were warmed rapidly and cell survival assessed using membrane integrity and metabolic activity. For single attached cells, the lower ice nucleation temperature, corresponding to increased incidence of IIF, resulted in decreased post-thaw cell recovery. In contrast, confluent monolayers in which IIF has been shown to be innocuous, show higher survival after cooling to temperatures as low as -40 degrees C, supporting the concept that intracellular ice confers cryoprotection by preventing cell dehydration during subsequent slow cooling.     

Differences among dogs in response of their spermatozoa to cryopreservation using various cooling and warming rates

Spermatozoa collected from the caudae epididymides of 16 dogs of various breeds were suspended in an isotonic salt solution (DIMI medium) containing 0.6 M glycerol, frozen in liquid nitrogen, and their "survival" was measured after thawing. In the first experimental series, duplicate samples of spermatozoa from each of 11 dogs were cooled at rates of 0.5, 3, 11, 58, or 209 degrees C/min, stored in liquid nitrogen, and the frozen samples warmed at approximately 830 or at 33 degrees C/min. Sperm "survival" was judged by microscopic assessments of motility and of membrane integrity, the latter as assayed with Fertilight, a double fluorescent stain. Motility of frozen spermatozoa that were thawed rapidly, averaged for 11 dogs, was low at low rates, increased to a maximum at 11 degrees C/min, and then decreased significantly at higher rates (P<0.01). This inverted V-shaped curve was also observed with slow thawing, although the apparent optimum cooling rate ranged from 3 to 11 degrees C/min. The integrity of sperm plasma membranes showed a similar dependence on cooling rate, although the percentages of spermatozoa with intact plasma membranes were higher than the percentages of motile spermatozoa. Motility of spermatozoa, as a function of cooling rate, varied considerably from male to male (P<0.01), whereas membrane integrity was much more consistent among the 11 dogs. In the second experimental series with spermatozoa from 5 dogs, motility of spermatozoa frozen at 0.5 degrees C/min and warmed at 3.6, 33, 140, or 830 degrees C/min also exhibited an inverted V-shaped survival curve, in this case as a function of warming rate. In summary, high survival of frozen-thawed canine epididymal spermatozoa depended on both cooling and warming rates, but spermatozoa from each dog exhibited their own sensitivity to cooling and warming rates.     

Cryopreservation of vascular endothelial cells as isolated cells and as monolayers

This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads. Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 degrees C. The cells were then cooled to -60 degrees C at controlled rates between 0.3 and 500 degrees C/min and stored below -180 degrees C. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution at 22 degrees C over 6 min. The recovery of cell suspensions was assayed by culturing aliquots in 24-well plates for 7-9 days and counting the number of colonies that contained >25 cells. Maximum survival was 45-50% at cooling rates of 0.3, 1.0, and 10 degrees C/min, but decreased to 20% at 50 degrees C/min and to <1% at 500 degrees C/min. Biosilon microcarrier beads were used for the attached cells. Confluent beads were cryopreserved by exactly the same technique and cell function was assayed by measuring active amino acid (leucine) transport at 37 degrees C. Control, untreated confluent beads gave approximately 73% of control uptake and negative controls (frozen without cryoprotectant) gave approximately 4% uptake. The cells attached to beads showed percentage uptakes that were numerically similar to the survival of cells in suspension at cooling rates between 10 and 500 degrees C/min, but at lower cooling rates the recovery of attached cells increased to 70% at 1 degrees C/min and to 85% at 0.3 degrees C/min. These results indicate a marked difference in the effect of cooling rate on ECV304 cells depending upon attachment.     

Quantitative cryomicroscopic analysis of intracellular freezing of granulocytes without cryoadditive

Purified human granulocytes were frozen in isotonic saline at different constant cooling rates down to -60 degrees C and subsequently thawed on the thermally defined cryostage of a cryomicroscope. Cells monitored on videotape were examined with respect to cooling rate threshold, type, and temperature of intracellular ice formation during cooling and recrystallization during warming. Two apparently different mechanisms of intracellular ice formation (iif) were distinguished during cooling, i.e., "twitching" (no visible ice front) and "darkening" (diffuse ice front). Both types of iif are related to cooling rate and hence also to dehydration. Cooling rate thresholds and temperatures of intracellular recrystallization were determined. It was found that twitching iif occurs just about 6.3 to 7.4 degrees C above the homogeneous nucleation temperature, suggesting that it might be catalyzed by nucleators present within the cells. Darkening iif, on the other hand, was observed at much higher temperatures, i.e., 23.4 to 28.3 degrees C above the homogeneous nucleation temperature, which could possibly indicate a nucleation induced by extracellular ice crystals (at a cooling rate of 30 degrees K/min, however, darkening iif was observed to occur at a temperature lower than that required for twitching iif). The proposed mechanisms of cryoinjury are related to membrane integrity measurements presented in M. W. Scheiwe, Ch. K?rber, and S. Englich, Cryo-Letters, 5, 300-306, 1984.     

Cryoinjury in endothelial cell monolayers

Developing successful cryopreservation strategies for corneas have proven to be more difficult than anticipated, because of the resulting loss of viability and detachment of endothelial cells from Descemet's membrane following cryopreservation of corneas. The objectives of this study are to develop a more detailed understanding of cryoinjury in human corneal endothelial cell (HCEC) monolayers and to examine the effects of storage temperature, cryoprotectant type and concentration, and cooling/warming rates on HCEC monolayers. Monolayers of endothelial cells attached to collagen-coated glass, immersed in an experimental solution (with and without cryoprotectant) were cooled at 1 degrees C/min to various temperatures (-5 to -40 degrees C), then thawed directly or cooled rapidly to -196 or to -80 degrees C before thawing. Cryoprotectants used were dimethyl sulfoxide and propylene glycol in concentrations of 1 and 2M. Monolayers were assessed for membrane integrity and detachment using SYTO/ethidium bromide fluorescent stain. The presence of cryoprotectants resulted in high recovery of membrane integrity and low monolayer detachment in monolayers thawed directly from temperatures down to -40 degrees C. In contrast, there was excessive detachment and loss of membrane integrity in monolayers cooled to -196 degrees C compared to monolayers cooled to -80 degrees C. Also, increasing cryoprotectant concentrations did not improve recovery of the monolayers. The higher recovery and lower detachment after storage at -80 degrees C compared to storage at -196 degrees C suggest that storage temperatures for corneas should be re-evaluated.