Selective destruction of leucocytes by freezing as a potential means of modulating tissue immunogenicity: membrane integrity of lymphocytes and macrophages

It is now known, when a tissue allograft is transplanted, that antigen recognition alone is not sufficient for lymphocyte activation in the host. "Passenger" leucocytes (antigen-presenting cells) present in the donor tissue are now recognized as a major immunogenic stimulus. Removal of these contaminating leucocytes, using a variety of procedures, has enabled the immunogenicity of allografts to be reduced, thus enhancing the survival of tissue allografts. This initial study explores the possibility of using a cryobiological approach to modulating the immunogenicity of tissues by virtue of the well-recognized differential susceptibility of different cell types to freezing injury. The investigation was prompted by demonstrations that pancreatic islets can secrete insulin in response to a graded glucose challenge after cryopreservation using relatively fast cooling rates which would be expected to be suboptimal for leucocyte survival. Batches of rat peripheral blood lymphocytes, or peritoneal exudate cells (macrophages) were cooled at 0.3, 1, 5, 20, 75, or 200 degrees C/min using three different cryopreservation protocols reported to yield viable pancreatic islets. Cell survival was evaluated in terms of the numbers of cells recovered after freezing as well as a fluorometric viability assay which assessed the membrane integrity of cells. Optimum survival of both lymphocytes and macrophages after freezing and thawing was found at cooling rates in the range of 0.3 to 5 degrees C/min. A significant number (10-40%) of these lymphoid cells survived freezing at 20 degrees C/min and only after cooling at rates greater than 75 degrees C/min was survival reduced to a negligible level.     

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.     

Cryogenic preservation of isolated islets of Langerhans: Two-step cooling

Rat islets of Langerhans were frozen to ?196 °C using a two-step freezing procedure. Islets isolated from the pancreases of Long Evans hooded rats were exposed to CMRL 1066 media containing 1 M dimethyl sulfoxide for 6 min at 4 °C. They were transferred directly to subzero holding baths ranging from ?20 to ?43 °C for 5 to 20 min prior to transfer to and storage in liquid nitrogen. After warming at ～7 °C/min, the islets were diluted with Hanks' balanced salt solution containing 10% fetal calf serum, washed, and cultured overnight. In general, maximum protection of the islets from the stress of cooling to ?196 °C was obtained after holding the islets at ?35 or ?40 °C for between 5 and 15 min. After thawing, islets frozen using an “optimized” two-step protocol released insulin in response to a glucose challenge at a rate equivalent to that of control islets.     

Ultrastructure before freezing,while frozen,and after thawing in assessing cryoinjury of mouse epididymal spermatozoa

Tails of mouse epididymides were treated as follows: control, unfrozen with and without cryoprotective agents (CPA); frozen (to below ?80 °C), slowly (8 °C/min), and rapidly (18 °C/sec), with and without CPA. Intracellular and/or extracellular location of CPA, at least glycerol, was influenced, respectively, by high (22 °C) or low (0 °C) exposure temperature. Standard procedures in electron microscopy were employed and the frozen state preserved by freeze-substitution. Motility before freezing and after thawing was the criterion of cryosurvival.Results showed no evidence of deleterious ultrastructural effects of freezing at rates compared, or of benefits of CPA, regardless of their cellular location. Differences were noted, however, in the appearance of spermatozoa in the frozen state, as a function of the rate of freezing but not as a function of the presence, absence, or location of either glycerol of DMSO. Rapidly frozen cells showed intracellular ice formation in the acrosome, neck, midpiece, and tail regions; there was no intranuclear ice, and extracellular ice artifacts were small. Slowly frozen cells showed large extracellular ice artifacts with evidence of shrinkage distortion due to the dehydration induced by extracellular ice. No spermatozoa survived any of the freezing treatments, showing the lethal effect of both extracellular ice during slow freezing and of intracellular and/or extracellular ice during rapid freezing.     

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.     

Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents.

Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp)[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp)[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions.     

Two-step freezing of cattle blastocysts with dimethylsulfoxyde (DMSO) or glycerol

Sixty five cattle blastocysts were frozen by the so-called two-step freezing method: The samples were seeded at -7 degrees C and then directly brought at -30 degrees C for 30 minutes before being taken into liquid nitrogen. Results in terms of survival rates at thawing and after short term cultures were compared to two controlled linear cooling rate procedures (i.e. 0.3 degrees C/min and 1.3 degrees C/min). The results demonstrate that: 1) two-step freezing yielded approximately the same survival rate as the two others techniques and 2) Glycerol yielded better survival rates than DMSO treatments (56 vs 31% after 24 hours in culture).     

Optimization of a Method for the Cryopreservation of Rabbit Corneas: Attempted Application to Human Corneas

The purpose of the present study was to set up and test a cryopreservation method for long-term storage of human corneas. Therefore the freezing solution was optimized in 264 rabbit corneas by testing the type of cryoprotectant, its concentration, addition and dilution pattern and exposure temperature. Then rabbit corneas were frozen in the optimum solution at different cooling rates and thawed in a water bath at different temperatures. Eight human corneas were cryopreserved with the method showing optimum results in rabbit corneas and four additional corneas were used as controls. Endothelial viability was assessed after each step by vital staining and scanning electron microscopy. Best results after exposure of rabbit corneas to the freezing solution were achieved when using a 10% cryoprotectant concentration, with direct addition/dilution and exposure at room temperature (3512 ±300 viable cellsmm² when using dimethylsulfoxide; 3403 ± 245 viable cellsmm² when using 1,2-propanediol). Cryopreserved rabbit corneas had the highest endothelial cell survival when frozen at 1°C/min and thawed at 37°C (2003 ± 372 viable cells/mm² when using dimethylsulfoxide and 1357 ± 667 viable cells/mm² when using 1,2-propanediol). Cryopreserved human corneas had 753 ± 542 viable cells/mm² when using dimethylsulfoxide and 56 ± 56 viable cells/mm² when using 1,2-propanediol. We can conclude that the method developed is easy to handle and shows optimum results in rabbit corneas, with an endothelial cell survival that is consistent with transplant acceptability criteria. The results obtained in human corneas are below prediction and are still unsatisfactory for successful use in eye banking.     

Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos.

The first successful freezing of early embryos to -196 degrees C in 1972 required that they be cooled slowly at approximately 1 degree C/min to about -70 degrees C. Subsequent observations and physical/chemical analyses indicate that embryos cooled at that rate dehydrate sufficiently to maintain the chemical potential of their intracellular water close to that of the water in the partly frozen extracellular solution. Consequently, such slow freezing is referred to as equilibrium freezing. In 1972 and since, a number of investigators have studied the responses of embryos to departures from equilibrium freezing. When disequilibrium is achieved by the use of higher constant cooling rates to -70 degrees C, the results is usually intracellular ice formation and embryo death. That result is quantitatively in accord with the predictions of the physical/chemical analysis of the kinetics of water loss as a function of cooling rate. However, other procedures involving rapid nonequilibrium cooling do not result in high mortality. One common element in these other nonequilibrium procedures is that, before the temperature has dropped to a level that permits intracellular ice formation, the embryo water content is reduced to the point at which the subsequent rapid nonequilibrium cooling results in either the formation of small innocuous intracellular ice crystals or the conversion of the intracellular solution into a glass. In both cases, high survival requires that subsequent warming be rapid, to prevent recrystallization or devitrification. The physical/chemical analysis developed for initially nondehydrated cells appears generally applicable to these other nonequilibrium procedures as well.     

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws

The interaction of glycerol concentrations of 0-10% and cooling rates from 1 to 1,500 degrees C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30 degrees C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0-1% glycerol after cooling at 30 degrees C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30 degrees C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1 degrees C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500 degrees C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30 degrees C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.     

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.     

Pancreatic islet banking: The transplantation of frozen-thawed rat islets transported between centers

This report describes the feasibility of islet banking for the purpose of transporting isolated islets from one center to another for transplantation. Adult rat islets survived freezing to ?196 °C when 0.25 °C/min was used as the cooling rate, 7.5 °C/min as the warming rate, and when the hyperosmotic protective agent was carefully removed. We were able to show that islets isolated and frozen in one center and transplanted in another center returned diabetic animals to clinical normalcy (fasting normoglycemia, aglycosuria, and weight maintenance). However, as measured by a glucose tolerance test three months after transplantation, these animals had an impaired early insulin release when compared with animals who received fresh islet transplants. Diabetic animals that received islets frozen at a cooling rate of 1.0 °C/min remained diabetic as measured by our clinical parameters. Thus specific definition of conditions used for cryopreservation is important in developing methods suitable for islet banking.     

Survival of Frozen Mycoplasmas

Cooling to -70 C killed a higher percentage of Acholeplasma laidlawii and Mycoplasma mycoides var. capri cells than cooling to -20 C. However, to preserve cell viability for prolonged periods storage at -70 C was much more preferable. The percentage of cells surviving freezing could be increased by increasing the initial cell concentration or by the addition of dimethyl sulfoxide or glycerol as cryoprotective agents. In the presence of 1.5 M of any one of these agents survival rates of up to 100% could be obtained. The optimal cooling rates for maximal survival of A. laidlawii under the experimental conditions tested were 11 C/min for cooling to -20 C and about 15 C/min for cooling to -70 C. Increasing the warming rate during thawing from 0.6 to 67 C/min increased survival by 3 log. Oleic acid enrichment of A. laidlawii membrane lipids, or reduction in the cholesterol content of M. mycoides var. capri membranes, increased the percentage of organisms surviving freezing. Hence, the composition of membrane lipids appears to have a marked influence on the susceptibility of mycoplasmas to freezing injury.     

Interaction of rate of latent heat removal during freezing and rates of subsequent cooling and warming on survival of the blood protozoan, Babesia rodhaini

Babesia rodhaini parasites in murine blood containing 1.5 m DMSO were frozen at two rates, as judged by the duration of the “freezing plateau”, then cooled to ?196 °C and rewarmed at two rates to detect interactions between the duration of the plateau and rates of subsequent cooling and rewarming. Infectivity tests showed that fast and slow freezing (plateau times of about 1 sec and 30 sec, respectively) had similar effects on parasite survival when cooling was at 130 °C/min and warming was at 800 °C/min. However, when either the cooling rate was increased to 3500 °C/min or the warming rate was decreased to 2.3 °C/min, fast freezing decreased parasite survival more than did slow freezing. It is suggested that fast freezing accentuated the damaging effects of fast cooling and slow warming by increasing intracellular ice formation.     

Viability studies on frozen--thawed rat islets of Langerhans.

In an attempt to determine logistical methods of curing diabetes mellitus in man, an investigation has been made on the viability, in vitro and in vivo, of deeply frozen (?150 °C) cryoprotected rat islets of Langerhans. It is found that rat islets, after recovery from a frozen bank of several syngeneic donors, secrete insulin, when thawed, cultured, and then subjected to a high glucose challenge. Cryoprotected frozen-thawed islets are also examined by electron microscopy. In vivo transplantation of recovered frozen islets has been studied for a period of 16 weeks in one streptozotocindiabetic Lewis rat. All normal tests indicated recovery. After sacrifice, staining procedures showed viable islets in the liver, the site of reimplantation, and only dead islet Beta cells in the pancreas.     

Freezing monolayers of cells without gap junctions

Cells in monolayers have been reported to be more susceptible to freezing injury than the same cell type frozen in dispersed suspensions. There appears to be an enhanced susceptibility to intracellular freezing in the monolayers, which is thought to be facilitated by the presence of gap junctions allowing the spread of ice between neighbouring cells. MDCK Type II cells do not form gap junctions in monolayer culture. When frozen at rates of 0.2 to 10 degrees C/min, monolayers in 10% (v/v) propane-1,2-diol or dimethyl sulphoxide showed little influence of cooling rate on survival. This suggested that, in the absence of gap junctions, cells in monolayers did not display enhanced susceptibility to intracellular freezing. In contrast, however, monolayers frozen in glycerol showed a marked increase in cell damage when cooled at rates higher than 0.5 degrees C/min. This does not necessarily counter the suggestion that lack of gap junctions mitigates intracellular freezing as there is evidence that glycerol may itself promote intracellular freezing.     

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.     

Direct freezing of cattle embryos after partial dehydration at room temperature

A new and simple method for freezing of bovine morulae and blastocysts was developed. Embryos were predehydrated at room temperature, frozen at -30 degrees C (cooling rate = 12 degrees C/min), and plunged into liquid nitrogen. This method was compared in vitro and in vivo to the slow freezing method (0.3 degrees C/min to -30 degrees C). Predehydration of the embryos in 1.5M glycerol was achieved by sucrose solution that makes the cells osmotically shrink. After the predehydrated morulae and blastocysts were frozen and thawed, 6 .4% (33 52 ) were developed in vitro for 48h and 44.2% (23 52 ) were hatched. Development obtained with slowly frozen embryos were 70.8% (17 24 ) and 58.3% (14 24 ) respectively. After transfer to recipient heifers, 33.3% (7 21 ) of the embryos frozen according this new method developed normally into viable foetuses or calves. This was the case for 48.5% (16 33 ) of the slowly frozen embryos.     

The effect of freeze rate,duration of phase transition,and warming rate on survival of frozen canine kidneys

The effect of cooling rate, warming rate, and duration of phase transition upon survival of frozen canine kidneys was investigated. In the present study, 11 kidneys out of 14 rapidly cooled (2–4 °C/min) to ?22 °C and thawed (70–110 °C/min) were viable following contralateral nephrectomy. The serum creatinine and BUN levels rose to a maximum of 8.4 and 30 mg%, respectively, on the eighth day post-contralateral nephrectomy. Average survival time was 10 days; however, two of the dogs in this group were allowed to survive, one for 3 months and one for over 2 years. Eight kidneys out of 16 slowly cooled (0.25–1.0 °C/min) and either rapidly or slowly warmed (20–30 °C/min) had function to produce small amounts of urine; however, they did not survive more than 5 days after contralateral nephrectomy.Cooling rates of 0.1 and 10 °C/min were too harmful to the kidney to have renal function after reimplantation.The minimum renal cell damage as assessed by LDH and GOT in the post-freeze perfusate was found in the 2–4 °C/min cooling rate following rapid warming (70 °–110 °C/min).Correlation of the duration of phase transition time to renal cell damage was linear for LDH and GOT (r = 0.93). This result suggests that the duration of phase transition time also is an important factor during the freezing process, affecting postthaw survival of canine kidneys.     

Impact of freezing/thawing procedures on the post-thaw viability of cryopreserved human saphenous vein conduits

BACKGROUND: Cryopreserved human blood vessels are important tools in reconstructive surgery. However, patency of frozen/thawed conduits depends largely on the freezing/thawing procedures employed. METHODS: Changes in tone were recorded on rings from human saphenous vein (SV) and used to quantify the degree of cryoinjury after different periods of exposure at room temperature to the cryomedium (Krebs-Henseleit solution containing 1.8M dimethyl sulfoxide and 0.1M sucrose) and after different cooling speeds and thawing rates following storage at -196 degrees C. RESULTS: Without freezing, exposure of SV to the cryomedium for up to 240 min did not modify contractile responses to noradrenaline (NA). Pre-freezing exposure to the cryomedium for 10-120 min attenuated significantly post-thaw maximal contractile responses to NA, endothelin-1 (ET-1) and potassium chloride (KCl) by 30-44%. Exposure for 240 min attenuated post-thaw contractile responses to all tested agents markedly by 62-67%. Optimal post-thaw contractile activity was obtained with SV frozen at about -1.2 degrees C/min and thawed slowly at about 15 degrees C/min. In these SV maximal contractile responses to NA, ET-1 and KCl amounted to 66%, 70% and 60% of that produced by unfrozen controls. Following cryostorage of veins for up to 10 years the responsiveness of vascular smooth muscle to NA was well maintained. CONCLUSION: Cryopreservation allows long-term banking of viable human SV with only minor loss in contractility.