Effect of warming rate on mouse embryos frozen and thawed in glycerol

Mouse embryos (8-cell) fully equilibrated in 1.5 M-glycerol were cooled slowly (0.5 degrees C/min) to temperatures between - 7.5 and - 80 degrees C before rapid cooling and storage in liquid nitrogen (-196 degrees C). Some embryos survived rapid warming (approximately 500 degrees C/min) irrespective of the temperature at which slow cooling was terminated. However, the highest levels of survival of rapidly warmed embryos were observed when slow cooling was terminated between -25 and -80 degrees C (74-86%). In contrast, high survival (75-86%) was obtained after slow warming (approximately 2 degrees C/min) only when slow cooling was continued to -55 degrees C or below before transfer into liquid N2. Injury to embryos cooled slowly to -30 degrees C and then rapidly to -196 degrees C occurred only when slow warming (approximately 2 degrees C/min) was continued to -60 degrees C or above. Parallel cryomicroscopical observations indicated that embryos became dehydrated during slow cooling to -30 degrees C and did not freeze intracellularly during subsequent rapid cooling (approximately 250 degrees C/min) to -150 degrees C. During slow warming (2 degrees C/min), however, intracellular ice appeared at a temperature between -70 and -65 degrees C and melted when warming was continued to -30 degrees C. Intracellular freezing was not observed during rapid warming (250 degrees C/min) or during slow warming when slow cooling had been continued to -65 degrees C. These results indicate that glycerol provides superior or equal protection when compared to dimethyl sulphoxide against the deleterious effects of freezing and thawing.     

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)     

Effect of cooling and warming rates during cryopreservation on survival of in vitro-produced bovine embryos

The effect of cooling and warming rates during cryopreservation on subsequent embryo survival was studied in 607 bovine morulae and 595 blastocysts produced by in vitro maturation, fertilization and culture (IVM/IVF/IVC). Morulae and blastocysts were prepared by co-culturing presumptive zygotes with bovine oviductal epithelial cells (BOEC) in serum-free TCM199 medium for 6 and 7 d, respectively. The embryos in 1.5 M ethylene glycol in plastic straws were seeded at -7 degrees C, cooled to -35 degrees C at each of 5 rates (0.3 degrees, 0.6 degrees , 0.9 degrees, 1.2 degrees, or 1.5 degrees C/min) and then immediately plunged into liquid nitrogen. The frozen embryos were warmed either rapidly in a 35 degrees C water bath (warming rate > 1,000 degrees C/min) or slowly in 25 degrees to 28 degrees C air (< 250 degrees C/mm). With rapid warming, 42.1% of the morulae that had been cooled at 0.3 degrees C/min developed into hatching blastocysts. The proportions of rapidly wanned morulae that hatched decreased with increasing cooling rates (30.4, 19.0, 15.8 and 8.9% at 0.6 degrees , 0.9 degrees, 1.2 degrees and 1.5 degrees C/min, respectively). With slow warming 25.9% of the morulae that had been cooled at 0.3 degrees C/min developed into hatching blastocysts, while <10% of the morulae that had been cooled faster developed. The hatching rate of blastocysts cooled at 0.3 degrees C/min and warmed rapidly (96.3%) was higher than those cooled at 06 degrees and 0.9 degrees C/min (82.7 and 84.6%, respectively), and was also significantly higher than those warmed slowly after cooling at 0.3 degrees, 0.6 degrees or 0.9 degrees C/min (69.1, 56.6 and 51.8%, respectively). Cooling blastocysts at 1.2 degrees or 1.5 degrees C/min resulted in lowered hatching rates either with rapid (71.2 or 66 0%) or slow warming (38.2 or 38.9%). These results indicate that the survival of in vitro-produced bovine morulae and blastocysts is improved by very slow cooling during 2-step freezing, nevertheless, slow warming appears to cause injuries to morulae and blastocysts even after very slow cooling.     

Effect of rapid addition and dilution of dimethyl sulfoxide on the viability of frozen-thawed rabbit morulae

The aim of the present study was to examine effects of altering thawing conditions and procedure of addition and dilution of Me2SO on the viability of frozen-thawed rabbit morulae. Five hundred and sixty two rabbit morulae were cooled from room temperature to -80 degrees C at 1 degree C/min in the presence of 1.5 M dimethyl sulfoxide (Me2SO) using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, cooled rapidly, and stored in liquid nitrogen. When Me2SO was added in a single step, the frozen embryos were thawed in ambient air at 40 degrees C/min and Me2SO was diluted in a single step, 99 of 107 (93%) embryos cultured for 48 hr and 12 of 32 (38%) embryos transferred to 6 recipients developed to expanding blastocysts and viable fetuses, respectively. When Me2SO was added in a single step and the frozen embryos were thawed at the same rate and transferred directly without removal of Me2SO to culture media or oviducts of 8 recipients, 67 of 75 (89%) embryos cultured and 12 of 40 (30%) embryos transferred developed to expanding blastocysts and viable fetuses, respectively. There were no significant differences between these survival rates and survival rates obtained by conventional method, i.e., frozen embryos were thawed at 4 degrees C/min by interrupted slow method and Me2SO was added and diluted in a stepwise manner.     

Gradual Thawing Improves the Preservation of Cryopreserved Arteries

This study was designed to test a slow, controlled, automated process for the thawing of cryopreserved arteries, whereby specimen warming is synchronized with the warming of its environment. Segments of minipig iliac artery, 4-5 cm in length, were subjected to controlled, automated cryopreservation in a biological freezer at a cooling rate of 1 degrees C/min to -120 degrees C, followed by storage in liquid nitrogen at -196 degrees C for 30 days. Following storage, the arterial segments were subjected to rapid (warming rate of approximately 100 degrees C/min) or gradual (1 degrees C/min) thawing. Thawed specimens were processed for light microscopy and for scanning and transmission electron microscopy, Cell death was determined by the TUNEL method. Metalloproteinase (MMP) expression was estimated by immunohistochemical analysis. Most of the cryopreserved vessels subjected to rapid thawing showed spontaneous fractures, mainly microfractures, whereas these were absent in slowly thawed specimens. In rapidly thawed vessels, the proportion of damaged cells was double that observed in those thawed more gradually. Increased intensity and extent of MMP-2 expression was shown by rapidly thawed specimens. The slow-thawing protocol tested avoids the formation of spontaneous fractures and microfractures and the accumulation of fluid within the arterial wall tissue. This results in improved tissue preservation.     

Effect of cooling rate and partial removal of yolk on the chilling injury in zebrafish (Danio rerio) embryos

High chilling sensitivity is one of the main obstacles to successful cryopreservation of zebrafish embryos. So far the nature of the chilling injury in fish embryos has not been clear. The aim of this study is to investigate the effect of cooling rate and partial removal of yolk on chilling injury in zebrafish embryos. Zebrafish embryos at 64-cell, 50%-epiboly, 6-somite and prim-6 stages were cooled to either 0 degrees C or -5 degrees C at three different cooling rates: slow (0.3 degrees C/min or 1 degree C/min), moderate (30 degrees C/min), and rapid (approximately 300 degrees C/min). After chilling, embryos were warmed in a 26 degrees C water bath, followed by 3-day culturing in EM at 26 +/- 1 degrees C for survival assessment. When embryos were cooled to 0 degrees C for up to 30 min, 64-cell embryos had higher survival after rapid cooling than when they were cooled at a slower rate. When 64-cell embryos were held at -5 degrees C for 1 min, their survival decreased greatly after both slow and rapid cooling. The effect of cooling rate on the survival of 50%-epiboly and 6-somite embryos was not significant after 1 h exposure at 0 degrees C and 1 min exposure at -5 degrees C. However, rapid cooling resulted in significantly lower embryo survival than a cooling rate of 30 degrees C/min or 1 degree C/min after 1 h exposure to 0 degrees C for prim-6 stage or 1 h exposure to -5 degrees C for all stages. Chilling injury in 64-cell embryos appears to be a consequence of exposure time at low temperatures rather than a consequence of rapid cooling. Results also indicate that chilling injury in later stage embryos (50%-epiboly, 6-somite and prim-6) is a consequence of the combination of rapid cooling and exposure time at low temperatures. Dechorionated prim-6 embryos were punctured and about half of yolk was removed. After 24 h culture at 26 +/- 1 degrees C after removal of yolk, the yolk-reduced embryos showed higher embryo survival than did control embryos after rapid cooling to -5 degrees C for 10 to 60 min. Results suggest that cold shock injury after rapid cooling can be mitigated after partial removal of yolk at the prim-6 stage. These findings help us to understand the nature of chilling sensitivity of fish embryos and to develop protocols for their cryopreservation.     

The effects of 1,2-propanediol as a cryoprotectant on the freezing of mouse oocytes

Cryopreservation of mammalian eggs has been successfully accomplished using 1,2-propanediol (PG). Effects of holding times of 0 and 30 min at -40 degrees C and storage times of 1 d and 1 mo at -196 degrees C were investigated in combination with various concentrations of PG (1.0, 1.5, and 2.0M) to determine the survival and fertilizability of mouse oocytes rapidly frozen and thawed in straws. A rapid one-step dilution using 0.5 M sucrose solution inside the straws was used following the thawing of oocytes. A significant effect of PG concentration was found between 1.0 M and 1.5 or 2.0 M (P<0.01), but no significance was discovered between 1.5 M and 2.0 M (P>0.05) on subsequent survival and fertilizability of frozen and thawed mouse oocytes. With 2.0 M PG, the best survival rate (58.3%) and fertilizability rate (19.0%) were obtained by holding at -40 degrees C for 30 min and by storage at -196 degrees C for 1 d. Thirty minutes of holding at -40 degrees C reduced oocyte damage during the procedure but not significantly (P>0.05). In addition, there was no significant difference in the various storage periods (P>0.05). This study demonstrated that mammalian oocytes can be cryopreserved in the presence of 1,2-propanediol by utilizing a rapid freezing and thawing procedure.     

Deep freezing of sheep embryos.

Sheep embryos, collected 1-8 days after oestrus, were placed in Dulbecco's phosphate-buffered saline medium (PBS). After treatment, the viability of the embryos was tested by temporary transfer to ligated rabbit oviducts. In Exp. 1, Days 5-8 embryos survived for at least 15 min at 0 degrees C in the presence of 1-5 M-DMSO. In Exp. 2, 12/14 Days 5-8 embryos survived after being frozen in 1-5 M-DMSO at 0-3 degrees C/min to temperatures ranging between-15 degrees and -60 degrees C and then thawed at 12 degrees C/min. In Exp. 3, Days 5-8 embryos were frozen in 1-5 M-DMSO at 0-3 degrees C/min to below-65 degrees C before being transferred to liquid nitrogen (-196 degrees C), and stored for 12 hr to 1 month. The embryos were thawed at 3 degrees C/min, 12 degrees C/MIN or 360 degrees C/min and, after transfer to rabbit oviducts, 0/4, 10/36 and 1/4, respectively, developed normally. The 11 embryos which were considered normal when recovered from the rabbit oviducts plus 1 slightly retarded embryo were transferred to 7 recipient ewes. Four ewes subsequently lambed, producing 5 lambs. In addition, 8 embryos were transferred to 4 ewes directly after thawing. Three of these ewes subsequently lambed, producing 3 lambs.     

Effect of post-thaw cell rehydration at 4 degrees C on survival of frozen and vitrified IVP-derived bovine embryos.

In vitro-produced bovine embryos (IVP) were either frozen in 10% glycerol in a phosphate-buffered saline solution (PBS) using conventional slow freezing or vitrified in 25% glycerol and 25% ethylene glycol in PBS. The results of viability and hatching rates were compared between frozen and vitrified embryos after thawing and dilution using one of three different protocols: (A) a three-step dilution procedure, (B) a one-step dilution procedure or (C) a procedure in which embryos were kept in situ inside the straw at 4 degrees C for 10 min during a one-step dilution procedure. No significant differences in embryo survival were found among protocols A, B and C for frozen embryos and between protocols A and B for vitrified embryos. Viability and hatching rates of vitrified embryos thawed and diluted by protocol C (73 and 62%) were significantly enhanced (P < 0.05) in comparison to those obtained with protocol A (55 and 41.6%) or protocol B (54.5 and 35.3%). These results indicate that for vitrified IVP bovine embryos, direct in-straw rehydration at 4 degrees C for 10 min improves embryo survival and it could be a practical procedure for use under field conditions where there is sometimes a longer interval between thawing and transfer.     

High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1-2 propanediol as cryoprotectant

A method for obtaining a high survival rate of frozen-thawed mouse embryos is presented. Eight-cell mouse embryos were frozen inside small plastic straws in the presence of 1-2 propanediol and stored at -196 C. After thawing, the embryos were diluted for only 5 min in a 1.0 M sucrose solution to remove the 1-2 propanediol from the cells. At high rate of thawing (is equivalent to 2500 C/min) more than 88% of the embryos survived in vitro to the blastocyst stage provided that the dilution of propanediol was performed rapidly during thawing. At a lower rate of thawing (is equivalent to 300 C/min), survival tended to be higher (94.7%) when dilution was done 5 min after thawing. When the frozen-thawed embryos were transferred to the oviducts of day 1 pseudopregnant recipients either directly after the dilution of 1-2 propanediol or after 24 or 48 hr of culture, a high proportion of them (65.9%) develop normally to viable fetuses.     

Cryopreservation of mouse spermatozoa]

The spermatozoa of cauda epididymis of mature mice were suspended in 3% skim milk in distilled water supplemented with 12, 15, 18 or 21% (W/V) raffinose. The suspension of spermatozoa were frozen in liquid nitrogen gas for 10 min, then stored in liquid nitrogen (-196 degrees C). The frozen suspensions of spermatozoa were thawed by rapid warming in water bath at room temperature. For removing the cryopreservative solution, a pair of syringes connected with a three stop cock and a filter unit (pore size 0.45 mu) were used. Highest sperm motility was obtained after 1 hr of thawing from the cryopreservative solution containing 18% raffinose and 3% skim milk. These cryopreserved spermatozoa were used for fertilization in vitro. The proportion of pronuclear oocytes was 35.9% (74/206) 6 hr after insemination, and the proportion of 2-cell embryos was 33.6% (42/125) 28 hr after insemination. All 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 45.2% (19/42) developed to normal young.     

Cryomicroscopic observations of cattle embryos during freezing and thawing

A cryomicroscope was used to observe changes in the appearance of day 6 1 2 to 7 1 2 cattle embryos during cooling and warming in 1.4M glycerol/PBS. Embryos were cooled at various rates between 0.2 and 25 degrees C/min to temperatures between -25 and -60 degrees C and then cooled rapidly ( approximately 250 degrees C/min) to temperatures below -140 degrees C. The volume of the embryos calculated from the cross-sectional area during slow cooling decreased at -25 degrees C to about 50% of the isotonic volume. Fracture planes could be observed in the extracellular ice matrix surrounding the embryos after rapid cooling to approximately -140 degrees C. The fracture planes often touched the zona pellucida and sometimes caused cracks in the zona. Cracks in the zona pellucida were observed more often after rapid cooling from temperatures between -20 to -35 degrees C (9 13 ) than from temperatures between -36 to -60 degrees C (2 7 ). When embryos were warmed rapidly ( approximately 250 degrees C/min) from temperatures below -140 degrees C, no change was observed in the appearance of either the embryo or its surroundings except the melting of the extracellular ice. However, when embryos were warmed slowly (2 or 5 degrees C/min), a series of events was observed; first, at approximately -70 degrees C the cytoplasm and the extracellular space gradually darkened and reached maximum darkness at approximately -55 degrees C. Then, on continued slow warming, the dark material gradually disappeared and finally the large extracellular ice crystals melted.     

Effects of methanol and developmental arrest on chilling injury in zebrafish (Danio rerio) embryos

Stage-dependent chilling sensitivity has been reported for many species of fish embryos. Most of these studies reveal that developmental stages beyond 50% epiboly are less sensitive to chilling, but the chilling sensitivity accelerates rapidly at subzero temperatures. In this study, the effects of methanol and developmental arrest on chilling injury were studied using zebrafish (Danio rerio) embryos at 64-cell, 50% epiboly, 6-somite, prim-6 and long-bud stages. Embryos were exposed to methanol or anoxic conditions before they were cooled to 0 or -5 degrees C with slow (1 degrees C/min), medium (30 degrees C/min) or fast ( approximately 300 degrees C/min) cooling rates and were held at these temperatures for different time periods. Embryo survival was evaluated in terms of the percentage of treated embryos with normal developmental appearance after 3-day culture. Experiments on the effect of methanol on chilling sensitivity of the embryos showed that the addition of methanol to embryo medium increased embryo survival significantly at all developmental stages and under all cooling conditions. Higher concentration of methanol treatment generally improved embryo survival when embryos were cooled at a fast cooling rate of 300 degrees C/min. Experiments on the effect of developmental arrest on chilling sensitivity of embryos showed that embryos at 50% epiboly and prim-6 stages underwent developmental arrest almost immediately after 15 min oxygen deprivation. After 4h in anoxia, the survival rates of the embryos were not significantly different from their respective aerobic controls. Anoxia and developmental arrest had no effect on the chilling sensitivity of zebrafish embryos.     

Survival of 16-celled and morula stage rabbit embryos frozen to -196 degrees C,.

Preimplantation stage (16-celled and morula) rabbit embryos were successfully frozen to -196 degrees C. The cooling rate (from a room temperature to 0 degrees C), the presence of the mucin layer surrounding embryos, the ice-seeding treatment and the thawing procedure were examined to determine their effects on the survival of the frozen embryos of Japanese white, New Zealand white and Dutch-Belted rabbits. A high proportion (51%; 16-celled, 69%; morula) of Dutch-Belted rabbit embryos developed in vitro, when they were frozen to -196 degrees C, applying the ice-seeding at -4 degrees C in the presence of 12.5% DMSO, after being cooled to 0 degrees C at the rate of 7-9 degrees C/min, and were diluted by a stepwise addition of 4 different strength PBS on thawing. The highest rate of in vitro development (81%; Japanese white, 75%; New Zealand white, 82%; Dutch Belted embryos) was obtained when the morula stage embryos were frozen to -196 degrees C applying seeding at -4 degrees C after being cooled to 0 degrees C at the rate of 1 degrees C/2.5 min and were diluted, on thawing, by stepwise addition of 6, 3 and 1% DMSO solution and a culture medium. No great difference was found in the survival rate between the embryos covered with the mucin layer and those which had not the coat. All the embryos frozen without applying seeding treatment failed to develop in vitro after being thawed and diluted. Nine out of 27 does each of which received 6 reimplantations of the embryos frozen-thawed became pregnant and were found to be carrying 37 normal fetuses on the 12th day of pregnancy.     

Some factors affecting the viability of mouse and cattle embryos frozen to −40°C before transfer to liquid nitrogen

A total of 1161 8- to 16-cell mouse embryos and 31 cattle early morulae and late blastocysts were frozen to ?40°C before transfer to liquid nitrogen. After thawing, mouse embryo viability was determined by in vitro development to the blastocyst stage and cattle embryo viability by both in vivo and in vitro development.Using glycerol as the cryoprotective agent, 88% of the mouse embryos developed to the blastocyst stage: thawing at 45 and 360° C/min gave the best results (88.8 and 84.8%, respectively). In another test with holding times at ?40°C of up to 60 min, about 70% of embryos developed to blastocysts with holding time 30–60 min.In cattle, 11 embryos frozen in DMSO and thawed at 360°C/min were transplanted to eight recipients. Four pregnancies (six fetuses) resulted. Thawing rates of 200 and 360°C/min resulted in the best in vitro development of cattle embryos.     

The viability of deep-frozen cow embryos

Day 7 cow embryos were frozen in 1.5 M-DMSO in PBS at 0.3 degrees C/min to -36 degrees C and at 0.1 degrees C/min between -36 and -60 degrees C before being plunged directly into liquid nitrogen. They were subsequently thawed (rapidly to -50 degrees C, at 4 degrees C/min from -50 to -10 degrees C, and rapidly again) to room temperature. Embryonic viability was tested by four different transfer techniques. Maximum pregnancy rate (8/12) was obtained with surgical transfer immediately after thawing and dilution of DMSO.     

The developmental potential of frozen-thawed hamster preimplantation embryos following embryo transfer: Viability of slowly frozen embryos following slow and rapid thawing

Hamster preimplantation embryos were slowly frozen (0.33°C/min) and seeded above 10°C in TC-199 containing 1.5 M-DMSO. These embryos were thawed either slowly (1.5°C/min) or rapidly (90°C/min). The thawed embryos were examined by morphology, trypan blue exclusion and viability after embryo transfer. Slow thawing gave significantly higher viability compared to rapid thawing. The early preimplantation embryos demonstrate higher sensitivity to freezing. The three tests of viability (morphology, trypan blue exclusion and embryo transfer) were found to be positively correlated.     

The effect of quick-freezing in ethylene glycol on morphological survival and in vitro development of mouse embryos frozen at different preimplantation stages

This study was conducted to examine the effect of a quick-freezing protocol on morphological survival and in vitro development of mouse embryos cryopreserved in ethylene glycol (EG) at different preimplantation stages. One-cell embryos were harvested from 6-to 8-wk-old CB6F1 superovulated mice, 20 to 23 h after pairing with males of the same strain and hCG injection. The embryos were cultured in human tubal fluid (HTF) containing 4 mg/ml BSA under mineral oil at 37 degrees C in 5% CO(2) plus 95% room air at maximal humidity. Twenty-four to 96 h after collection, the embryos were removed from culture and frozen at the 2 cell, 4 to 8-cell, compact morula, early blastocyst, expanding blastocyst and expanded blastocyst stages. To perform the quick-freeze procedure, embryos were equilibrated in Dulbecco's phosphate buffered saline (DPBS) + 10 % fetal bovine serum (FBS) + 0.25 M sucrose + 3 M ethylene glycol (freeze medium) for 20 min at room temperature (22 to 26 degrees C) and loaded in a single column of freeze medium into 0.25-ml straws (4 to 5 embryos per straw). The straws were held in liquid nitrogen vapor for 2 min and immersed in liquid nitrogen. Embryos were thawed by gentle agitation in a 37 degrees C water bath for 20 sec and transferred to DPBS + 10 % FBS + 0.5 M sucrose (re-hydration medium) for 10 min at room temperature, rinsed 2 times in HTF plus 4 mg/ml BSA and then cultured for 24 to 96 h. Survival of embryos was based on their general morphological appearance after thawing and their ability to continue development upon subsequent culture in vitro. Survival of blastocysts after thawing also required expansion or reexpansion of the blastocoel after several hours in culture. Significant differences were found in the survival and development of mouse embryos at different developmental stages quick-frozen in ethylene glycol and sucrose: 2-cell embryos 43/84 (51%), 4 to 8-cell embryos 44/94 (47%), morulae and early blastocysts 56/70 (80%; P相似文献   

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 degrees 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 degrees 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 degrees C), significant differences (p < 0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at -80 degrees C followed by rapid thawing led to greatest endothelial cell loss (61.36+/-9.06% covered endothelial surface), while a temperature of -145 degrees 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 degrees 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.     

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.