An isolated perfused rat mesentery model for direct observation of the vasculature during cryopreservation

An intact vasculature is essential for successful hypothermic perfusion and cryopreservation of solid organs, but few studies have specifically assessed the vascular effects of these procedures. A technique was therefore developed for continuous, direct observation of an isolated vascular bed during hypothermic perfusion with cryoprotectants, and during freezing and thawing. The isolated rat mesentery was spread across a controlled low temperature microscope stage and perfused with solutions containing fluorescein isothiocyanate (FITC)-Dextran 70 as an indicator of macromolecular permeability of the vessels. Hypertonic citrate washout, HP-5 perfusion (23), rapid and slow addition and removal of glycerol, and freezing/thawing were studied. Control perfusion with HP-5 produced slow FITC-Dextran leakage, reflecting normal physiological macromolecular permeability of vessels. Rapid addition of glycerol dramatically increased vascular permeability, consistent with osmotic damage to vessels. Rapid removal stopped flow through capillaries and decreased vascular dimensions, suggesting overhydration of endothelial cells and extravascular tissue. Venules and capillaries were the most susceptible vessels to osmotic stress. Slow addition and removal of glycerol (80 mmol/liter/min) produced results similar to control perfusions. During slow freezing (0.5 degree C/min to -5 degrees C) extravascular ice compressing vessels was more obvious than intravascular ice. Glycerol afforded some protection to the microvasculature during freeze/thaw cycles since flow was reestablished in venules and arterioles after thawing, although FITC-Dextran leakage indicated that damage had occurred.     

Cooling of rabbit kidneys permeated with glycerol to sub-zero temperatures.

The aims of this study were to investigate if kidney preservation could be enhanced by cooling of the organs to high sub-zero temperatures after depression of their freezing points by addition of glycerol, and to study whether the added amounts of this compound would confer protection to the organs during freezing and thawing at slow rates.Glycerol was added and removed gradually by continuous, hypothermic perfusion, and the post-preservation viability was assessed by autotransplantation.Brief cooling to ?5 °C of kidneys perfused with 3 m glycerol was found to be compatible with life-sustaining posttransplant function, whereas no kidneys stored at that temperature for 5 days survived.Slow cooling af kidneys glycerolized to 3 m to ?80 °C was associated with a marked increase in vascular resistance after thawing, and none of such frozen kidneys functioned after transplantation. They showed immediately after revascularization severe impairment of the circulation, and vascular damage was observed by light microscopy. The use of 5 m glycerol for cryoprotection attenuated this rise in vascular resistance and reduced the release of the endocellular enzyme, lactate dehydrogenase after thawing, indicating less cellular damage although no kidneys functioned after grafting.It is suggested that the mechanical effect of interstitial and intravascular ice formation is a major factor in damage to intact organs during freezing, and that further injury is produced by incomplete removal of the cryoprotectant before transplantation.     

Cryoprotection in hearts as measured by CPK

Rat hearts were treated with a cryoprotectant solution composed of glycerol and DMSO in concentrations ranging from 10–25% $\text{v}\text{v}$, and then frozen in liquid nitrogen. Creatine phosphokinase activity was then measured spectrophotometrically and activities were compared with activities of frozen-untreated hearts, unfrozen-treated hearts, and unfrozen-untreated hearts. Independently freezing or treating increased enzyme activity, but when hearts were treated and then frozen, activity diminished below that of the unfrozen-untreated group. Evidence suggests (1) DMSO-glycerol solution either has a toxic effect that increases with concentration and freezing additionally aggravates this effect, or the concentrations of cryoprotectants used do not adequately protect the tissue during freezing which causes damage to the contractile mechanism; and (2) the anoxic state of the tissue causes depletion of ATP and creatine phosphate such that creatine phosphokinase is inactive or not able to function.     

Freezing preservation of the mammalian cardiac explant. II. Comparing the protective effect of glycerol and polyethylene glycol.

We compared the cryoprotective ability of glycerol and polyethylene glycol (PEG) during freezing. Isolated rat hearts were flushed with one of three cardioplegic solutions (CP-14, CP-15, and CP-16), frozen at -1.4 degrees C, and reperfused after thawing to assess function. After 3 h freezing, cardiac output (CO) in CP-14-flushed hearts recovered to 58.1% of control. CP-16 (CP-14 with 5% PEG) improved CO to 77.5%. Five hours of freezing abolished recovery in CP-14 hearts, but CP-15 (CP-14 with 50 mM glycerol) and CP-16 hearts produced 40.0 and 49.0% CO, respectively. With 6 h freezing, CP-15 hearts did not recover, whereas CP-16 hearts recovered 37.5% CO. In CP-14 hearts frozen for 3 h, 37.4% of the tissue water was ice that increased to 44.7% with 5 h freezing. CP-15 and CP-16 hearts had 34.4 and 30.9% tissue ice, respectively, after 5 h freezing. Tissue water contents in CP-14 and CP-15 hearts (3.83 to 3.96 g H2O/g dry) were 14 to 24% higher than that in CP-16 hearts. Six hours of freezing elevated AMP and ADP contents and reduced ATP levels in CP-15 and CP-16 hearts. Total adenine nucleotide (TAN) content of CP-15 hearts was 72% of control, while that of CP-16 hearts was normal. In conclusion, both glycerol and PEG offered cryoprotection by reducing tissue ice formation. PEG was superior by reducing tissue ice content further via dehydration and by better preserving TAN content.     

Seleno-DL-methionine and the protection of human platelets against freezing injury

The effect of seleno-DL-methionine, which has antioxidative properties, on the recovery of human platelets after freezing with 0.5 mol/liter glycerol or 0.7 mol/liter (5% v/v) dimethyl sulfoxide was investigated. Incubation of platelets with 2 mumol/liter seleno-DL-methionine for 30 min at room temperature before equilibration with the protective additives improved the post-thaw uptake of 5-hydroxytryptamine and the percentage reversal in the hypotonic stress test. The effect was small, but in view of the ability of seleno-DL-methionine to inhibit lipid peroxidation in membranes, the results suggest that oxidative damage is implicated in freezing injury. The dimethyl sulfoxide protocol apparently afforded greater protection to the platelets than the glycerol protocol. But, if the platelets were incubated for 24 hr at 37 degrees C after thawing, there was a marked improvement in the response of cells in the hypotonic stress test, particularly in the samples frozen with glycerol, and there was no longer any difference between the two additives. There was, however, a concomitant loss of almost half the number of cells in the thawed suspensions during the prolonged incubation at 37 degrees C.     

The process of freezing and the mechanism of damage during hepatic cryosurgery

Experiments were performed to correlate the structures of liver tissue frozen during cryosurgery, liver frozen at various constant cooling rates, and unfrozen, dried normal liver. The results show that during freezing of tissue ice forms and propagates along the vascular system, expanding during freezing at low cooling rates. This expansion occurs over most of the region frozen during cryosurgery and may be one of the mechanisms of damage to tissue during cryosurgery.     

Factors contributing to inactivation of isolated thylakoid membranes during freezing in the presence of variable amounts of glucose and NaCl.

During freezing of isolated spinach thylakoids in sugar/salt solutions, the two solutes affected membrane survival in opposite ways: membrane damage due to increased electrolyte concentration can be prevented by sugar. Calculation of the final concentrations of NaCl or glucose reached in the residual unfrozen portion of the system revealed that the effects of the solutes on membrane activity can be explained in part by colligative action. In addition, the fraction of the residual liquid in the frozen system contributes to membrane injury. During severe freezing in the presence of very low initial solute concentrations, membrane damage drastically increased with a decrease in the volume of the unfrozen solution. Freezing injury under these conditions is likely to be due to mechanical damage by the ice crystals that occupy a very high fraction of the frozen system. At higher starting concentrations of sugar plus salt, membrane damage increased with an increase in the amount of the residual unfrozen liquid. Thylakoid inactivation at these higher initial solute concentrations can be largely attributed to dilution of the membrane fraction, as freezing damage at a given sugar/salt ratio decreased with increasing the thylakoid concentration in the sample. Moreover, membrane survival in the absence of freezing decreased with lowering the temperature, indicating that the temperature affected membrane damage not only via alterations related to the ice formation. From the data it was evident that damage of thylakoid membranes was determined by various individual factors, such as the amount of ice formed, the final concentrations of solutes and membranes in the residual unfrozen solution, the final volume of this fraction, the temperature and the freezing time. The relative contribution of these factors depended on the experimental conditions, mainly the sugar/salt ratio, the initial solute concentrations, and the freezing temperature.     

In vitro maturation and fertilization of goat oocytes frozen at the germinal vesicle stage

The ability of frozen immature goat oocytes to undergo in vitro maturation (IVM) and fertilization (IVF) was investigated. Fully grown germinal vesicle stage (GV-stage) goat oocytes were submitted to different variables of cryopreservation: 1) exposure to propanediol before maturation but without freezing to detect the level of damage attributable to propanediol alone, 2) removal of cumulus cells to mimic damage attributable to osmotic stress during cryoprotectant exposure or freezing procedure, and 3) rapid freezing with propanediol. Maturation and fertilization rates were 82.1, 71, 65.3 and 23.7% and 71.2, 40, 58.4 and 23.1% for control, exposed, denuded and frozen oocytes, respectively. These results indicate that freezing sticto sensu (i.e., cooling and warming phases) have detrimental effects on IVM of GV-stage oocytes, whereas the reduced IVF rates of post-thaw matured oocytes are imputable to a cryoprotectant effect.     

Evaluation and preservation of vascular architectures in decellularized whole rat kidneys

Organ transplantation is the gold standard treatment for end-stage organ failure. Due to the severe shortage of transplantable organs, only a tiny fraction of patients may receive timely organ transplantation every year. Decellularization-recellularization technology using allogeneic and xenogeneic organs is currently conceived to be a promising solution to generate functionally transplantable organs in vitro. This approach, however, still faces tremendous technological challenges, one of them being the ability to evaluate and preserve the integrity of vascular architectures upon decellularization and cryostorage of the whole organ matrices so that the off-the-shelf organ grafts are available on demand for clinical applications. In the present study, we report a Micro-CT imaging method for evaluating the integrity of vasculature of the decellularized whole organ scaffolds with/without freezing/thawing. The method uses radiopaque Microfil perfusion and x-ray fluoroscopy to acquire high-resolution angiography of the organ matrix. The whole rat kidney is decellularized using a new multistep perfusion protocol with the combined use of Triton X-100 and DNase. The decellularized kidney matrix is then cryopreserved after the pretreatment with different cryoprotectant solutions. The reconstructed tomographic images from Micro-CT confirm various structural alterations in the vasculature of the whole decellularized kidney matrix with/without frozen storage. The freezing damage to the vascular architectures can be reduced by perfusing cryoprotectant solutions into the whole kidney matrix. Ice-free cryopreservation with the vitrification solution VS83 can successfully preserve the integrity of the whole kidney matrix's vasculature after frozen storage.     

An investigation of the frost hardiness of grapevine (Vitis vinifera) during bud break

The aim of this investigation was to assess ice nucleation and frost resistance of two varieties of grapevine (Siegrebbe and Madeleine Angevine) during bud burst under radiative freezing conditions analogous to those during Spring in the UK. During bud burst, grapevines were almost entirely resistant to freezing during frosts of less than -3°C by virtue of their ability to supercool. The risk of frost damage increased significantly as bud development progressed, and once buds had passed growth stage DS3 they became more sensitive to freezing and freezing damage was more extensive. The two varieties did not differ in frost resistance but, because of its earlier developing habit, variety Siegrebbe could be more prone to frost damage in the field. Buds were more prone to damage after freezing once bud burst had commenced and the damage could not be reversed by acclimating plants for periods of 7 to 21 days at 4°C in an 8 h photoperiod. Such acclimation appeared to predispose frozen buds to more extensive damage.     

Ultrastructure-function correlative studies for cardiac cryopreservation. V. Absence of a correlation between electrolyte toxicity and cryoinjury in the slowly frozen,cryoprotected rat heart

Hearts were frozen to ?17 °C in the initial presence of 2.1 m DMSO. Attempts were made to prevent or minimize the consequences of an osmotic shock based on Lovelock's classical hypothesis of freezing injury. Substitution of mannitol or potassium for NaCl before freezing did not improve the results, nor did perfusion of thawed hearts with hyperosmotic perfusate. It was found that freezing and thawing resulted in a significant attenuation of coronary flow and that, as a result of this, DMSO was apparently retained within the heart after thawing. DMSO was also difficult to remove at 30 °C in the absence of prior freezing and caused a significant drop in coronary flow upon institution of DMSO washout with balanced salt solution. The blanching of freezing and thawing was also seen, in milder form, in nonfrozen hearts. For both frozen-thawed and nonfrozen hearts, the blanching was associated with DMSO washout with balanced salt solution. Flow was improved by perfusion with hyperosmotic perfusate in both nonfrozen and in frozen-thawed hearts, but the improvement was largely temporary. Evidence from earlier studies indicates that electrolyte concentrations during freezing cannot be correlated with cardiac cryoinjury, in support of the present findings. It is suggested instead that cryoprotectant toxicity may be the chief agent of injury under the conditions studied.     

Acetaminophen in the post-ischemia reperfused myocardium

Acetaminophen was administered acutely at the onset of reperfusion after 20 min of low-flow, global myocardial ischemia in isolated, perfused guinea pig hearts (Langendorff) to evaluate its influence in the postischemia, reperfused myocardium. Similarly prepared hearts were treated with vehicle or with uric acid (another phenol for comparison). Functionally, acetaminophen-treated hearts (0.35 mM) achieved significantly greater recovery during reperfusion. For example, left ventricular developed pressures at 40 min reperfusion were 38 +/- 3, 27 +/- 3, and 20 +/- 2 in the presence of acetaminophen (P < 0.05, relative to the other two groups), vehicle, and uric acid, respectively. Coronary perfusion pressures and calculated coronary vascular resistances, in the acetaminophen-treated hearts, were significantly lower at the same time (e.g., coronary perfusion pressures in the three groups, respectively, were 40 +/- 2 [P < 0.05], 51 +/- 3, and 65 +/- 12 mm Hg). Under baseline, control conditions, creatine kinase ranged from 12-15 units/liter in the three groups. It increased to 35-40 units/liter (P < 0.05) during ischemia but was significantly reduced by acetaminophen during reperfusion (e.g., 5.3 +/- 0.8 units/liter at 40 min). Oxidant-mediated chemiluminescence in all three treatment groups during baseline conditions and ischemia was similar (i.e., approximately 1.5-2.0 min for peak luminescence to reach its half maximal value). It took significantly more time during reperfusion for the oxidation of luminol in the presence of acetaminophen (>20 min, P < 0.05) than in its absence (3-8 min in uric acid- and vehicle-treated hearts). These results suggest that administration of acetaminophen (0.35 mM), at the onset of reperfusion, provides anti-oxidant-mediated cardioprotection in the postischemia, reperfused myocardium.     

The effect of glycerol on aggregation and ultrastructure of human platelets

Cryopreservation of platelets depends on the use of cryoprotectants to reduce freezing damage. However, the cryoprotectants may in themselves be harmful, and it is important to determine the amount of damage caused by these compounds. Platelets were incubated at 37 °C in plasma containing 0, 0.5 and 1.0 mol/liter glycerol. The aggregation response to 10 and 5 μmol/liter ADP was determined after 2, 15, 30, 60, and 120 min of incubation. Samples were prepared for electron microscopy after 30 min at 37 °C. Glycerol at a concentration of 0.5 mol/liter had no effect on the extent of aggregation, whereas 1.0 mol/liter glycerol caused a progressive decline in the response. However, platelet ultrastructure appeared to be undisturbed by 1.0 mol/liter glycerol. The results demonstrated a lack of toxicity of 0.5 mol/liter glycerol and support the use of glycerol at concentrations less than 1.0 mol/liter for cryopreservation.     

Analysis of “solution effects” injury: Rabbit renal cortex frozen in the presence of dimethyl sulfoxide

Slices of rabbit renal cortex were frozen in 0.64 or 1.92 M dimethyl sulfoxide (Me₂SO) to various subzero temperatures, thawed, and assayed for viability. Salt and Me₂SO concentrations were calculated and correlated with the injury taking place during freezing. In separate experiments, slices were treated with NaCl or Me₂SO in concentrations sufficient to simulate the exposure brought about as a result of freezing. The effects of these treatments on cortical viability were compared with the results of freezing to equivalent concentrations of either NaCl or Me₂SO. The results show that whereas slices will tolerate exposure to at least six times the isotonic concentration of NaCl at 0 °C, they are unable to tolerate even three times the isotonic salt concentration when frozen in 1.92 M Me₂SO. They can, however, tolerate 3 × NaCl when frozen in 0.64 M Me₂SO. Freezing damage did not depend upon the amount of ice formed per se, since slices frozen in the low concentration of Me₂SO tolerated removal of about 75% of the initial fluid content of the system, whereas slices frozen in 1.92 M Me₂SO did not tolerate an identical removal of unfrozen solution. It was found that treatment of slices with high concentrations of Me₂SO at subzero temperatures in accordance with Elford's application (14) of Farrant's method (20) produced damage which correlated approximately with the damage observed when the same concentrations of Me₂SO were produced by freezing. It is concluded that most of the damage caused by freezing in 1.92 M Me₂SO is produced either directly or indirectly by Me₂SO. Possible mechanisms for this injury are discussed.     

Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing

Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze-substitution and freeze-quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.     

A mathematical model for the freezing process in biological tissue

A mathematical model has been developed to study the process of freezing in biological organs. The model consists of a repetitive unit structure comprising a cylinder of tissue with an axial blood vessel (Krogh cylinder) and it is analysed by the methods of irreversible thermodynamics. The mathematical simulation of the freezing process in liver tissue compares remarkably well with experimental data on the structure of tissue frozen under controlled thermal conditions and the response of liver cells to changes in cooling rate. The study also supports the proposal that the damage mechanism responsible for the lack of success in attempts to preserve tissue in a frozen state, under conditions in which cells in suspension survive freezing, is direct mechanical damage caused by the formation of ice in the vascular system.     

Freezing damage of bovine erythrocytes: Simulation using glycerol concentration changes at subzero temperatures

When a cell is frozen and thawed, it is exposed to (i) lowered temperature, (ii) increased solute concentration during freezing, and (iii) decreased solute concentration during thawing. Without actually freezing the cells, an attempt has been made to simulate physical-chemical changes to which bovine erythrocytes are exposed when frozen and thawed in glycerol solutions. Experimentally, the study consisted of suspending erythrocytes in 1, 2, or 3 glycerol at 20 °C for various times and then exposing them to each of several dilution sequences. The dilution sequences were: (i) transfer from the initial glycerol concentration at 20 °C into the same concentration at −5 °C, (ii) transfer into an increased glycerol concentration at 20 °C, (iii) transfer into an increased followed by a decreased glycerol concentration at 20 °C, (iv) transfer into an increased glycerol concentration at −5 °C, and (v) transfer into an increased followed by a decreased glycerol concentration at −5 °C. This last sequence is analogous to the exposure that cells undergo at subzero temperatures to increased solute concentration during freezing and decreased solute concentration during thawing. This dilution sequence yielded a survival pattern very similar to that obtained when bovine erythrocytes are frozen and thawed, and thus does appear to mimic freezing damage. It is concluded that a major factor in freezing damage is the extent to which a cell must shrink or swell to achieve osmotic equilibrium at subzero temperatures in partially frozen or thawed solutions.     

Cryopreservation of immature embryos of Theobroma cacao

Immature, white zygotic embryos of Theobroma cacao L. (cacao) retained the ability to produce callus and to undergo somatic embryogenesis after slow hydrated freezing and desiccated fast freezing in liquid nitrogen. The highest rate of somatic embryogenesis occurred in embryos which were precultured on a medium containing 3% sucrose, frozen slowly with cryoprotectants before exposure to liquid nitrogen, and recovered on a medium containing 3 mg/liter NAA. Embryos precultured on media containing sucrose increasing to 21% had a higher rate of survival but were less embryogenic after freezing. These results suggest that immature embryos might be used for long-term germplasm storage of T. cacao germplasm.     

Evidence for an early free radical-mediated reperfusion injury in frostbite

Frostbite is characterized by acute tissue injury induced by freezing and thawing. Initial complete ischemia is followed by reperfusion and later, tissue necrosis. These vascular events support the hypothesis that free radical-mediated reperfusion injury at thawing might contribute to tissue necrosis after frostbite in a manner similar to that seen after normothermic ischemia. To test this hypothesis, rabbit ears were frozen at -21 degrees C for 30, 60, 90, or 120 s and rewarmed at room temperature (22 degrees C). Rabbits were treated "blindly" with saline alone, highly purified, pharmaceutical grade superoxide dismutase (SOD), allopurinol, or deferoxamine. The area of ear necrosis was determined 3 weeks after frostbite by "blinded" morphometry. The administration of SOD at the time of thawing significantly improved viability in ears frozen for 60 and 90 s, but not in those frozen for 30 or 120 s. Deferoxamine also improved viability in ears frozen for 60 s. Allopurinol did not significantly affect ear survival. Electron micrographs showed the appearance of severe endothelial cell injury beginning during freezing and extending through early reperfusion. Later, neutrophil adhesion, erythrocyte aggregation, and microvascular stasis were seen. These findings suggest that free radical-mediated reperfusion injury has a role in frostbite, and quantitate the proportion of the injury that is due to this mechanism.     

Functional status of cryopreserved rat liver mitochondria

Mitochondria from the rat liver have been frozen down to -196 degrees C under protection of dimethylsulfoxide, after which they were subjected to periodical increase and decrease in temperature (cycling) in the ranges of -105 degrees C to -196 degrees C or -135 degrees to -196 degrees C. There have been observed some non-lethal damages of mitochondria which were revealed in the decrease in the respiratory control and in the ADP/O ratio after the cycling. The damage rate increased with the increase of the number of temperature decrease-increase cycles. The damages were also greater in the case when the upper limit of cycling temperature was higher than the glass transition temperature of the freezing medium (Tg = -126 degrees C). The non-lethal damages of mitochondria are explained by the influence of electric fields, appearing in the frozen sample during the temperature increase or decrease.