Cold Storage Solution Research Articles

移植心の心機能回復時間に関する実験的研究 単純浸漬保存心における保存時間別検討

移植心の心機能回復時間に関する実験的研究 単純浸漬保存心における保存時間別検討

PROLONGED PANCREAS PRESERVATION USING A SIMPLIFIED UW SOLUTION CONTAINING POLYETHYLENE GLYCOL

The University of Wisconsin solution is considered the most effective universal flush and cold storage solution to date, and is now being widely applied clinically in organ transplantation. The results of this study show that Cardiosol, a modified cardioplegic solution containing 5% polyethylene glycol (PEG20M, MW 17,000 daltons), is significantly superior to UW (P less than 0.001) in the flush perfusion and hypothermic storage of pancreases for more than 36 hr prior to transplantation into streptozotocin-induced diabetic rats. When the pancreases were stored in Cardiosol, the 1-week survival rate was 7 of 10 (70%) after 24 hr of preservation; 7 of 12 (58%) after 36 hr; and 3 of 10 (30%) after 48 hr. In contrast, when the pancreases were stored in UW solution, the 1-week survival rate was 8 of 12 (67%) after 24 hr of preservation; after 36 hr, no animal survived (0 of 14). Intravenous glucose tolerance test K-values (decline in glucose concentration, percentage per minute) were normal in both groups receiving 24-hr-preserved pancreases, ranging from 2.60 to 4.16. Of interest, the peak insulin response 1 min following intravenous glucose was significantly higher (P less than 0.01) in the Cardiosol-preserved organs (303 +/- 29.8 microU/ml) (+/- SEM) than in the glands preserved in UW solution (112 +/- 47.9 microU/ml). We conclude that Cardiosol allows prolonged whole organ pancreas preservation in the rat transplant model.

Kupffer cell activation and endothelial cell damage after storage of rat livers: Effects of reperfusion

Reperfusion injury characterized by loss of endothelial cell viability occurs after cold ischemic storage of livers for transplantation surgery. Here, ultrastructural changes in stored rat livers were examined by scanning and transmission electron microscopy. With increasing times of storage in Euro-Collins solution (4 to 24 hr) followed by 15 min of reperfusion at 37 degrees C, a sequence of structural alterations was observed involving endothelial and Kupffer cells. Widening of endothelial fenestrations occurred after 4 hr and progressed over 8 to 24 hr to retraction of cellular processes, ball-like rounding, sinusoidal denudation and ultrastructural derangements consistent with loss of cell viability. Kupffer cells exhibited progressive rounding, ruffling of the cell surface, polarization, appearance of wormlike densities, vacuolization and degranulation over a similar time course. By contrast, the structures of parenchymal and fat-storing cells were relatively undisturbed by cold storage and reperfusion. Alterations to endothelial and Kupffer cells were also studied as a function of time of reperfusion. After 24 hr of storage, endothelial cells showed retraction of cytoplasm before reperfusion that progressed quickly to loss of viability and denudation during reperfusion. Kupffer cell activation (ruffling, degranulation) during reperfusion was slower and occurred after deterioration of endothelial cells. Livers stored in Euro-Collins solution were also compared with livers stored in University of Wisconsin cold storage solution, an improved preservation medium for transplantation. University of Wisconsin solution provided better preservation of endothelial structure and markedly reduced parenchymal cell blebbing and swelling before reperfusion. University of Wisconsin solution also reduced Kupffer cell activation and release of lysosomal enzymes. In conclusion, endothelial cell deterioration followed by Kupffer cell activation occurred after increasing times of cold ischemic storage and reperfusion of rat livers. Both changes may contribute to the pathophysiology of graft failure caused by reperfusion-mediated storage injury.

Endothelial cell toxicity of solid-organ preservation solutions

Endothelial cell damage caused by myocardial cardioplegic solutions (Bretschneider HTK and St. Thomas' Hospital No. 2) or renal and hepatic cold storage solutions (modified Collins and University of Wisconsin solution) was assessed in monolayer cultures of adult human venous endothelial cells at 4 ° to 10 °C with phase-contrast microscopy. St. Thomas' Hospital solution caused the cells to contract, resulting in disruption of monolayer integrity and opening of intercellular gaps, and resulted in a 24-hour postexposure survival of 51.0% ± 2.4%. Bretschneider HTK solution altered cellular morphology less and produced the best postexposure survival (80.2% ± 2.6%; p < 0.001). Although morphology was altered the least with University of Wisconsin solution, postex-posure survival with this solution, which was similar to that with modified Collins solution, was superior to that with St. Thomas' ( p < 0.01) but inferior to that with Bretschneider HTK ( p < 0.05). The superior protection provided by Bretschneider HTK was due to its additives histidine, tryptophan, and KH-2-oxygluterate ( p < 0.005), and to its low chloride content ( p < 0.005). Furthermore, modifying St. Thomas' solution by decreasing its chloride content improved cell survival to 71.2% ± 2.3% ( p < 0.001). Normothermic (37 °C) exposure to Bretschneider HTK, modified Collins, and University of Wisconsin solution was cytotoxic, whereas normothermic exposure to St. Thomas' cardioplegia was not. In conclusion, the preservation solution that is the least harmful to endothelial cells at hypothermia is Bretschneider HTK cardioplegic solution.

Intraoperative Protektion freier mikrochirurgischer Transferlappen: „Cold Storage” in HTK-Lösung*

There is a rule of thumb that an ischaemia time of 6 hours should not be exceeded when preserving a free flap. Microsurgical reconstructions sometimes require a long operation time and a long tolerance of ischaemia of the free flap. Using Bretschneider's HTK solution for the intraoperative cold storage of free flaps with a temperature of 5-7 degrees C, an ischaemia time of 12 hours should not affect the free flap, and hence the time limit of the ischaemic tolerance looses its importance. Based on Bretschneider's research results of cardioplegia and of the ischaemic tolerance of the kidney and the liver, we chose the HTK solution for the intraoperative protection of free microsurgical flaps.

KIDNEY PRESERVATION WITH THE UW AND EURO-COLLINS SOLUTIONS

This is a preliminary report of a European Multicenter Trial of the efficacy and safety of the UW solution in kidney preservation. The results obtained with the UW solution are compared with those obtained with Euro-Collins solution in a prospectively randomized study. To date 257 patients have been evaluated, with 128 receiving kidneys preserved in UW solutions and 129 receiving kidneys preserved in Euro-Collins solutions. Demographic characteristics of donors and recipients were identical in both groups. Median (and range) preservation times were similar (24 hr in EC and 24 hr in UW). Maximum preservation time in each group was about 48 hr. The results show that the UW solution is a safe preservation solution for kidneys, with postoperative renal functions, at least, equivalent to those seen in patients transplanted with kidneys preserved in EC solution. In this preliminary analysis of 257 kidneys, use of the UW solution resulted in a more rapid reduction in postoperative serum creatinine, higher creatinine clearance rate, and less postoperative dialysis (21% vs. 31%) when compared with kidneys preserved in EC solution. This study indicates that the UW solution is an effective preservation medium for clinical kidney transplantation. It supports the use of UW solution as a general flushout and cold storage solution for all intraabdominal organs used for transplantation.

Hypothermic preservation of hepatocytes: I. Role of cell swelling

Hepatocytes from isolated rat livers were hypothermically incubated (5 °C) in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). The incubation solution was either a tissue culture medium (L-15), an organ preservation perfusate (UW gluconate), or a simple cold-storage solution used for organ preservation (UW lactobionate). Hepatocyte viability was assessed from the release of lactate dehydrogenase (LDH) into the incubation medium. Cell swelling (due to the uptake of water) was also measured. Within 24 hr, hepatocytes hypothermically stored in each of the three incubation solutions became swollen (30 to 40% water gain) and lost a significant amount of LDH (as much as 60%). The addition of polyethylene glycol (PEG; relative molecular mass 8000; 5 g%) to the solutions suppressed cell swelling and allowed the incubated hepatocytes to remain relatively well preserved (30% LDH release) for as long as 120 hr. Adding either dextran (relative molecular mass 10,000 to 78,000; 5 g%) or saccharides (100 mmol/liter) instead of PEG neither prevented cell swelling nor prevented the cells from dying. The results of this study suggest (i) there is a direct correlation ( r = 0.873) between hypothermia-induced cell swelling and cell death (i.e., the suppression of cell swelling prevents cell death); (ii) the mechanism by which PEG prevents cell swelling (and thus maintains cell viability) is not related to the osmotic or oncotic properties of the molecule but instead is apparently related to some unknown interaction between PEG and the cell, an interaction that provides stability during hypothermie incubation; and (iii) hypothermia-induced cell swelling must be prevented if isolated hepatocytes are to be used as a model for studying the mechanism by which cell damage occurs during hypothermic organ preservation. By eliminating cell death due to cell swelling, the biochemical mechanisms of cell death can be studied.

Improvement of liver preservation quality with UW solution by chlorpromazine pretreatment of the donor in an experimental model.

We investigated the effect of donor pretreatment with chlorpromazine (CPZ), in rabbit livers cold-stored in University of Wisconsin (UW) cold storage solution for 48 hr. Three groups of livers were investigated: livers flushed with Perfadex and immediately thereafter reperfused on an isolated circuit (controls), and livers cold stored in UW solution for 48 hr, with or without donor pretreatment with CPZ, 3 mg/kg. After preservation, reperfusion was performed in vitro, using an isolated circuit (IPL). The reperfusion medium consisted of an oxygenated Krebs-Henseleit bicarbonate solution supplemented with 5 mM glucose, 50 mg/L of streptomycin and penicillin G, and 3.5% Dextran 60 for oncotic support. Livers that were not pretreated with CPZ produced 5.3 +/- 1.2 ml bile/100 g (mean +/- SD) during 2 hr of IPL reperfusion. CPZ donor pretreatment significantly improved the bile flow to 17.1 +/- 6.9 ml (P less than 0.01, Wilcoxon). This figure was not different from that in control livers without a storage period (18.3 +/- 3.8 ml). Alanine aspartate aminotransferase (ASAT) released into the perfusate was measured, and levels were increasing during 2 hr of reperfusion. ASAT values were moderately increased in the preserved groups compared with controls (P less than 0.01), with no discernible differences between livers with and without CPZ pretreatment. It is concluded that CPZ pretreatment of the donor improves preservation quality, as evidenced by improved bile formation. The present results suggest that 48 hr cold storage in UW solution may be safe for clinical preservation, if donors are pretreated with chlorpromazine.

Reperfusion injury to endothelial cells following cold ischemic storage of rat livers.

Storage of donor livers in Euro-Collins solution for human transplantation surgery is limited to about 8 hr. Here, tissue damage to isolated rat livers stored under the same conditions as human livers was characterized following reperfusion. The purpose of this work was to determine the importance of nutritional status on injury due to cold storage and reperfusion, to establish whether lethal injury occurs during cold storage or only after reperfusion, and to identify the cell types most vulnerable to damage. Rat livers were perfused with Krebs-Henseleit-bicarbonate buffer, stored 8 to 48 hr in Euro-Collins solution and reperfused with warm, oxygenated Krebs-Henseleit buffer for 15 min. Nuclear trypan blue uptake and lactate dehydrogenase release were used as indices of cell death. After 8 hr of cold storage and reperfusion, little loss of parenchymal or nonparenchymal viability occurred. After 24 or 48 hr, virtually all parenchymal cells remained viable. However, severe damage to nonparenchymal cells was observed, and about 40% of nonparenchymal cells were trypan blue positive. Nutritional status (fed vs. fasted) did not affect the extent of cell damage. Nonparenchymal cell killing was accompanied by cellular rounding, nuclear pyknosis and protrusion of cells into sinusoidal lumens. Scanning electron micrographs demonstrated denudation of the sinusoidal lining. Rounding and pyknosis were not observed in 24-hr-stored livers which were not reperfused, and trypan blue uptake did not occur in stored livers infused with cold, anoxic Euro-Collins solution. Based on cytochemistry and electron microscopy, lethal cell injury occurred predominantly to endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Newly Approved Organ Preservation Solution Puts Longer Life in Liver Grafts

AN ORGAN preservation medium, recently approved by the Food and Drug Administration for clinical use in the United States, is making liver transplantation a more leisurely procedure, according to several reports at the recent meeting of the American Society of Transplant Surgeons in Chicago, Ill. The University of Wisconsin (UW) cold storage solution, developed by surgeon Folkert O. Belzer, MD, and biochemist James Southard, PhD, contains a combination of metabolic and preservative agents concocted by the two over a period of several years. It has been licensed by the university to E.I DuPont de Nemours &amp; Company, Wilmington, Del, and is marketed under the trade name ViaSpan. The solution has been evaluated in clinical trials in the United States and Europe since 1986, which, almost uniformly, have demonstrated that it has doubled the cold storage time for harvested livers. More recently, studies have been directed at establishing whether organs stored

The effect of type of preservation solution and hemodilution of the recipient on postischemic erythrocyte trapping in kidney grafts. An experimental study in the rat.

The role of recipient hemodilution for postischemic renal medullary red cell trapping was investigated after different periods of cold storage in a conventional cold storage solution (Sacks'). At all cold storage times investigated (4, 12, 24, and 48 hr) medullary red cell trapping was reduced by isovolemic hemodilution, with about 50% reduction of recipient hematocrit. Trapping was also reduced when a modification of a new preservation solution (University of Wisconsin solution [UW]) was used and compared with flush-out and storage in a standard preservation solution (Sacks'). The combination of hemodilution and preservation in modified UW solution had additional capacity to reduce medullary red cell trapping. Thus, even after 48 hr of cold storage, only a moderate trapping was observed. The results also indicate that measurements of medullary red cell trapping offers an accurate method of grading postischemic renal damage.

Organ flush out solutions and cold storage preservation solutions: effect on organ cooling and post ischemic erythrocyte trapping in kidney grafts. An experimental study in the rat.

Three flush out and cold storage solutions were tested for flow rate during flush out, ability to decrease organ temperature, degree of tissue edema and degree of preservation damage of the kidney as measured by the amount of post transplantation erythrocyte trapping in the renal medulla. The solutions tested were a modification of a new perfusion solution (University of Wisconsin; mUW), a standard preservation solution (Sacks') and an extracellular histidine solution (Frödin-Wolgast; FW). The flow rate was significantly higher for FW compared to mUW and Sacks' and consequently a more rapid decrease of organ temperature was achieved. The results also show that edema formation as well as the amount of erythrocyte trapping was lower in kidneys stored in mUW, indicating that mUW is better cold storage solution than Sacks' or FW.

Use of UW Solution in Pancreas Transplantation

We have recently reported successful 72-h preservation of the canine pancreas with a new cold-storage solution developed at the University of Wisconsin (UW solution). Over 10 mo, we performed 11 combined pancreas-kidney and 4 isolated-pancreas transplants with this solution. In situ cooling of the donor pancreas was performed with 1000 ml of UW solution followed by ex vivo perfusion with an additional 250-500 ml. Graft preservation times ranged from 3 to 19 h (mean 10.2 h). Pancreas transplants were vascularized whole-organ grafts with pancreaticoduodenocystostomy. Early graft function was excellent as assessed by immediate insulin independence, high urinary amylase and low serum amylase levels, and a technetium perfusion index indicating good pancreatic blood flow. There were no episodes of primary nonfunction, graft pancreatitis, or vascular thrombosis. Actuarial patient and graft survival at 1 mo was 92.9%. We conclude that UW solution provides excellent early graft function for up to 19 h of cold storage. Based on previously reported data on its efficacy in liver and kidney preservation, UW solution seems ideally suited as a universal intra-aortic flush and cold-storage solution.

PRESERVATION OF THE CANINE LIVER FOR 24–48 HOURS USING SIMPLE COLD STORAGE WITH UW SOLUTION

The results of a series of 29 orthotopic liver transplants in the dog are described. The livers were preserved in a new cold storage fluid, UW solution, and were successfully transplanted after periods of storage of 24, 30, 36, and 48 hr. All six animals transplanted after 24 hr survived beyond 5 days after transplantation and had excellent graft function. Four of six survived for at least 5 days after 30 hr of cold storage, and five of five after 36 hr. Five of six consecutive dogs that received transplants that had been cold-stored for 48 hr survived for 5 or more days. This solution represents a substantial advance over all existing cold storage solutions for liver preservation.

A comparison of cold storage solutions for hepatic preservation using the isolated perfused rabbit liver

Rabbit livers were stored cold for periods of 6 or 24 hr and tested using the isolated perfused liver model. Five solutions were tested: Eurocollins (EC), Ross and Marshall's hypertonic citrate (HC), modified plasma protein fraction (Cambridge PPF), Ringer lactate, and the recently developed “University of Wisconsin” (UW) solution. After storage livers were perfused with an erythrocyte-free oxygenated Krebs-Henseleit solution containing 4% bovine serum albumin at 38 °C for 2 hr. Bile production proved to be the most sensitive index of liver function for discriminating between the various storage solutions and the different preservation times. After 6 hr of cold storage, bile production was similar to control liver bile production (9.8 ± 2.4 ml/2 hr/100 g) in livers stored in HC (8.8 ± 2 ml), PPF (9.9 ± 2.2 ml), and UW (10.3 ± 1.9 ml); it was slightly depressed in EC (6.7 ± 2.5 ml, P = 0.06), and markedly depressed in Ringer lactate (4.3 ± 0.8 ml, P < 0.05). After 24 hr of cold storage bile production in UW-stored livers was near normal (9.3 ± 0.7 ml) but significantly depressed (3.5–6.2 ml) in all other solutions tested. Release of enzymes into the normothermic perfusate was also measured (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). In this small series the differences between cold storage solutions did not always reach statistical significance although the trend was for less enzyme release in livers stored in UW solution. This technique permits rapid assessment and refinement of new storage methods and new solutions for liver preservation prior to testing in a large animal transplant model. The results suggest that UW solution is superior to other preservation solutions and would permit successful 24-hr storage of livers.

Successful 72-hour cold storage of dog kidneys with UW solution.

Effects of three cold-storage solutions on kidney function in dogs were examined with the isolated perfused (IPK) kidney model and the autotransplant model. EuroCollins' (EC) solution, phosphate-buffered sucrose solution, and a new solution developed at the University of Wisconsin (UW) were studied. Kidneys were cold-stored for 48 hr or 72 hr. With the IPK model, cold storage for 48 hr or 72 hr in each of the three solutions caused creatinine clearance to decrease by 80%-90%. More protein was excreted by kidneys stored for 48 hr in PBS solution than by kidneys stored in EC or UW solution; protein excretion after 72 hr of storage was similar for kidneys stored in EC or UW solution. Sodium reabsorption decreased after 48 hr or 72 hr of storage, but was higher in kidneys stored in UW solution (83% and 56%, respectively) than in EC solution (52% and 22%, respectively). With the autotransplant model, 40% of the kidneys were viable after 48-hr storage in PBS solution, but 80% viable when stored in EC solution and 100% were viable when stored in UW solution. All kidneys were viable when stored for 72 hr in UW solution; none were viable when stored for 72 hr in EC solution. These results suggest that UW solution effectively preserves kidneys for 72 hr. We previously reported successful 72-hr pancreas preservation. Recently UW solution was able to preserve canine livers for 30 hr. Thus, this single solution appears to be effective for preserving all intraabdominal organs and may simplify cold storage of organs for transplantation.

Cold storage of the rat heart for transplantation: Two types of solution required for optimal preservation

Two solutions, our cardioplegic solution and Collins' solution, were tested with regard to preservation of the heart under deep hypothermia before transplantation. The setup used was the isolated perfused working rat heart model and 4 hours of preservation at 0 degree C. The following three groups were prepared: Group 1: the heart was arrested with the cardioplegic solution (potassium: 20 mmol/L, sodium: 87 mmol/L) and then flushed with and stored in Collins' solution (potassium: 117 mmol/L, sodium: 10 mmol/L); Group 2: the heart was arrested with and stored in Collins' solution; and Group 3: the heart was arrested with and stored in the cardioplegic solution. The recovery of cardiac function was more satisfactory in Group 1 than in Groups 2 and 3. The increase in lactate was greater, and adenosine triphosphate and total adenine nucleotide were more depleted during storage in Group 2 than in Groups 1 and 3. In Group 3 myocardial sodium accumulation and potassium depletion during storage were greater than in Groups 1 and 2, and myocardial sodium and calcium overload after reperfusion were greater than in Group 1. Myocardial calcium overload after reperfusion in Group 2 was also greater than that in Group 1. These findings plus coronary vascular resistance analysis revealed that Collins' solution damages the heart during arrest procedures and that the cardioplegic solution is less effective for storage of the arrested heart under deep hypothermia. Therefore the heart should be first arrested with the cardioplegic solution and then flushed with and kept in Collins' solution for simple cold storage.

Development of a cold storage solution for pancreas preservation

Canine pancreas tissue slices were incubated at 5 °C for 24 hr in solutions containing different saccharides (raffinose, sucrose, mannitol, or glucose). At the end of incubation tissue water (TW expressed as kg H 2O kg dry wt) was determined as a measure of tissue edema. Tissue edema was greatest in slices stored in Eurocollins (EC) solution (TW = 4.96 ± 0.14) which contains glucose for osmotic pressure. The degree of edema was decreased by saccharides in proportion to their molecular mass: mannitol (MW = 180, TW = 3.84 ± 0.08), sucrose (MW = 348, TW = 3.54 ± 0.08), and raffinose (MW = 594, TW = 3.30 ± 0.07). Tissue edema was also greatest in slices incubated in solutions containing the smallest molecular mass anions: Cl − (TW = 4.02 ± 0.16), gluconate (TW = 3.69 ± 0.10), and lactobionate (TW = 3.28 ± 0.13). Cold storage of the intact pancreas in EC solution for 24 hr did not induce as much edema as in slices (TW = 2.88 ± 0.10). However, on isolated reperfusion at normothermia (37 °C) the pancreas became edematous (TW = 3.33 ± 0.12). Storage of the pancreas in a lactobionate-raffinose solution did not induce edema after 90 min of normothermic reperfusion. The suppression of tissue edema in the pancreas may be essential to obtaining long-term preservation (24–72 hr) of this organ which is currently limited to about 6–8 hr in EC solution. The newly developed lactobionate-raffinose solution appears to control tissue edema in both tissue slices and the intact-flushed out organ.

Effects of short-term hypothermic perfusion and cold storage on function of the isolated-perfused dog kidney

The isolated-perfused dog kidney was used as a model to measure the effects of short-term hypothermic preservation on renal function and metabolism. Kidneys were cold-stored in Collins' solution, hypotonic citrate, or phosphate-buffered sucrose for 4 and 24 hr, or were continuously perfused for 4 and 24 hr with a synthetic perfusate. Following preservation kidneys were perfused with an albumin-containing perfusate at 37 °C for 60 min for determination of renal function. The results indicate that many of the effects of short-term preservation on renal function in dog kidneys are similar to results reported for rat and rabbit kidneys. Cold storage for 4 hr resulted in a large decrease in GFR (57%), but only a small decrease in Na reabsorption (from 97 to 87%). Cold storage for 24 hr caused a further decline in renal function (GFR = 95% decrease, Na reabsorption = 49–64%). Results were similar for all cold storage solutions tested. Perfusion for 4 hr was less damaging to renal function than cold storage. The GFR decreased only 14% and urine formation and Na reabsorption were practically normal. After 24 hr of hypothermic perfusion, the GFR was reduced by 79%, urine flow was normal, and Na reabsorption was 78%. There were no obvious biochemical correlates (adenine nucleotides, tissue edema, or electrolyte concentration) with the loss of renal function during short-term preservation. The results suggest that the isolated-perfused dog kidney can be used to test the effects of preservation on renal function, and yields results similar to those obtained using small animal models. Dog kidneys preserved by the methods used in this study were fully viable on transplantation, suggesting that the initial renal injury, as revealed by the isolatedperfused system, did not compromise renal viability.

Pulmonary Sarcoidosis

The Food and Drug Administration (FDA) held an open public workshop in September 2011 to discuss the current state of science related to the effects of ischemia reperfusion injury (IRI) on outcomes in kidney transplantation. Topics included the development of IRI and delayed graft function (DGF), histology and biomarkers, donor factors, recipient factors, organ quality and organ preservation by means of cold storage solutions or machine perfusion. Various mechanisms of injury and maladaptive response to IRI were discussed as potential targets of intervention. Animal models evaluating specific pathophysiological pathways were presented, as were the limitations of extrapolating animal results to humans. Clinical trials of various drug products administered in the peri-transplant period were summarized; a few demonstrated early improvements in DGF, but none demonstrated an improvement in late graft function. Clinical trial design for IRI and DGF were also discussed.