Cold Storage In UW Research Articles

Cold flush after dynamic liver preservation protects against ischemic changes upon reperfusion - an experimental study.

Summary Ex vivo machine perfusion of the liver after cold storage has found to be most effective if combined with controlled oxygenated rewarming up to (sub)‐normothermia. On disconnection of the warm graft from the machine, most surgeons usually perform a cold flush of the organ as protection against the second warm ischemia incurred upon implantation. Experimental evidence, however, is lacking and protective effect of deep hypothermia has been challenged for limited periods of liver ischemia in other models. A first systematic test was carried out on porcine livers, excised 30 min after cardiac arrest, subjected to 18 h of cold storage in UW and then machine perfused for 90 min with Aqix‐RSI solution. During machine perfusion, livers were gradually rewarmed up to 20 °C. One group (n = 6) was then reflushed with 4 °C cold Belzer UW solution whereas the second group (n = 6) remained without cold flush. All livers were exposed to 45 min warm ischemia at room temperature to simulate the surgical implantation period. Organ function was evaluated in an established reperfusion model using diluted autologous blood. Cold reflush after disconnection from the machine resulted in a significant increase in bile production upon blood reperfusion, along with a significant reduction in transaminases release alanine aminotransferase and of the intramitochondrial enzyme glutamate dehydrogenase. Interestingly, free radical‐mediated lipid peroxidation was also found significantly lower after cold reflush. No differences between the groups could be evidenced concerning histological injury and recovery of hepatic energy metabolism (tissue content of adenosine triphosphate). Post‐machine preservation cold reflush seems to be beneficial in this particular setting, even if the organs are warmed up only to 20 °C, without notion of adverse effects, and should therefore be implemented in the protocol.

(420) - Cold Storage Stabilization of Gap Junctions Reduces Post Transplant Ischemia Reperfusion Injury

Organ procurement, cold storage and ischemia reperfusion injury (IRI) promote inflammation, which induces endothelial cell (EC) activation and dysfunction post transplantation. EC gap junctions (GJs) breakdown as a consequence of these injuries and play a key role in graft injury post transplantation. Here we explore the therapeutic potential of adding a novel gap junction (GJ) stabilizing peptide, ACT1, to UW preservation solution as a therapeutic agent to improve endothelial cell health. ACT1, is a small peptide Cx43 mimetic, which impairs the association of ZO-1 with Cx43 thus promotes GJ integrity. Mouse cardiac ECs (MCECs) were exposed to 6 hrs of cold storage in UW or UW/ACT1 solution followed by reperfusion to mimic clinical cold storage and reperfusion. Efficacy was determined by trans-endothelial electrical resistance (TEER), a measure of GJ function, cell viability assays, and ELISAs for pro-inflammatory cytokines. In-vivo, utilizing a cardiac allograft model, Balb/c donor hearts were stored in UW or UW/ACT1 for 6 hrs prior to transplantation into C57Bl/6 recipients. Grafts were harvested 48 hrs post-transplant and cardiac graft injury determined by serum cardiac troponin I and histological analyze. Graft inflammation was assessed by immunohistochemistry specific for neutrophil and macrophages. In-vitro studies demonstrate that UW/ACT1 solution significantly reduced EC injury and inflammation, as measured by TEER, cell viability and ELISA. In-vivo studies similarly showed that ACT1 pretreatment of the donor organ led to a reduction in IRI, as noted by reduced serum troponin and histological analysis. Subsequent analysis of neutrophil and macrophage infiltration showed pretreatment significantly reduced graft infiltrates. Taken together these novel findings propose a role for GJ in the pathogenesis of cold storage IRI, and further demonstrate that stabilization of GJ with a novel connexin 43 mimetic, ACT1, significantly inhibits post transplant IRI.

Improved Islet Yield From UW Solution Preserved Pancreas With Dimethyl Fumarate Supplementation.

Background: One of the major obstacles in clinical islet transplantation is the insufficient number of viable islets derived from preserved pancreas. During pancreas preservation and islet isolation, islets suffer from hypoxia and oxidant stress. In responses to oxidative stress, Nuclear factor erythroid-derived 2 factor (Nrf2) promotes expression of genes encoding antioxidant and detoxifying enzyme. Dimethyl fumarate, a novel Nrf2 activator, is also an intermediate in the citric acid cycle used by cells to produce energy in the form of ATP. We hypothesized that supplementation of preservation solution with DMF may improve islet yield by protecting islet during pancreas preservation. Method: 8-week old male Sprague Dawley rats were used as pancreas donors. The common bile duct was cannulated and the pancreas was removed en bloc with duodenum and spleen and preserved in UW (UW group) or UW + DMF, 50uM (DMF group) at 4°C for 24 hours after flushing by same preservation solution via aorta. After 24 hour cold storage, the ADP/ATP ratio and expression of antioxidant proteins in pancreatic tissue were evaluated. Pancreas was then digested by collagenase and isolated rat islets were examined by assessing islet yield and cure rates after transplantation to diabetic nude mice. Results: The islet yield (IEQ) in DMF group was significantly higher than UW group (969.3 ± 273.7 vs 412 ± 80.8; respectively, P<0.05). The cure rate after transplantation in DMF group (57.1%) was similar with UW group (66.7%, P>0.05). Likewise the percentage of viable and 8-OHDG positive cells in the isolated islets were comparable in the two groups. After 24 hour preservation, the ADP/ATP ratio of pancreas in DMF group was 4-fold lower than in the UW group. Enhanced expressions of HO-1 and NQO1 of pancreas were detected in DMF group. Conclusion: UW supplementation with DMF significantly improved the islet yield compared with UW cold storage alone. The islet function and viability were comparable in both groups. Thus DMF may protect islets and reduce islet loss during preservation and isolation procedure by enhancing the citric acid cycle and antioxidant defense system.

Normothermic Acellular Ex Vivo Liver Perfusion (NEVLP): Development of a New Method for the Storage, Assessment, and Repair of Marginal Liver Grafts

Livers retrieved after cardiac death (DCD) are often declined for transplantation because of the increased risk for graft failure and bile duct injury. The current cold static preservation technique is associated with a high risk of biliary complications and does not offer the opportunity to assess graft injury and function. We compared the novel normothermic acellular ex vivo liver perfusion (NEVLP) with conventional cold static organ preservation for livers retrieved after cardiac death. We implemented spectrophotometric imaging as tool for online monitoring of blood flow and oxygen transport to tissue during NEVLP to predict organ function post reperfusion. Methods: First, pig livers were perfused (n=5) for 12 hours in a cell free, oxygenated perfusion solution (STEEN), followed by 12hr perfusion with whole blood as a model of transplantation. ALT and histology were evaluated as parameters of the liver injury, urea synthesis, bile production and oxygen consumption were determined as marker of the liver function. In a second approach, pig livers either subjected to 1hr warm ischemia plus 12hrs cold storage in UW (n=5) or 1hr warm ischemia plus 4hr cold storage plus 8hr NEVLP. After 12hr organ preservation the livers were perfused for 8hr in a pig liver transplant model. Spectrophotometric imaging was used to monitor blood flow and oxygen supply to liver tissue and bile duct tissue. Results: 12hr normothermic acellular ex vivo liver perfusion followed by 12hr whole blood reperfusion was not associated with liver injury. Serum ALT (mean 27U/L) remained normal, and histology did not show any evidence of necrosis (< 1%). Bile production (mean 3cc/hr), oxygen consumption (400mmHg), and BUN synthesis (1.8mmol/l) were within normal limits. In a second approach, NEVLP was compared to cold static storage in a DCD model using 1hr warm ischemia and 12hr preservation. NEVLP was associated with significantly decreased serum AST (26U/L vs 285U/L), decreased necrosis (10% vs 35%, p< 0.05), and increased oxygen consumption (400mmHg vs 230mmHg, p< 0.05). Cold static storage after DCD retrieval was associated with loss of peripheral arterial blood supply, while arterial blood flow was maintained in NEVLP preserved grafts. Accordingly, UW preserved grafts had massive biliary necrosis (90%), while bile ducts were normal in NEVLP preserved livers. Spectrophotometric imaging showed decreased oxygen supply especially in peripheral parts of liver and bile duct tissue. Conclusion: NEVLP allows prolonged organ storage with normal liver metabolism without inducing preservation injury. Bile duct injury after DCD liver retrieval develops in cold static, but not NEVLP preserved organs. NEVLP is a novel preservation technique for the assessment of marginal grafts. The implemented spectrophotometric imaging can monitor oxygen transport to tissue and seems to be a promising tool to predict organ function post reperfusion.

Modified Two-Layer Preservation Method (M-Kyoto/PFC) Improves Islet Yields in Islet Isolation

Islet allotransplantation can achieve insulin independence in patients with type I diabetes. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and UW solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. However, UW solution has several disadvantages, including the inhibition of Liberase activity. In this study, we investigated the features of a new solution, designated M-Kyoto solution. M-Kyoto solution contains trehalose and ulinastatin as distinct components. Trehalose has a cytoprotective effect against stress, and ulinastatin inhibits trypsin. In porcine islet isolation, islet yield was significantly higher in the M-Kyoto/PFC group compared with the UW/PFC group. There was no significant difference in ATP content in the pancreas between the two groups, suggesting that different islet yields are not due to their differences as energy sources. Compared with UW solution, M-Kyoto solution significantly inhibited trypsin activity in the digestion step; moreover, M-Kyoto solution inhibited collagenase digestion less than UW solution. In conclusion, the advantages of M-Kyoto solution are trypsin inhibition and less collagenase inhibition. Based on these data, we now use M-Kyoto solution for clinical islet transplantation from nonheart-beating donor pancreata.

Two-Layer Method in Short-Term Pancreas Preservation for Successful Islet Isolation

Background We sought to determine whether the two-layer method (TLM) offers advantages over UW storage solution for locally procured pancreata with cold ischemia time of <8 hours for successful islet isolation. Methods From October 2003 through February 2005, 22 human pancreata were procured locally from cadaveric donors and preserved using UW solution (n = 11) or TLM (n = 11). Results Donor characteristics were similar in the two groups, with no statistical difference. Cold ischemia time was 4.5 ± 0.6 (2.5 to 8) hours in the UW and 5.1 ± 0.5 (3 to 8) hours in TLM group ( P > .05). Organs preserved with TLM were exposed to PFC for 4 ± 0.5 (2 to 7.5) hours. After TLM preservation, 8 of 11 (72%) pancreata yielded >300,000 IEQ pancreatic islets, which met all criteria for clinical transplantation; after UW cold storage, only 3 of 11 isolations were equally successful (27%) ( P < .05). Mean IEQ was higher in the TLM than in the UW group: 349,000 ± 37,000 vs 277,800 ± 34,000; IEQ/g was also higher at 5100 ± 760 vs 3000 ± 570, respectively ( P < .05). Islet quality, characterized by purity, viability, and insulin SI, did not differ statistically in the two groups: 67 ± 4 vs 74 ± 4%, 87 ± 2 vs 83 ± 4%, and 4 ± 0.7 vs 4.8 ± 1, respectively ( P > .05). Conclusions The Two Layer Method for locally procured human pancreata with cold ischemia time lower than 8 hours offers significant advantage over UW cold storage increasing the pancreatic islet isolation yield and the isolation success rate.

Machine perfusion preservation of the non–heart-beating donor rat livers using polysol, a new preservation solution

Aims The increasing shortage of donor organs has led to a focus on extended criteria donors, including the non–heart-beating donor (NHBD). An optimal preservation method is required to facilitate successful transplantation of these ischemically damaged organs. The recent literature has shown clear advantages of hypothermic machine perfusion (MP) over cold storage (CS). For MP, modified University of Wisconsin perfusion solution (UW-G) is often used, which, however, is known to cause microcirculatory obstruction, is difficult to obtain, and is expensive. Therefore, Polysol was developed as a MP preservation solution that contains specific nutrients for the liver, such as amino acids, energy substrates, and vitamins. The aim of this study was to compare Polysol with UW-G in a NHBD rat liver model. Methods After 24 hours hypothermic MP of NHBD rat livers using UW-G or Polysol, liver damage and function parameters were assessed during 60 minutes of reperfusion with Krebs-Henseleit buffer. Control livers were reperfused after 24 hours CS in UW. Results Liver enzyme release was significantly higher among the CS-UW group compared to MP using UW-G or Polysol. Flow during reperfusion was significantly higher when using Polysol compared to UW-G. Bile production and ammonia clearance were highest when using Polysol compared to UW-G. There was less cellular edema after preservation with Polysol compared to UW-G. Conclusions MP of NHBD rat livers for 24 hours using UW-G or Polysol resulted in less hepatocellular damage than CS in UW. MP of NHBD livers for 24 hours using Polysol is superior to MP using UW-G.

Ischemic preconditioning improves rat kidney graft function after severe ischemia/reperfusion injury

Ischemic preconditioning (IP) has been shown to ameliorate renal ischemia reperfusion injury. Using a rat kidney transplantation model we determined if IP improves graft function after prolonged cold storage. Materials and methods Syngeneic rat kidneys were divided into two groups. Prior to 42 hours of cold storage in UW and transplantation, one group ( n = 10) received IP (15 minutes of warm ischemia/10 minutes of reperfusion), whereas another group ( n = 10) received no treatment. Early graft function and 1-week recipient survival were assessed. Results Recipient survival was not significantly different between groups [70% (IP) vs 40% (non-IP); P = .28]. IP treatment led to a quicker recovery of renal function. On PODs 3 and 6, serum creatinine levels in the IP group were significantly lower compared with the untreated group. In conclusion, one cycle of IP (15/10) accelerates recovery of renal graft function after severe ischemia reperfusion injury. This simple treatment modality may improve outcomes of renal transplants with prolonged cold storage.

Improved rat liver preservation by hypothermic continuous machine perfusion using polysol, a new, enriched preservation solution

For experimental machine perfusion (MP) of the liver, the modified University of Wisconsin solution (UW-G) is most often used. In our search for an enriched MP preservation solution, Polysol was developed. Polysol is enriched with various amino acids, vitamins, and other nutrients for the liver metabolism. The aim of this study was to compare Polysol with UW-G for MP preservation of the liver. Rat livers were preserved during 24 hours with hypothermic MP using UW-G (n = 5) or Polysol (n = 5). Hepatocellular damage (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], alpha-glutathione-S-transferase [alpha-GST]) and bile production were measured during 60 minutes of reperfusion (37 degrees C) with Krebs-Henseleit buffer. Control livers were reperfused after 24 hours of cold storage in UW (n = 5). MP using UW-G or Polysol showed less liver damage when compared with controls. Livers machine perfused with Polysol showed less enzyme release when compared to UW-G. Bile production was higher after MP using either UW-G or Polysol compared with controls. In conclusion, machine perfusion using Polysol results in better quality liver preservation than cold storage with UW and machine perfusion using UW-G.

Hypothermic machine perfusion of liver grafts for transplantation: Technical development in human discard and miniature swine models

Introduction Cold storage (CS) is the standard preservation technique for liver transplantation (LTx). Hypothermic machine perfusion (HMP) is an alternative preservation technique that provides a continuous supply of substrates and removes waste products. HMP improves early graft function in kidney transplantation, especially for marginal organs: To our knowledge there have been no reports HMP in human LTx. The aim of this study was to develop a reproducible technique for liver HMP prior to initiating a clinical trial. Methods For the discard protocol, between May 2001 and March 2002, 10 nontransplantable human livers were obtained. We designed a model of atraumatic, centrifugal HMP of the portal vein (PV) and hepatic artery (HA) via donor vascular conduit. Livers were perfused at 3°C to 5°C with Vasosol solution for 5 to 10 hours using a modified Medtronic Portable Bypass System. Perfusion variables (temp, flow, pressure) where recorded every 30 minutes. During the study, we also validated our techniques in an animal model. For the animal protocol; six swine were used as liver donors and randomized to 12 hours of CS in UW ( n = 3) or 12 hours of HMP using Vasosol solution ( n = 3). LTx was performed in six swine. Animals survived until postoperative day 5. Results For the discard protocol, mean HMP time was 6.7 ± 1.8 hours. Target flow was 0.7 mL/g liver/min. PV and HA pressure ranged from 3 to 5 and 12 to 18 mm Hg, respectively. All grafts were maintained at 3°C to 5°C during HMP. For the animal protocol, all recipients had good liver function and survived to postoperative day 5. AST and TBili were similar between CS and HMP. Conclusions Our method of liver HMP appears to be a safe and reliable method to preserve livers. A clinical trial is now underway to evaluate this technique in human LTx.

Short-term storage of the ischemically damaged human pancreas by the two-layer method prior to islet isolation.

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata in the canine pancreas transplant model. In this study, we applied a short-term preservation of the TLM to human pancreata after prolonged cold ischemia prior to islet isolation, and investigated the mechanisms of resuscitation of the ischemically damaged human pancreas by the TLM. Human pancreata were procured from cadaveric donors and preserved by the TLM for 3.2 +/- 0.5 h after 11.1 +/- 0.9 h of cold storage in UW (TLM group), or by cold UW alone for 11.0 +/- 0.3 h (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro functional viability of isolated islets were significantly increased in the TLM group compared with the UW group. According to the criteria of the Edmonton protocol, 10/14 cases (71%) in the TLM group were transplanted to patients with type I diabetes mellitus compared with only 5/21 cases (24%) in the UW group. In the metabolic assessment of human pancreata, levels of energetic parameters (ATP, total adenylates, and energy charge) were significantly increased, and malondialdehyde (MDA) levels were significantly decreased after the TLM preservation. There was no observable change in the incidence or degree of mitochondrial injury after the TLM preservation. Additional short-term storage by the TLM resuscitates the ischemically damaged human pancreas by regenerating the energetic status and prevents further damage by oxidative stress, ultimately leading to improvements of islet recovery and in vitro function. Use of the TLM following prolonged storage in UW provides an excellent adjunctive protocol for treating human pancreata for the rigors of the islet isolation process.

Liver mitochondrial calcium uptake following cold ischemia is regulated by phospholipase C

Introduction: Altered mitochondrial Ca2+ (MCa2+) uptake has been implicated in graft dysfunction following cold ischemia (CI), when excessive cytosolic Ca2+ enters mitochondria through the MCa2+ uniporter. The actual mechanisms governing uniporter activity are unknown. Ca2+ uptake by the cell is regulated by Phospholipase C (PLC). We therefore hypothesized that PLC exists in mitochondria and regulates MCa2+ uptake following CI. Methods: Rat livers were perfused with UW solution and harvested. Half was homogenized immediately; the other half was first subjected to 24 hour cold storage in UW. Mitochondria were isolated and incubated in a buffer containing 1mM ATP (known [ATP] during ischemia) and 0.2 μM 45Ca2+. Ruthenium Red (RR, [10 μM]) was used as a negative control. A selective PLC inhibitor, U-73122, and its inactive analog, U-73343, were added to determine their effects on MCa2+ uptake. Western blots were performed using anti-PLC antibodies. Mitochondrial transmembrane potential (MΔψ) was evaluated using Mitotracker Red fluorescence. Results: 1) Western blot confirmed the presence of PLC in isolated mitochondria and mitochondrial membranes. 2) MCa2+ uptake was significantly increased after 24 hour cold storage in UW. 3) U-73122 and RR, but not U-73343, significantly and dose-dependently decreased Ca2+ uptake in mitochondria from both ischemic and non-ischemic livers, without affecting MΔψ. Conclusions: These data demonstrate for the first time that PLC exists in liver mitochondria. The effects of U-73122 indicate that PLC is essential for MCa2+ uptake during both physiologic and CI conditions. Inhibition of mitochondrial PLC therefore represents a novel target in the study of CI damage during liver transplantation.

Human islet transplantation from pancreases with prolonged cold ischemia using additional preservation by the two-layer (UW solution/perfluorochemical) cold-storage method.

A two-layer (University of Wisconsin solution/perfluorochemical [UW/PFC]) cold-storage method delivers sufficient oxygen to the pancreas during preservation and restores the ischemically damaged pancreas. In this study, we determined whether the additional preservation by the two-layer method could improve islet recovery from human pancreases with prolonged cold storage in UW. Human pancreases were procured from cadaveric organ donors and preserved by the two-layer method (UW/PFC) for 2.9+/-0.7 hours (mean+/-SEM) at 4 degrees C after 11.8+/-1.5 hours of cold storage in UW (UW/PFC group, n=7), or by cold UW alone for 11.3+/-0.3 hours (UW group, n=14). The selected pancreases met the criteria of having at least 10 hours of cold storage in UW. All were processed by using a standard protocol of Liberase perfusion with Pefabloc by way of the duct, gentle mechanical dissociation, and Ficoll gradient purification. Transplanted islets were selected with the criteria of the Edmonton protocol (>5,000 islet equivalents [IE]/kg recipient body weight). The islet recovery was significantly increased in the UW/PFC group compared with the UW group (349.2+/-44.1 x 10 and 214.0+/-31.0 x 10 IE, respectively; <0.05). This resulted in islet yields of 4.6+/-1.0 x 10 IE/g of pancreas in the UW/PFC group compared with 2.0+/-0.3 x 10 IE/g of pancreas in the UW group ( <0.05). Five of 7 cases (71%) in the UW/PFC group and 5 of 14 cases (36%) in the UW group were transplanted. The islet grafts in the UW/PFC group improved the ability of glycemic control and decreased exogenous insulin administration in all recipients. Improvements in methods to preserve and recover ischemically damaged human pancreases before islet isolation and transplant could be extremely beneficial to the field of clinical islet transplantation. This preliminary study shows that additional short preservation by the two-layer (UW/PFC) cold-storage method can significantly improve islet recovery and increase opportunities of islet transplantation from human pancreases after prolonged cold ischemia.

Functional and Compositional Studies of Arteries Stored in University of Wisconsin Solution Compared with Krebs-Bülbring Buffer

Endothelium-dependent and -independent vasorelaxation in ring segments of rabbit thoracic aorta is reduced and noradrenaline-induced vasoconstriction unaltered after prolonged storage in University of Wisconsin solution (UW) compared to arteries stored in extracellular-type solutions such as Krebs-Bülbring buffer (KBB). The aims of the present study were to determine whether angiotensin-II-induced vasoconstriction, alterations in myosin light chains, protein synthetic capacity, and subcellular structures are altered after 8 days of UW storage at 4°C. The present study showed reduced contractility to angiotensin II, following 8 days of cold storage in UW, that was not reversed in the presence of a nitric oxide synthase inhibitor, NG-nitro-l-arginine methyl ester (100 μM). Measurements of contractile protein ratios in the same tissues after cold storage in UW or KBB did not show any significant alterations in smooth muscle myosin light chains or protein synthetic capacity (reflected by total RNA). It is concluded that reductions in vasoconstriction in UW-stored tissue are unlikely to be due to increased release of nitric oxide nor reduced availability of myosin light chains for phosphorylation and vasoconstriction.

Electroporation of antibodies, DNA, and other macromolecules into cells: a highly efficient method

While antibodies are a major extracellular tool of the highest specificity to answer important biomedical questions, the improvements in electroporation discussed below may make it feasible to also use antibodies as an intracellular deletion tool to study (a) viruses inside the cell, (b) cancer cells, (c) signal transduction, (d) genetics, (e) metabolism, and (f) other structures and mechanisms. Already, others have succeeded in depositing macromolecules, including antibodies (Abs), and nucleic acids inside cells, using many techniques, including electroporation (EP). However, EP has limitations that have precluded its widespread use, particularly its high kill rate for cells and the low percentage of cells that are able to incorporate macromolecules. If these limitations could be overcome for Abs and nucleic acids, then it would be practical to use them as highly specific probes for intracellular molecules. In our experiments using EP, we were able to largely prevent lethality for cells during EP by employing a commercially available cold-storage solution for organ transplants containing high K + and Mg ++ (ViaSpan, Belzer UW cold-storage solution, DuPont Pharmaceuticals). This solution decreased cell death after standard EP by an average of 50% for a number of cell lines. Viability of WISH cells after EP approached 100%. In transfection studies, ViaSpan medium strongly increased both P3HR1 cell survival as well as the total number of cells transfected with DNA for green fluorescent protein (GFP). In additional experiments with Abs, we were able to strongly increase the percent of cells that incorporated Ab by using two serial EPs. This enhanced the intracellular protection by Abs against viruses in Vero cells from 64% to a maximum of 98%. We were able to further simplify the EP technique by using unpurified antiserum in place of purified IgG. Thus, this EP technique offers multiple advantages: simplicity, high cell viability, high effectiveness, high specificity, rapid action, usefulness with adherent or non-adherent cells, and no requirement for purification of antibodies from antiserum.

High-Na+ low-K+ UW cold storage solution reduces reperfusion injuries of the rat liver graft.

The isolated perfused rat liver model was used to assess graft viability after 24 h of cold preservation. Two solutions were compared for liver preservation: Belzer's original UW solution (high-K+ UW) and a solution containing the same components but with inverted concentrations of sodium and potassium (high-Na+ UW). During the 120 min of normothermic reperfusion, livers preserved in the high-Na+ UW solution released lower levels of creatine kinase-BB isoenzyme, transaminases (ALT and AST), and potassium than those preserved in the high-K+ UW solution. Bile flow and biliary excretion of indocyanine green increased when livers were preserved in the high-Na+ UW solution. We found no statistical differences for oxygen consumption and tissue ATP concentration. The results of this study support the concept that a high-Na+ UW solution is a more effective means of preserving rat livers, at least after 24 h of cold-storage and 120 min of reperfusion in the isolated perfused model, than the original high-K+ UW solution. Liver preservation in the high-Na+ UW solution reduces damage to sinusoidal endothelial and hepatocellular cells. The use of an extracellular-like Belzer cold storage solution eliminates potassium-related problems in cold preservation and subsequent normothermic reperfusion while keeping all the qualities of the original UW solution.

High-Na+low-K+UW cold storage solution reduces reperfusion injuries of the rat liver graft

Abstract The isolated perfused rat liver model was used to assess graft viability after 24 h of cold preservation. Two solutions were compared for liver preservation: Belzer's original UW solution (high-K+UW) and a solution containing the same components but with inverted concentrations of sodium and potassium (high-Na+UW). During the 120 min of normothermic reperfusion, livers preserved in the high-Na+UW solution released lower levels of creatine kinase-BB isoenzyme, transaminases (ALT and AST), and potassium than those preserved in the high-K +UW solution. Bile flow and biliary excretion of indocyanine green increased when livers were preserved in the high-Na +UW solution. We found no statistical differences for oxygen consumption and tissue ATP concentration. The results of this study support the concept that a high-Na +UW solution is a more effective means of preserving rat livers, at least after 24 h of cold-storage and 120 min of reperfusion in the isolated perfused model, than the original high-K +UW solution. Liver preservation in the high-Na +UW solution reduces damage to sinusoidal endothelial and hepatocellular cells. The use of an extracellular-like Belzer cold storage solution eliminates potassium-related problems in cold preservation and subsequent normothermic reperfusion while keeping all the qualities of the original UW solution.

Role of free radicals in primary nonfunction of marginal fatty grafts from rats treated acutely with ethanol.

Acute treatment with one large dose of ethanol, which mimics binge drinking, causes marginal fatty liver and decreases survival significantly after liver transplantation in rats, yet mechanisms remain unclear. Therefore, we evaluated the possible role of free radicals in primary nonfunction caused by acute ethanol. Female donor rats were administered ethanol (5 g/kg orally) 20 hr before explantation, and grafts were stored in UW cold storage solution for 24-42 hr before implantation. Free radicals were trapped with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone after transplantation, and adducts were detected using electron spin resonance spectrometry. Ethanol increased a carbon-centered radical adduct in bile approximately 2-fold and elevated serum lipid hydroperoxides approximately 4-fold. Ethanol also increased transaminase release 3.7-fold and decreased bile production by 55%. Catechin, a free radical scavenger, minimized the increase in free radicals, blunted transaminase release, and elevated bile production significantly, indicating that free radical production plays an important role in ethanol-induced fatty graft injury. GdCl3 (20 mg/kg intravenously), a selective Kupffer cell toxicant, largely blocked the increases in free radical and lipid hydroperoxide production caused by ethanol. In addition, ethanol nearly doubled white blood cell adhesion after transplantation, leading to increased superoxide production in fatty grafts. GdCl3 largely blocked leukocyte adhesion as well as superoxide production. Allopurinol, an inhibitor of xanthine oxidase, also diminished free radical production, blunted transaminase release, and improved bile production in fatty grafts significantly. Taken together, we conclude that free radical formation increases in ethanol-induced fatty grafts due mainly to activation of Kupffer cells and increased adhesion of white blood cells. Antioxidants can effectively block free radical formation and minimize injury to marginal fatty grafts caused by binge drinking.

Excellence of the two-layer method (University of Wisconsin solution/perfluorochemical) in pancreas preservation before islet isolation

Background. In islet transplantation pancreatic preservation before islet isolation is an obstacle compromising islet yield and viability. We tested the feasibility of a two-layer method (University of Wisconsin solution [UW]/perfluorochemical) for pancreatic preservation before islet isolation. Methods. Dog pancreases were processed into pure islets by the method of Ricordi preceded by five different preservations (groups 1-a and 1-b, the two-layer method far 3 and 24 hours; groups 2-a and 2-b, simple cold storage in UW for 3 and 24 hours; group 3, without preservation). Islet yields and functional success after autotransplantation into the liver were compared among the groups. Results. Postpurification islet equivalents (IE)/gm pancreas and functional success rate were 5600 (mean), 83% in group 1-a; 4000, 56% in group 1-b; 4700, 33% in group 2-a; 1300, 0% in group 2-b; and 5000, 89% in group 3 (p < 0.05; 2b versus 1-a, 1-b, and 3), respectively. There was no statistical difference among groups 1-a, 1-b, and 3 in terms of islet yield and function (p > 0.2). Conclusions. The two-layer method is more effective than conventional simple cold storage in UW for pancreatic preservation before islet isolation. Clinical trials with the two-layer method are warranted.

Protective effect of preservation of canine pancreas by the two-layer (University of Wisconsin solution/perfluorochemical) method against rewarming ischemic injury during implantation.

Rewarming ischemia during implantation severely compromises posttransplant pancreas graft survival because the graft has already been subjected to warm and cold ischemia before implantation. The purpose of this study was to examine whether preservation of the pancreas graft by the two-layer method ameliorates rewarming ischemic injury of the graft during implantation using a canine model. After flushing with cold University of Wisconsin solution (UW), the pancreas grafts were preserved by the two-layer (UW/perfluorochemical [PFC]) method (group 1) or simple cold storage in UW (group 2) for 24 hr and then autotransplanted. In control, the pancreas grafts were flushed out with cold UW and immediately autotransplanted without preservation (group 3). After completion of vascular anastomosis, vascular clamp was not released until 90, 120, or 150 min of rewarming ischemia, including anastomosis time, had elapsed. After 90 min of rewarming ischemia, graft survival rates were 5/5, 100%, 5/5, 100%, and 5/5, 100%, in groups 1, 2, and 3, respectively. After 120 min, all the grafts in groups 2 and 3 failed (0/5, 0%, and 0/5, 0%, respectively); however, all the grafts in group 1 survived (5/5, 100%). Even after 150 min, 1 of 3 grafts in group 1 survived (1/3, 33%). After 24 hr preservation, tissue ATP levels of the grafts in group 1 were about 2-fold the reference values before harvesting (8.23 +/- 0.72 vs. 4.44 +/- 0.49 mumol/g dry weight, P < 0.05) and significantly higher compared with group 2 (8.23 +/- 0.72 vs. 1.76 +/- 0.52 mumol/g dry weight, P < 0.01). After 120 min of rewarming ischemia, tissue ATP levels in group 1 were 84% of the reference values and significantly higher compared with group 2 (3.75 +/- 0.25 vs. 1.57 +/- 0.48 mumol/g dry weight, P < 0.05). Two hours after reperfusion, ATP levels in group 1 were 42% of reference values but significantly higher compared with group 2 (1.86 +/- 0.36 vs. 1.03 +/- 0.18 mumol/g dry weight, P < 0.05). We conclude that the two-layer (UW/PFC) method ameliorates rewarming ischemic injury of the pancreas graft during implantation by increasing tissue ATP contents during preservation and consequently maintaining tissue ATP levels during implantation.