Maintenance of glucose-sensitive insulin secretion of cryopreserved human islets with University of Wisconsin solution and ascorbic acid-2 glucoside

Normal human islet cells are an ideal source for pancreas-targeted cell therapies, but the availability of human donor pancreata for islet isolation is severely limited. To effectively utilize such scarce donor organs for cell therapies, it is crucial to develop an excellent isolation, effective cryopreservation, and efficient gene transfer techniques for the transportation of isolated cells. In the present study, we investigate the effect of University of Wisconsin (UW) solution and ascorbic acid-2 glucoside (AA2G) on the cryopreservation of human islets. We also evaluate the gene transfer efficiency of a lentiviral vector expressing the E. coli LacZ gene, Lt-NLS/LacZ, in human islets. Human islets were isolated with a standard digestion method at the University of Alberta. Isolated islets were transported to Japan for 40 h and then subjected to cryopreservation experiments. The following preservation solutions were tested: UW solution with 100 micro g/mL of AA2G, UW solution, 100% fetal bovine serum (FBS), and CMRL supplemented with 10% FBS. Following three months of cryopreservation, the islets were thawed and analyzed for viability, glucose-sensitive insulin secretion, proinsulin gene expression profile, and in vivo engraftment. The islets were also subjected to monolayer formation with 804G-cell-line-derived extracellular matrix (ECM), followed by Lt-NLS/LacZ transduction. The viability, morphology, glucose-sensitive insulin secretion, proinsulin gene expression, and monolayer formation efficiency of the thawed cryopreserved islets are significantly better maintained by the use of UW solution. When AA2G (100 microg/mL) is combined with UW, such parameters are further improved. The adequate engraftment of UW + AA2G-cryopreserved human islets is achieved in the liver of nude mice. Efficient Lt-NLS/LacZ transduction is identified in monolayered islets cryopreserved with UW solution with AA2G. The present work demonstrates that the combination of UW solution with AA2G (100 microg/mL) would be a useful cryopreservation means for human islets. Human islets monolayer-cultured with 804G-derived ECM are efficiently transduced with a lentiviral vector Lt-NLS/LacZ.     

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.     

Warm preflush with streptokinase improves microvascular procurement and tissue integrity in liver graft retrieval from non-heart-beating donors

BACKGROUND: Apart from the warm ischemic insult, integrity of liver grafts from non-heart-beating donors (NHBD) is additionally affected by microvascular alterations including erythrocyte aggregation and thrombus formation, which might hamper appropriate equilibration of the preservation solution to the grafts' microvasculature precluding cold preservation. Thus, the objective of our study was to characterize microvascular perfusion quality of University of Wisconsin (UW) solution during initial flushout of livers from NHBD rats, and to analyze the effects of an additional warm preflush with Ringer's lactated solution (RL) and with RL containing streptokinase (SK). METHODS: Cardiocirculatory arrest was induced by phrenotomy. Subsequent to 30 min of warm ischemia, livers were perfused via an aortic catheter by gravity of 100 cm H2O either with 4 degrees C 100 ml UW solution (UW, n=7), or with 25 degrees C 30 ml RL preflush followed by 4 degrees C 100 ml UW solution (RL+UW, n=7), or with 25 degrees C 30 ml SK- (7500 IU) containing RL preflush and 4 degrees C 100 ml UW solution (SK/RL+UW, n=6). Liver microperfusion was quantified using in situ fluorescence epi-illumination microscopy. Liver microcirculation of sham-operated living animals (n=4) served as controls. Enzyme release after a 24-hr cold preservation period was used as an indicator of graft integrity. RESULTS: After 30 min of warm ischemia, microvascular perfusion of UW solution was found markedly altered when compared with that of sham-operated living controls, as indicated by a significant reduction (P<0.05) of acinar and sinusoidal perfusion. Preflush with RL (RL+UW) only slightly attenuated the acinar and sinusoidal perfusion deficits, whereas the addition of SK to RL (SK/RL+UW) resulted in a significant improvement of microvascular graft perfusion (P<0.05). Accordingly, the increased enzyme release observed in solely UW-flushed livers after 24 hr cold preseravtion was only slightly influenced by preflush with RL, but markedly attenuated (P<0.05) by pre-flush with RL containing SK. CONCLUSION: The additive fibrinolytic therapy using SK is effective to improve microvascular procurement of livers after warm ischemia and may thus represent a promising approach to attenuate parenchymal cell injury in liver graft retrieval from NHBD.     

Effect of S-nitrosoglutathione (GSNO) added to the University of Wisconsin solution (UW): I) Morphological alteration during cold preservation/reperfusion of rat liver

Cold liver preservation in the University of Wisconsin solution (UW) followed by reperfusion alters hepatic parenchyma and stroma. In this study we demonstrated the benefit of adding S-nitrosoglutathione (GSNO) to the UW solution before cold storage, as an effective Nitric Oxide (NO) donor to prevent hepatic injury. Wistar adult rat livers were stored in UW solution (4 degrees C-48Hs) and then reperfused 60 minutes in the isolated perfused rat liver model (IPRL). Normal untreated livers and perfused livers, but not preserved were used as controls. Parenchymal damages were evaluated with Hematoxylin-Eosin stain and an inmunohistochemistry assay for albumin was used as functional test. To study the stroma, collagen type III and I networks were analyzed using Picro-sirius Red stain and Gordon Sweets' method for reticulin. After 48 Hs of cold preservation in UW solution livers showed few rounded endothelial cells inside sinusoidal lumen and extended areas of cell vacuolation. Albumin distribution was evident only around central veins and middle zones of the hepatic lobule. Collagens III and I networks were disorganized. When preserved with the addition of 100 microM GSNO and then reperfused, the hepatic morphology, in general, was conserved showing little vacuolation, fewer endothelial cells inside sinusoids and good albumin distribution around central veins and middle zones. The stroma had organized networks of collagen III and I. We concluded that the addition of 100 microM GSNO as a NO donor, can improve UW solution properties to preserve rat liver by maintaining the hepatic morphology and avoiding hepatic injury post cold preservation/reperfusion.     

Influence of preservation solution on the isolation and culture of human hepatocytes from liver grafts

A major problem for the isolation and transplantation of hepatocytes is the lack of resources for obtaining viable hepatocytes. Improving this situation would enhance hepatic cell transplantation programs. Our objective was to evaluate the influence of the preservation solutions used during organ retrieval on the quality of hepatocytes isolated from liver tissue. We compared the results of the collagenase perfusion technique for isolation of hepatocytes in human livers flushed with University of Wisconsin (UW) and Celsior preservation solutions. Yield (number of viable cells per gram of tissue), cellular viability, efficiency of cells to attach to culture plates and form a monolayer, and drug metabolizing competence of the hepatocytes were measured. Successful isolation was achieved in 63% of the procedures using the UW solution and 100% of the procedures using the Celsior solution. In the UW group, significantly lower cell viability (38 +/- 41% vs. 79 +/- 14%, p < 0.05), yield of cells (4.0 +/- 5.2 x 10(6) vs. 8.2 +/- 5.6 x 10(6) cells/g, p < 0.05), and protein content at 24 h of culture (0.6 +/- 0.6 vs. 1.2 +/- 0.3 mg protein per plate, p < 0.05) than in Celsior solution were found. However, similar values of P450 activities were found in both groups. The more successful isolation, better yield, and higher cell viability obtained from human liver grafts preserved in Celsior solution, in comparison to UW solution, suggest Celsior solution as the most appropriate for preserving cadaveric hepatic tissue to be used for hepatocyte harvesting.     

The function of a colloid in liver cold-storage preservation

The value of colloid in preservation of the liver by cold storage has not yet been fully clarified. Therefore, we studied the effects of colloid on cell swelling, liver weight, and bile production after cold storage in rat liver tissue slices and isolated rabbit liver. In rat liver tissue slices cold-stored for 24 hr in UW solution, total tissue water (TTW) was the same as in the control freshly unpreserved tissue and omitting the colloid (hydroxyethyl starch) from the UW solution did not affect the TTW. However, after cold storage for 24 hr in Perfadex, TTW was markedly increased (by 100%, P less than 0.001). Omitting the colloid in this solution, dextran, or replacing it with hydroxyethyl starch, did not affect this increase in TTW. Thus, the hypothermia-induced cell swelling evident after preservation in Perfadex was not prevented by colloid. Rabbit liver cold-stored in UW solution for 24 hr lost 15.4 +/- 4.7% of weight, but omitting the colloid from UW solution decreased this weight loss to 3.1 +/- 3% (P less than 0.01). In contrast, rabbit livers cold-preserved in colloid-free Perfadex gained 23.3 +/- 5.7% in weight. Adding colloid, either dextran or hydroxyethyl starch, decreased significantly this weight gain, to 9 +/- 3.7% and 10.4 +/- 1.8%, respectively (P less than 0.01), probably as a result of colloid osmotic pressure, preventing the interstitial edema. Rabbit livers preserved for 24 hr in UW solution, with or without colloid, produced the same amount of bile as control unpreserved livers. In contrast, livers preserved in colloid-free Perfadex for 24 hr had a markedly impaired bile production (3.9 +/- 0.9 ml/100 g) as compared with control livers (15.5 +/- 2.6 ml/100 g, P less than 0.01). Colloid partially restored this impaired bile production, to 8 +/- 1.4 mg/100 g by dextran and to 8.5 +/- 1.7 ml/100 g by hydroxyethyl starch, respectively (P less than 0.01). Thus, although colloids do not prevent the hypothermia-induced cell swelling, they prevent the development of interstitial edema, and, hence, improve the liver function.     

Glycogen storage of the liver: a determining factor of initial function of the hepatic graft]

In this study we have investigated the effects of hepatocytes glycogen storage on the quality of livers for transplantation. Rats were fed or fasted for 24 h and hepatocytes isolated and cold stored in UW solution for 24 and 48 hours. Viability of the cells was analyzed by LDH release after 2 hours incubation in L15 with O2. Also, rabbits were fed, fasted (48 h) or glucose fed (48 h) and livers cold stored for 6, 24 and 48 h in UW solution. Functions of the livers were analyzed by isolated perfusion for 2 hours. Hepatocytes from fasted rats released significantly more LDH than hepatocytes from fed rats after 24 and 48 h cold storage. In rabbit livers, fasting depleted glycogen by 85% but had no effect on ATP or glutathione concentration. Livers from fasted rabbits produced similar amount of bile, released similar concentrations of lactate dehydrogenase and aspartate transaminase into the perfusate, maintained similar concentrations of glutathione after 24 hours preservation when compared to fed animals. After 48 h preservation livers from fasted animals were less viable than livers from fed animals and the decrease of liver functions in livers from fasted animals preserved for 48 hours was prevented by feeding glucose. This study shows that liver glycogen storage in hepatocyte is an important metabolite for successful liver preservation. Glycogen may be a source for ATP and antioxydant synthesis during the early period of reperfusion.     

SWH液对大鼠肝脏保存中肝细胞凋亡的影响

目的　通过与UW液进行比较 ,观察SWH保存液对大鼠肝细胞凋亡及其对肝移植受者存活率的影响。方法　应用大鼠原位肝移植模型 ,通过HE染色、电镜和TUNEL法检测肝细胞凋亡以及观察移植术后 7d动物存活率。结果　随着保存时间的延长 ,两组的凋亡指数 (AI)均无明显升高 ,保存 18h后 ,两组的AI亦无明显改变 ,仅在行肝移植恢复血流循环后AI才明显上升 ,移植前后存在显著性差异 ,P <0 .0 1,SWH液组略低于UW液组 ;SWH液组移植术后 7d动物存活率有高于UW液组的趋势。结论　有效地防止肝细胞凋亡可以提高肝脏的保存效果 ,在防止肝细胞凋亡方面 ,SWH液优于UW液。     

Inhibition of free radical generation and improved survival by protection of the hepatic microvascular endothelium by targeted erythrocytes in orthotopic rat liver transplantation

The capacity of specifically targeted erythrocytes to inhibit free radical-mediated injury to the endothelial cell after cold preservation, and improve liver function was studied in two experimental models: An isolated perfused rat liver (IPRL) system and syngeneic orthotopic rat liver transplantation. In the IPRL model, livers were preserved in University of Wisconsin solution for 24 h at 4 degrees C. At the end of the preservation period, livers were flushed with lactated Ringer's (control), immunoerythrocytes (IES), or blank intact erythrocytes prior to warm reperfusion for 2 h using an assanguinous Krebs-Henseleit buffer. Production of superoxide (O2-) anion during warm reperfusion in the IES-treated liver was reduced by 65% as compared with controls (P less than 0.001) and by 74% (P less than 0.001) when compared with blank erythrocyte-treated livers. Endothelial cell preservation, as assessed by levels of purine nucleoside phosphorylase (PNP), was much better in the IES-treated group (P less than 0.001) when compared with untreated livers. Hepatocellular preservation was markedly improved in the IES-treated livers. In the syngeneic liver transplantation model, livers were preserved in UW solution for 24 h at 4 degrees C. Prior to implantation, livers were flushed with 5 ml of cold lactated Ringer's or immunoerythrocytes. Survival after three weeks was 60% in the IES-treated group and 30% in the untreated group. Survival in the IES-treated group was not significantly different from a control (no preservation) group. IES-treated livers in both models demonstrated better endothelial cell integrity and ultimate liver function. IES treatment therefore appears to protect the hepatic microvascular endothelial cell from reperfusion injury and could prove to be an easy reproducible method of donor organ preparation after cold preservation.     

Short-term treatment with mycophenolic acid increases bile flow in continuously perfused and cold-preserved rat livers and does not affect hepatic ischemia-reperfusion injury

Mycophenolate mofetil (MMF) is a new immunosuppressive agent which has been used successfully after kidney and heart transplantation. Experience with MMF after liver transplantation is still limited. In particular, there is no information about influence on ischemia-reperfusion injury (IRI). Therefore, the aim of this investigation was to assess the effects of mycophenolic acid (MPA), the pharmacologically active metabolite of MMF, in the cold-preserved or normal rat liver. Livers of male Sprague-Dawley rats were subjected to cold ischemia in University of Wisconsin (UW) solution (24 h, 4 degrees C) and reperfused for 2 h in the absence or presence of MPA (100 microg/ml, n=5-6 each). Another group received MPA pretreatment for 20 min prior to ischemia ( n=7). In further experiments, livers were perfused with a bile salt-free Krebs-Henseleit buffer in a continuous fashion (controls, n=5). MPA was infused from 20-40 min after starting perfusion in therapeutic concentrations (5 microg/ml, 10 microg/ml, 40 microg/ml, and 100 microg/ml; n=3-6 each). There was no significant influence of MPA on portal pressure nor on postischemic efflux rates of LDH. MPA pretreatment resulted in a significant improvement of bile flow during reperfusion (0.32+/-0.05 microl/min x g liver) compared with controls (0.17+/-0.04 microl/min x g liver, mean+/-SEM). In contrast, postischemic bile flow was not influenced by continuous administration of MPA during the reperfusion period only (0.18+/-0.07 microl/min x g liver). In continuously perfused livers, MPA increased bile salt-independent bile flow (1.00+/-0.06 microl/min x g liver) in a dose-dependent manner, reaching half-maximal effects around 5 microg/ml (1.66+/-0.15 microl/min x g liver) and maximal effects at 40 microg/ml (2.61+/-0.28 microl/min x g liver). In conclusion, neither preischemic nor postischemic administration of MPA influences IRI to hepatocytes significantly after hypothermic liver preservation in UW solution. In contrast to other immunosuppressive agents, MPA exhibits strong choleretic effects, which are related to a stimulation of bile salt-independent bile formation.     

Cryopreservation of primarily isolated porcine hepatocytes with UW solution

Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at -80 degrees C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.     

Sequential cold storage and normothermic perfusion of the ischemic rat liver

Extending transplant criteria to include livers obtained from donors after cardiac death (DCD) could increase the liver donor pool, but conventional simple cold storage of these ischemic organs can lead to poor graft function after transplantation. Experimental normothermic machine perfusion has previously proven to be useful for the recovery and preservation of DCD livers, but it is more complicated than conventional cold storage, and, therefore, is perhaps not practical during the entire preservation period. In clinical situations, the combined use of simple cold storage and normothermic perfusion preservation of DCD livers might be more realistic, but even a brief period of cold storage prior to normothermic preservation has been suggested to have a negative impact on graft viability. In this study we show that rat livers subjected to 45 minutes of ex vivo warm ischemia followed by 2 hours of simple cold storage can be reclaimed by 4 hours of normothermic machine perfusion. These livers could be orthotopically transplanted into syngeneic recipients with 100% survival after 4 weeks (N = 10), similar to the survival of animals that received fresh livers that were stored on ice in University of Wisconsin (UW) solution for 6 hours (N = 6). On the other hand, rats that received ischemic livers preserved on ice in UW solution for 6 hours (N = 6) all died within 12 hours after transplantation. These results suggest that normothermic perfusion can be used to reclaim DCD livers subjected to an additional period of cold ischemia during hypothermic storage.     

Improved liver preservation for transplantation due to calcium channel blockade.

Intracellular calcium is an important determinant for cell death in organ hypothermic preservation for transplantation. In this study, we show that prevention of calcium entry improves the result of liver cold storage in UW solution. The isolated perfused rabbit liver was used. After 48 hr of cold storage in UW solution, bile production was reduced by 70% (P less than 0.005). However, by adding the calcium channel blockers verapamil or nifedipine (40 microM) to the UW solution, this reduction was abolished, and the livers produced the same amount of bile as unpreserved livers. Furthermore, addition of the calcium channel activator, BAY K8644 (40 microM), to the UW solution, reduced bile production by 50% (P less than 0.01) already after preservation for 24 hr. We conclude that calcium entry is of importance for liver function after preservation and cold storage, and that including a calcium channel blocker to the preservation solution makes long-term liver preservation safer.     

Influence of preservation solution on human islet isolation outcome

BACKGROUND: The influence of the preservation solution used for in situ perfusion of the donor and pancreas storage on islet isolation has received little attention. METHODS: In this prospective controlled trial, we compared the outcome of human islet isolation from pancreata perfused with University of Wisconsin (UW) solution or Celsior, an alternative colloid-free extracellular solution. RESULTS: At the 1-year interim analysis, the viability and insulin secretion of islets isolated from donors perfused with UW (n=19) or Celsior (n=5) were identical. However, total islet recovery (IEQ) and isolation yield (IEQ/g) were 1.8-fold and 2.1-fold inferior in the Celsior group (P<0.05 vs. UW). Overall, 13 (68%) of islet preparations were effectively transplanted from the UW group vs. none from the Celsior group (P=0.01). The clinical study was discontinued and the causes of these differences were further explored in the pig (n=14). In contrast to UW, Celsior induced cell swelling and pancreas edema after only four hours of cold storage. These abnormalities were delayed when the donor was perfused with Solution de Conservation d'Organes et de Tissus (SCOT), an extracellular solution containing polyethylene glycol. CONCLUSIONS: Our results suggest that colloid-free preservation solutions might be suboptimal for pancreas perfusion and cold storage prior to islet isolation and transplantation. Because pancreata are now frequently recovered for islet transplantation, preliminary experimental and clinical data about islet isolation should be obtained prior to the routine implementation of new preservation solutions for abdominal perfusion during multiorgan recovery.     

Celsior solution compared with University of Wisconsin solution (UW) and histidine–tryptophan–ketoglutarate solution (HTK) in the protection of human hepatocytes against ischemia–reperfusion injury

Celsior, a new preservation solution in thoracic organ transplantation was evaluated for efficacy in cold preservation of human hepatocytes and compared with University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). Human hepatocyte cultures were preserved at 4 degrees C in Celsior, UW and HTK for 2, 6, 12, 24 and 48 h with 6 h of reperfusion. Levels of lactate dehydrogenase (LDH; cell necrosis), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; mitochondrial function), and adenosine 5'-triphosphate (ATP; loss of intracellular energy) were measured. Cell necrosis, mitochondrial dysfunction, and loss of ATP were significantly ( P<0.001, P<0.001, P<0.002, respectively) lower in Celsior than in HTK. The amount of cell necrosis and mitochondrial dysfunction in Celsior solution (CS) and UW was equal ( P=n.s.) up to 24 h and significantly lower in UW after 48 h ( P<0.001). Additionally, the intracellular level of ATP was significantly higher after ischemia ( P<0.001) and reperfusion from long-term ischemia (24, 48 h) ( P<0.002). We can conclude that Celsior was superior to HTK and equal to UW in the protection of human hepatocytes against cold preservation injury from ischemia and reperfusion. Furthermore, Celsior was effective in long-term preservation of human hepatocytes.     

大鼠肝脏保存中不同器官保存液对肝细胞凋亡的影响

目的 研究３种目前国内常用的器官保存液对供肝细胞凋亡的影响。方法 分别用ＵＷ液、ＨＣ－Ａ液和ＷＭＯ－１号液灌洗并保存大鼠肝脏,于保存后０、１２、２４ｈ用原位末端标记法检测供肝细胞凋亡情况,并将保存２４ｈ的肝及行大鼠原位全肝移植,观察受者的３ｄ存活率。结果 ３种保存液保存的肝脏均在保存１２ｈ时出现细胞凋亡,ＨＣ－Ａ液级瑟ＷＭＯ－１号液组的凋亡指数（ＡＩ）高于ＵＷ液组（Ｐ〈０．００１）,而ＨＣ－Ａ液组     

Delivery of the bioactive gas hydrogen sulfide during cold preservation of rat liver: effects on hepatic function in an ex vivo model

The insults sustained by transplanted livers (hepatectomy, hypothermic preservation, and normothermic reperfusion) could compromise hepatic function. Hydrogen sulfide (H₂S) is a physiologic gaseous signaling molecule, like nitric oxide (NO) and carbon monoxide (CO). We examined the effect of diallyl disulfide as a H₂S donor during hypothermic preservation and reperfusion on intrahepatic resistance (IVR), lactate dehydrogenase (LDH) release, bile production, oxygen consumption, bromosulfophthalein (BSP) depuration and histology in an isolated perfused rat liver model (IPRL), after 48 h of hypothermic storage (4°C) in University of Wisconsin solution (UW, Viaspan). Livers were retrieved from male Wistar rats. Three experimental groups were analyzed: Control group (CON): IPRL was performed after surgery; UW: IPRL was performed in livers preserved (48 h—4°C) in UW; and UWS: IPRL was performed in livers preserved (48 h—4°C) in UW in the presence of 3.4 mM diallyl disulfide. Hypothermic preservation injuries were manifested at reperfusion by a slight increment in IHR and LDH release compared with the control group. Also, bile production for the control group (1.32 µL/min/g of liver) seemed to be diminished after preservation by 73% in UW and 69% in UW H₂S group at the end of normothermic reperfusion. Liver samples analyzed by hematoxylin/eosin clearly showed the deleterious effect of cold storage process, partially reversed (dilated sinusoids and vacuolization attenuation) by the addition of a H₂S delivery compound to the preservation solution. Hepatic clearance (HC) of BSP was affected by cold storage of livers, but there were no noticeable differences between livers preserved with or without diallyl disulfide. Meanwhile, livers preserved in the presence of H₂S donor showed an enhanced capacity for BSP uptake (k_ACON = 0.29 min^?1; k_AUW = 0.29 min^?1; k_AUWS = 0.36 min^?1). In summary, our animal model suggests that hepatic hypothermic preservation for transplantation affects liver function and hepatic depuration of BSP, and implies that the inclusion of an H₂S donor during hypothermic preservation could improve standard methods of preparing livers for transplant.     

HX—3液和UW液保存大鼠肝脏效果的比较

采用大鼠肝脏非循环离体灌注模型比较自制的ＨＸ－３液和ＵＷ液对大鼠肝脏的保存效果。实验结果显示，经ＨＸ－３液原位灌洗并保存４８小时的肝脏肝组织含水量正常，而同等条件下换用ＵＷ液，肝组织的含水量虽无明显变化，但都低于正常值；随着保存时间的延长，两组肝窦内皮细胞死亡率逐渐上升，但在２４小时以内两组肝窥内皮细胞死亡率的差异不显著．     

Protection by vascular endothelial growth factor against sinusoidal endothelial damage and apoptosis induced by cold preservation

BACKGROUND: It is well known that sinusoidal endothelial cell (SEC) damage during cold preservation of liver tissue is closely involved in early graft failure. The objective of this study was to investigate the involvement of apoptosis in the SEC damage induced by cold preservation and to demonstrate the protective effect of vascular endothelial growth factor (VEGF) on SEC injury, including apoptotic changes. METHODS: Isolated SECs and liver tissue of Wistar rats were cold-preserved in University of Wisconsin (UW) solution, and the protective effect of VEGF was then investigated. Isolated SECs were cultured for 24 hr, and divided into the following 3 groups: Group A, in which the cells were cultured for an additional 27 hr, Group B, in which the cells were cold-preserved in UW solution for 3 hr, and then recultured for 24 hr, and Group C, in which 20 ng/ml of VEGF was added to both the culture medium and the UW solution of cells cultured according to the Group B protocol. Each group of SECs was morphologically examined using the phase contrast microscopic method and the transmission electron microscopic method (TEM), and quantitatively analyzed using the WST-1 assay. Rat livers were cold-preserved in UW solution and divided into the VEGF(+) group and the VEGF(-) group, depending on whether VEGF was added or not. Each group of livers were analyzed by scanning electron microscopic method (SEM) after 24 hr of preservation. The hyaluronic acid uptake rate (HUR) was also determined after 6 hr of preservation. After 24 hr of preservation and 6 hr of reperfusion, tissues were examined by TEM and by the terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay. RESULTS: The phase contrast microscopic method and the WST-1 assay showed a protective effect of VEGF against the injury to isolated SECs during cold preservation and subsequent reculturing. Apoptosis was detected immediately by TEM after isolation of SECs, and the number of apoptotic cells increased with the incubation time. This increase was accelerated after cold preservation. The scanning electron microscopic method and the hyaluronic acid uptake rate showed a protective effect of VEGF against SEC damage in the cold-preserved livers. In the liver tissue, the TEM and the TUNEL assay detected apoptosis of SECs only after cold preservation and subsequent reperfusion. VEGF suppressed the apoptosis of SECs induced by cold preservation in both isolated cells and liver tissue. CONCLUSIONS: We demonstrated that SEC damage in the cold preservation of liver tissue was caused mainly by apoptosis, which required subsequent reperfusion. Moreover, isolated SECs showed spontaneous occurrence of apoptotic changes during culture, and these changes were accelerated by the preceding cold preservation. This is the first report to demonstrate the apoptotic changes of SECs seen here were inhibited by VEGF.     

Comparison of UW solution and Euro-Collins solutions for cold preservation of human liver grafts

University of Wisconsin solution, a new organ preservation medium, is reported to extend the period of cold storage. In order to evaluate the efficacy of UW solution in human liver preservation we compared 58 donor liver grafts preserved in Euro-Collins (EC) solution. All livers were harvested in a similar manner. Donor and recipient characteristics in the two groups were comparable. The mean preservation time of the UW solution was 11.5 +/- 4.2 hr (range 3-20 hr), significantly longer than the EC mean preservation time of 4.9 +/- 1.6 hr (2-9.6 hr) (P = 0.0001). Evaluation of mean postoperative liver function tests and coagulation factors on days 1-7 showed no statistical difference between the two groups. There was one primary graft nonfunction in the EC group and none with the UW organs. Hepatic artery thrombosis was similar in each group. The incidence of early retransplantation was similar. Three-month graft survival was 81% in the UW group vs. 73% in the EC group. Patient survival at three months was 87% with the UW organs and 84% with the EC organs. We conclude that cold storage of liver grafts in the UW solution has allowed for significantly longer preservation, permitting transplantation to be performed under semielective conditions and procurement of organs from much further distances. Grafts stored in UW solution perform as well as those stored in Euro-Collins, with no significant difference in liver function abnormalities postoperatively.