Slow Thawing Research Articles

Modelling and validation of robust partial thawing of frozen convenience foods during distribution in the cold chain

In collaboration with two commercial distributors we have tested a new concept for distribution, where convenience products for the food service industry are prepared, frozen and packed in cardboard boxes, but distributed in the chill chain at +5°C instead of in the frost chain. This will lead to a partial thawing before reaching the end user. The high thermal buffering capacity of the product under thawing ensures that the product temperature remains stable at subzero temperatures, also in cases of temperature abuse for some hours. To investigate and validate the concept, cardboard boxes packed with small blocks of a frozen model food (23 pct. Tylose® gel) and quipped with temperature loggers were distributed by trucks operating in the cold chain. In addition, controlled storage and temperature abuse experiments were conducted. To predict the product temperature–time relationship we developed a new thawing model for the slow thawing of food pieces packed in a box. The model is based on enthalpy balances under quasi-stationary conditions; a condition that is fulfilled in the practical distribution experiments. The experiments confirmed the model predictions that the products were still partially frozen even after two days or more of distribution at +5oC, and that the temperatures inside the product and in the middle of the box were quite stable against the normal oscillations of the ambient temperature in the cold chain. The product temperature was also robust against temperature abuse, as predicted. The new distribution method not only has the potential of prolonging product shelf life but also gives the distributors an increased flexibility in the distribution chain.

Histologic and ultrastructural features of cryopreserved ovine ovarian tissue: deleterious effect of 1,2-propanediol applying different thawing protocols

Ovarian cortical fragments were frozen in presence of 1.5 M PROH, thawed at different temperatures (37 degrees, 30 degrees, or 4 degrees C) and submitted to histologic and ultrastructural analysis. Thawing at 30 degrees C minimized stromal and follicular damage when compared with 4 degrees and 37 degrees C.

Cod and rainbow trout as freeze-chilled meal elements

'Meal elements' are elements of a meal, e.g. portions of pre-fried meat, sauces, frozen fish or pre-processed vegetables typically prepared industrially. The meal elements are distributed to professional satellite kitchens, where the staff can combine them into complete meals. Freeze-chilling is a process consisting of freezing and frozen storage followed by thawing and chilled storage. Combining the two would enable the manufacturer to produce large quantities of frozen meal elements to be released into the chill chain according to demand. We have studied the influence of freeze-chilling on the quality attributes of cod and rainbow trout portions. Sensory profiling and chemical analyses were used to determine the changes in quality after slow thawing and subsequent chill storage and to find the high-quality shelf life. Cod had a consistent and high sensory quality during the first 6 days of chilled storage, and the corresponding time for rainbow trout was 10 days. After this period the sensory quality decreased and chemical indicators of spoilage were seen to increase. The consistent quality during storage and the high-quality shelf life are practically applicable and cod and rainbow trout seem potential candidates for freeze-chilled meal elements.

Interactions among pre-cooling, cryoprotectant, cooling, and thawing for sperm cryopreservation in rhesus monkeys

The present study evaluated the interactions among pre-cooling, cryoprotectant, cooling, and thawing for rhesus monkey sperm using a four-way factorial design. Specifically, pre-cooling and thawing were evaluated for two conditions: slow vs. fast. Cooling was evaluated at four rates of 5, 29, 200, and 400°C/min. The types of cryoprotectant involved combinations of egg yolk and glycerol, egg yolk and ethylene glycol, and egg yolk alone without permeable cryoprotectants or buffer alone with glycerol but without egg yolk. Our findings showed strong interactions among cryoprotectants, cooling, and thawing rates, but not pre-cooling rate, on post-thaw motility and forward progression. The optimal combination of cooling and thawing for maximum post-thaw survival depended on the types of cryoprotectant. When glycerol was used as a permeable cryoprotectant in the presence of egg yolk, slow thawing yielded similar success as fast thawing in some males. However, when glycerol was replaced with ethylene glycol for the same treatment, post-thaw motility was significantly lower in samples that were thawed slowly than those that were thawed rapidly. In the absence of permeable cryoprotectant but the presence of egg yolk, fast cooling was always favorable. On the contrary, in the absence of egg yolk but the presence of permeable cryoprotectant (glycerol), post-thaw motility was significantly reduced especially when samples were thawed slowly. Generally, fast thawing was superior to slow thawing regardless of the types of cryoprotectant or cooling rates, and glycerol in the presence of egg yolk yielded the highest post-thaw motility in all treatment groups.

Effect of cooling, freezing and thawing rates and storage conditions on preservation of human spermatozoa.

Human spermatozoa was relatively resistant to cooling shock. However, when diluted semen was cooled faster than 10 degrees C per minute from room temperature (RT) to 5 degrees C and rewarmed to RT, percentage motility and percentage alive of spermatozoa decreased when compared to the slower cooling rates (less than 5 degrees C/min). The optimum cooling rate from RT to 5 degrees C resulting in maximum survival of human spermatozoa was found to be 0.5 to 1 degree per minute when cooled from RT to 5 degrees C and subsequently frozen-thawed in liquid nitrogen (LN2). The optimal freezing rate of 10 degrees C per minute, from 5 degrees to -80 degrees C, resulted in higher survival of human spermatozoa than slower (1.1 degrees C/min) or faster (87.1 degrees C/min) freezing rates. Slow thawing in 20 or 35 degrees C air, on a dry bench, resulted in better survival than the other slower or faster thawing methods used. The temperature at which human semen samples were transferred to LN2 significantly influenced spermatozoa survival. Survival was higher when transferred at -30 degrees C or lower when compared with samples transferred at -15 degrees C or higher. However, maximal spermatozoa survival was obtained when the samples were transferred at -80 degrees C or lower. Transfer of human semen from LN2 to -25 to -30 degrees C and storage for 24 hours significantly reduced spermatozoa viability when compared with storage at 196 degrees C or -80 to -85 degrees C. No significant differences were found between storage temperatures of -80 to -85 degrees C and -196 degrees C in the maintenance of spermatozoa viability for up to 90 days.

Investigation of Freezing- and Thawing-Induced Biological, Chemical, and Physical Changes to Enoxaparin Solution

This study investigated the effect of freezing and thawing on the biological, physical, and chemical properties of enoxaparin solution. Solutions were frozen and thawed under different conditions, in the presence or absence of dimethyl sulfoxide (DMSO) or 1,2-propanediol (1,2-PD), and the antifactor Xa (AFXa) activity was determined. Enoxaparin solution lost more than 60% of its AFXa activity when thawed rapidly after freezing at −196°C. The loss of AFXa activity was less with higher freezing temperatures and increased with the number of freeze/thaw cycles, but was independent of the duration of freezing. Slow freezing to −196°C with rapid thawing, or rapid freezing with slow thawing, resulted in negligible loss of AFXa activity. The loss of AFXa activity did not involve the loss of N-sulfate groups, the breakdown of glycosidic bonds or the glassy state transition. Controlling the freezing or thawing conditions, dilution with water or addition of a small percentage of DMSO ameliorated the loss of enoxaparin AFXa activity. The loss in AFXa activity was found by size exclusion chromatography to be primarily due to aggregation and was reversed by sonication in the presence of DMSO. These results may provide insight into solutions for the long-term storage of concentrated or diluted enoxaparin.

The Effect of Cryopreservation on the Mouse Embryos at Various-pronuclear Stages

This study was carried out to establish an appropriate condition for the efficient cryopreservation of the mouse pronuclear embryo. in vitro cryopreservation ot pronuclear embryos was carried out by slow Ireezing or vitrification methods and development rate of 2-cell, blastocyst and hatched blastocyst was measured as well as survival rate of the thawed pronuclear embryo. After slow freezing, vitrification and thawing of mouse pronuclear embryos, the survival rate and blastocyst development rate for the vitrification group was 97.3 and 53.4%, respectively, which was significantly higher as compared to the slow freezing group with 88.6 and 23.9%, respectively (p<0.05). Blastocyst developmental rate in each experimental group was significantly higher for 21 h in the post-hCG group at 40.5-57.0% than the 24 h post-hCG group at 40.5% (p<0.05). ICM (Inner cell mass) cell numbers of blastocyst-stage embryos during the different stages of mouse pronuclear embryos, slow freezing and vitrification period in the control and vitrification groups were 22.1±2.7 and 17.0±3.1∼22.0±3.2, respectively; hence, the slow freezing group (10.2±2.0) had significantly higher cell numbers than those of the other two groups (p<0.05). Trophoblast (TE) cell number in the control group, 65.8±12.6, was significantly higher than in the slow freezing group, 41.6±11.1 (p<0.05). The total cell numbers in the control group and 21 h post hCG group were 87.9±13.6 and 81.8+14.1, respectively, and were significantly higher than for the slow freezing group (51.8±12.6; p<0.05).

Rhesus monkey sperm cryopreservation with TEST-yolk extender in the absence of permeable cryoprotectant

Recently, there has been increased interest in ultra-rapid freezing with mammalian spermatozoa, especially for vitrification in the absence of cryoprotectants. Sperm cryopreservation in non-human primates has been successful, but the use of frozen–thawed sperm in standard artificial insemination (AI) remains difficult, and removal of permeable cryoprotectant may offer opportunities for increased AI success. The present study intended to explore the possibility of freezing rhesus monkey sperm in the absence of permeable cryoprotectants. Specifically, we evaluated various factors such as presence or absence of egg yolk, the percentage of egg yolk in the extenders, and the effect of cooling and thawing rate on the success of freezing without permeable cryoprotectants. Findings revealed that freezing with TEST in the absence of egg yolk offers little protection (<15% post-thaw motility). Egg yolk of 40% or more in TEST resulted in decreased motility, while egg yolk in the range of 20–30% yielded the most motile sperm. Cooling at a slow rate (29°C/min) reduced post-thaw motility significantly for samples frozen with TEST-yolk alone, but had no effect for controls in the presence of glycerol. Similarly, slow thawing in room temperature air is detrimental for freezing without permeable cryoprotectant (<2% motility). In addition to motility, the ability of sperm to capacitate based on an increase in intracellular calcium levels upon activation with cAMP and caffeine suggested no difference between fresh and frozen–thawed motile sperm, regardless of treatment. In summary, the present study demonstrates that ejaculated and epididymal sperm from rhesus monkeys can be cryopreserved with TEST-yolk (20%) in the absence of permeable cryoprotectant when samples were loaded in a standard 0.25-mL straw, cooled rapidly in liquid nitrogen vapor at 220°C/min, and thawed rapidly in a 37°C water bath. This study also represents the first success of freezing without permeable cryoprotectant in non-human primates.

Effect of the Developmental Stage and Thawing Temperature on the Survival and Development of the Vitrified Embryos

The best developmental stage and the best thawing protocol suitable for cryopreservation of the early embryos are not well documented. The present study aimed at evaluating the effect of the ultra rapid cryopreservation (vitrification) technique, followed by slow or fast thawing protocol, on the fertilized ova, 4-cell embryos and morula. The vitrification method included equilibrating the ova in the vitrification solution (EFS40; consisted of 40% ethylene glycol, 30% Ficoll, 0.5 M sucrose in D-PBS) for 2 minutes before immersion in liquid nitrogen. Slow and fast thawing were done and the cryoprotectants were withdrawn by a hyperosmolar sucrose solution, which was then gradually diluted and replaced by culture medium. The best results were obtained with vitrification of the 4-cell embryos both with slow and fast thawing, which gave survival rate of 86% and 94%, and in vitro development rate of 74% and 80%, respectively. Fast thawing showed better survival rates (80%, 94%, 77%) and better in vitro development rates (60%, 80%, 63%) than those of slow thawing, following vitrification of the fertilized ova, 4-cell embryos and morula, respectively. These criteria of vitrification/ thawing could be inferred to the human 4-cell embryos in the IVF protocol.

The effects of different thawing temperatures on morphology and collagen metabolism of −20 °C dealt normal human fibroblast

The purpose of present study is to investigate the effects of two different thawing temperatures on normal human fibroblast which dealt with −20 °C, hoping to provide a clue for further study in reducing excessive collagen formation after cryotherapy on skin diseases in vitro, as well as in differentiation disorders. In order to elucidate its action mechanism, a programmable freezing device was developed to apply freezing temperatures on cell cultures. The effects of two different thawing temperatures on frozen fibroblast proliferation, viability, collagen synthesis and alpha smooth muscle actin (α-SMA) expressing were investigated. We found that compared with 37 °C, thawing with 20 °C yielded same motility. But there are significant differences in terms of the α-SMA expression ( P < 0.05) of fibroblast and collagen I, III synthesis ( P < 0.01) between two groups after 72 h. The results suggest that comparing with slow thawing; rapid thawing cannot only keep the same cell’s damage, but also can modify collagen synthesis and differentiation of fibroblasts. It may be more suitable for the cryosurgical treatment of keloids and benign skin diseases.

Effect of modified starch concentration and freezing and thawing rates on properties of mashed potatoes (cv. Kennebec)

AbstractEffects of modified cornstarch concentration, freezing rate and thawing mode on quality properties of mashed potatoes were examined. The product was tested by oscillatory rheometry, instrumental texture profile analysis (ITPA) and cone penetration, colour, dry matter (DM) and sensory analyses. Oscillatory parameters showed that increasing starch concentration resulted in a softer product in which gel strength decreased in direct proportion to concentration in quick‐frozen and microwave‐thawed product. Quick freezing made for firmer mashed potatoes, whereas oscillatory, ITPA and penetration parameters were lower in the samples thawed at 4 °C. Starch concentration and instrumental consistency and firmness were directly proportional to one another. Adding starch at the highest concentration, quick freezing and slow thawing produced a lighter‐coloured mashed potato, although the loss of colour in the processed product was not detected by panellists in the microwave‐thawed samples. DM was higher in the quick‐frozen samples and lower in air‐thawed samples. The factors studied affected different sensory texture parameters but had hardly any effect on attributes perceived during final and residual phases of mastication. Only starch concentration had a significant effect on sensory acceptability, but interactions between factors showed that starch addition followed by quick freezing was judged undesirable by the panellists due to excessive softening. Oscillatory parameters correlated well with sensory texture attributes and proved quite precise. They would therefore appear to be the best test to monitor properties of frozen mashed potatoes. Copyright © 2007 Society of Chemical Industry

Viabilidad de espermatozoides criopreservados de macha Mesodesma donacium (Mollusca, Bivalvia)

The viability of the surf clam spermatozoa was evaluated using the percentage of fertilization obtained with cryopreserved spermatozoa and different protocols. In total, 42 protocols combined three types of cryoprotectants (DMSO, Methanol, and Prophylen-glycol) at three different concentrations (0.5 M, 1.0 M, and 1.5 M), four freezing rates (-5, -10, -15, and -206oC·min -1 ), and two thawing rates (slow: 72oC·min -1 and fast: 312oC·min -1 ). The best sperm motility was obtained with DMSO frozen at -15oC·min -1 and with a slow thawing rate. The best fertilization percentages were obtained with DMSO frozen at -15oC·min -1 but with a fast thawing rate.

P-176: A computer assisted sperm motility based study on rapid vs. slow thawing temperatures on human sperm motility parameters

In animal studies it has been shown that very rapid thawing at 65°C for 7.5 seconds provided optimal preservation of sperm motility and acrosome integrity. Cellular damage may be caused during the thawing process as the ice melts or re-crystallizes. Slow thawing is most likely to induce injury, as it allows time for consolidation of microscopic ice crystals into larger forms, which are known to be damaging. Some workers have reported that thawing human sperm at 37°C for 10 min was superior to room temperature for 30 min. The purpose of this study was to compare the difference in sperm characteristics in post thaw semen samples undergoing slow (37°C) and rapid thaw (55°C) procedure. Prospective study. The study includes 15 semen samples of men attending the Andrology Laboratory. Each semen sample after liquefaction was divided into two equal parts and frozen using glycerol-based cryoprotectant (Sperm Freeze, Ferti Cult™, Belgium). For the comparative study on thawing temperature and survivability, aliquots of the same samples were thawed at 37°C (10 minutes) and 55°C (3 minutes). Post-processing samples were analyzed using CASA (Hamilton-Thorn, CEROS) for motility parameters. Semen samples thawed at 55°C for 3 minute had higher % motility (12.0 ± 10.8 vs. 19.8 ± 12.7, P-value = 0.010) and % rapid forms (9.8 ± 9.8 vs. 15.3 ± 13.1, P = 0.030) compared to the sample thawed at 37°C. There were no significant differences in other CASA velocity parameters like VAP, VSL, VCL, ALH, BCF and linearity between the two groups. Our results indicate that a short exposure to higher temperature may be beneficial to recover higher percentage of motile sperms from cryopreserved human semen. This may be due to a shortening of the re-crystallization time, an event that can have considerable impact on cell damage due to the physical and osmotic forces exerted at this stage.

Effect of thawing rate and post-thaw culture on the cryopreserved fetal rat islets: Functional and morphological correlation

The ability of the fetal pancreatic islet cells to multiply rendered them a potential tissue for transplantation studies to cure diabetes. A bank of fetal islets could be created with proper storage in liquid nitrogen. The aim of this study is to evaluate the effect of thawing rate and post-thaw culture on the structural and functional integrity of isolated cryopreserved islets of rat fetuses. Fetal rat islets were isolated by the collagenase digestion, cultured for three days, and then cryopreserved using dimethylsulphoxide as cryoprotectant and the step-rate cooling to − 40 °C before immersing them in liquid nitrogen. The islets were thawed by the slow or fast warming rates using hyperosmolar sucrose solution and then cultured for 1 or 2 days. Insulin and C-peptide contents of the slow thawed islets were higher than those of the control. In the fast thawed islets the contents were similar to those of the control. Insulin and C-peptide release in response to glucose for the slow thawed islets were lower than those of the control and in the fast thawed islets they were similar to that of the control. Histological examination showed irregular periphery and fragmented central part of the large slowly thawed islets, which showed also variable immunohistochemical reaction to anti-insulin serum, ranging from strongly positive reaction to markedly weak reaction. Fast thawed islets showed mostly regular periphery and their reaction to the anti-insulin serum was slightly weaker than that of the control islets. It was concluded that fast thawing and post-thaw culture is much better than slow thawing, as indicated by nearly normal insulin and C-peptide content and release and intact structural integrity.

Frost heave on earth hummocks (pounus) in Finnish Lapland

Frost heave, frost and snow depths on two earth hummocks (pounus) were monitored from 1992 to 2002 in Finnish Lapland. Frost heave was measured with motometers, frost depth with Gandahl's frost tube containing methylene blue, and snow depth with frost tubes above the ground surface. The annual range of frost heave was from 1 mm to 94 mm. The mean annual frost heave on a peat pounu was up to twice that on a mineral-cored pounu. Frost heave values on the pounus did not differ much from those from the surrounding mire. During the early summer, slow thawing of frost keeps the surface of pounus higher than the surrounding mire surface and this supports the formation of earth hummocks.

Infiltrative squamous cell carcinoma on the scalp after treatment with 5% imiquimod cream

Infiltrative squamous cell carcinoma on the scalp after treatment with 5% imiquimod cream

Effects of cryopreservation and thawing on the structure of vascular segment

The objective of this study was to analyze the importance of the quality of the vascular segment to be cryopreserved and the influence of storage in a gas phase, a liquid phase, or after accidental immersion in liquid nitrogen. In addition, we investigated the effects of rapid versus slow thawing on the occurrence of fractures and changes in the structure of the vessel wall. The tissue sources were whole thoracic and abdominal aortas from 15 pigs. Each aorta was cut into equal segments and randomly assigned to each study group. One segment of fresh unfrozen aorta of the same size was used as a control. The samples were cryopreserved using a programmed apparatus. After 2 weeks the arterial segments were thawed rapidly or slowly. A great variation in the results was obtained depending on the quality of the control. Although endothelial cells were better preserved in the liquid phase, the internal elastic lamina and elastic lamelli showed better preservation and fewer microfractures in the gas phase. The internal elastic lamina showed a greater number of microfractures when an accidental immersion in liquid nitrogen had taken place. Furthermore, better preservation of the structure of the vascular segment was observed with a slow thawing method. In general, the conditions of storage and the method of thawing seem to damage the structure of vascular segments. It is necessary to use a severe protocol of donor and vascular segment selection to optimize the post-thaw quality of the cryopreserved samples.

Determination of tetramine in marine gastropods by liquid chromatography/electrospray ionization-mass spectrometry

Tetramine (tetramethylammonium ion) is found at high levels (several mg/g) in the salivary gland of buccinid gastropods and has been involved in numerous poisoning incidents after ingestion of those gastropods. A sensitive and selective determination method for tetramine, which is based on a combination of liquid chromatography (LC) and electrospray ionization-single quadrupole mass spectrometry (ESI-MS), was developed. Following separation by LC on a cation-exchange column, tetramine was easily detected by simultaneous monitoring of a molecular ion ( m/ z 74) at a cone voltage of 30 V and a fragment ion ( m/ z 58) at 70 V. A linear calibration curve was obtained in the range of 0.1–100 ng by plotting the peak areas of the molecular ion versus the amounts of tetramine. Spiking experiments demonstrated that tetramine in gastropod tissues can be determined by the LC/ESI-MS method, without being affected by sample matrices as well as the extration procedure. Applications of the new method to gastropod samples revealed that a small amount of tetramine is contained even in mid-gut gland and muscle and that tetramine in the salivary gland diffuses to other tissues during boiling and slow thawing.

O0060 EFFECT OF SERA FROM CHILDREN WITH ACUTE LIVER FAILURE DUE TO NON A-E HEPATITIS ON CRYOPRESERVED HUMAN HEPATOCYTES

Introduction: Acute liver failure (ALF) serum has been shown to have inhibitory effects on DNA and protein synthesis in cultured human HepG2 cells. Isolation and culture of hepatocytes has provided a source of functioning hepatocyte mass either for cell transplantation or for use in bioartificial liver assist devices but there are concerns about the effect of toxic substances accumulating in the ALF patients on the function of the hepatocytes. Our aim was to assess the effect of ALF sera from non A-E hepatitis patients on cryopreserved human hepatocytes. Methods: Cryopreserved human hepatocytes were thawed using a slow thaw technique. Collagen-coated 96 well culture plates were seeded with hepatocytes (30,000 cells /well) and incubated overnight at 37°C, 5% CO2. Sera obtained from 10 children with ALF due to non A-E hepatitis were pooled. The controls consisted of pooled sera from 10 age-matched normal children. The culture media was replaced with fresh media containing pooled normal or ALF sera at concentrations ranging from 0% to 80%. Plates were re-incubated for 24 h and cell activity and function were analysed using MTT, [14 C]-leucine incorporation and [3H]- thymidine incorporation assays. Results: There was no difference between the effects of ALF and normal serum on overall cell activity as determined by the MTT assay (20% ALF serum: mean 0.092 ± SD 0.01 OD units/well; 20% normal serum: 0.096 ± 0.004 OD units/well), and on the protein synthesis as assessed by [14C]-leucine incorporation assay (20% ALF: 94.9 ± 13.5 cpm/well; 20% normal sera: 97.1 ± 12.6 cpm/well). However, when the hepatocytes were incubated with both ALF and normal sera inhibitory effects were observed on DNA synthesis as assessed by the [3 H]-thymidine incorporation assay. The inhibitory effect of both normal and ALF sera increased with serum concentration, but greater effect was observed in the presence of ALF sera (20% ALF sera: 17.05 ± 3.9 cpm/well; normal sera: 229.87 ± 96.5 cpm/well; p<0.003). Conclusion: Sera from children with ALF due to non A-E hepatitis did not have toxic effects on overall cell activity and protein synthesis of cryopreserved human hepatocytes. As the main goal of hepatocyte transplantation or use of bioartificial livers in ALF is the immediate metabolic support, therefore the observed inhibitory effect of ALF sera on DNA synthesis on human hepatocytes is unlikely to be important.

New approach to improving endothelial preservation in cryopreserved arterial substitutes

The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig ( n=20) were frozen at a temperature reduction rate of 1 °C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (−80, −145 or −196 °C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens ( p<0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at −145 °C), significant differences ( p<0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at −80 °C followed by rapid thawing led to greatest endothelial cell loss (61.36 ± 9.06% covered endothelial surface), while a temperature of −145 °C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38 ± 16.67% surface cover). In conclusion, storage at a temperature of −145 °C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.