UW solution: a promising tool for cryopreservation of primarily isolated rat hepatocytes

Cryopreserved hepatocytes are a ready source of metabolic and synthetic functions for hepatocyte transplantation and bioartificial livers. In this study, we evaluated a cytoprotective effect of University of Wisconsin (UW) solution during cryopreservation of rat hepatocytes. We also investigated the feasibility of lentivirus‐based gene transfer into thawed hepatocytes after cryopreservation. Primary rat hepatocytes were isolated using a two‐step collagenase perfusion technique, and the resulting hepatocytes with more than 85% viability assessed by a trypan blue exclusion test were subjected to the present study. These cells were subjected to the present study. Cells were cryopreserved with UW solution containing 10% fetal bovine serum (FBS) with 12% dimethylsulfoxide (DMSO) (group 1, G1), Cellbanker solution (group 2, G2), and 10% FBS‐containing Dulbecco modified Eagle medium (DMEM) with 12% DMSO (group 3, G3). After thawing the cryopreserved hepatocytes, cell viability, plating efficiency, morphological appearance, and ammonia clearance activity were determined for each group. The efficacy of lentivirus‐mediated Escherichia coli LacZ gene delivery was evaluated. Hepatocyte viabilities after 3‐ and 7‐day cryopreservation were 73.2% and 62.5% for G1, 57.5% and 46.5% for G2, and 57.3% and 41.5% for G3, respectively. Plating efficiency and ammonia clearance activity were improved in G1 hepatocytes compared to G2 and G3 cells. Lentiviral transfer of a LacZ gene was confirmed in the thawed hepatocytes after cryopreservation by an X‐gal stain assay.     

New preservation strategies for preventing liver grafts against cold ischemia reperfusion injury

BACKGROUND: In spite of improvements in University of Wisconsin (UW) preservation solution, the injury from grafts during cold storage is an unresolved problem in liver transplantation. The aim of the present study was to evaluate the beneficial effect on ischemia-reperfusion injury associated with liver transplantation of the inversion of K(+) and Na(+) concentrations and the replacement of hydroxyethyl starch (HES) by polyethylene glycol (PEG) in UW preservation solution. METHODS: Using an orthotopic liver transplantation model, the effects on rat liver preservation of a modified preservation solution (UW-PEG) were evaluated, based on the inversion of K(+) and Na(+) concentration and the replacement of HES by PEG 35 kDa (0.03 mmol/L) in UW preservation solution. RESULTS: The use of UW-PEG preservation solution ameliorated the biochemical and histological parameters of hepatic damage. Thus, at 24 h after transplantation, transaminase levels were reduced significantly when livers were preserved during 8 h in UW-PEG preservation solution compared with the original UW solution. In addition, histological findings revealed fewer and smaller areas of hepatocyte necrosis. The benefits of UW-PEG solution cannot be explained by modifications in oxidative stress or neutrophil accumulation associated with liver transplantation. However, the results of hepatic and portal blood flow indicated that the benefits of this modified preservation solution, UW-PEG were associated with improvements in the microcirculatory disorders after reperfusion. CONCLUSIONS: The UW-PEG solution, while retaining all the advantages of UW solution, improved hepatic ischemia-reperfusion injury associated with liver transplantation.     

心脏移植供心的心肌保护研究

目的总结4例心脏移植的供心保护经验。方法缩短热缺血时间,切除供心时先以冷晶停跳液500 ml灌注,使供心迅速停搏,心包内放入冰屑使供心降温,切除供心后,再灌注0-4℃UW保存液1000 ml(6-8分钟灌注完),用浸泡法低温保存供心,4例供心热缺血时间分别为3、4、6、6分钟,冷缺血时间分别为220、415、215、226分钟。结果升主动脉开放后,3例自动复跳,1例电击除颤后复跳,围手术期供心功能良好,术后患者康复顺利,现心功能均为Ⅰ级。结论用此方法可使供心得到良好的心肌保护。     

Advantages of Celsior solution in graft preservation from non-heart-beating donors in a canine liver transplantation model.

BACKGROUND: The optimal method for preserving livers from non-heart-beating donors (NHBD) is still unknown. We compared the Celsior solution, a new extracellular-type, low-potassium, low-viscosity preservation solution, with the University of Wisconsin (UW) solution in a canine orthotopic liver transplantation from NHBD. MATERIALS AND METHODS: Fourteen adult mongrel dogs, weighing 9 to 17 kg, were divided into two groups: the Celsior or the UW group (n = 7 each). The thoracic descending aorta and supradiaphragmatic inferior vena cava were cross-clamped for 20 min to induce warm ischemia as a NHBD model. The liver was flushed with the respective cold preservation solution and then stored at 4 degrees C for 4 h. The grafts were transplanted using the piggy-back technique under portal decompression by leaving the native right lobe as a temporary shunt. RESULTS: The duration of liver flushing out (min) was shorter (P < 0.05), and the serum glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, lactate dehydrogenase, lactate, and alpha-glutathione S-transferase concentrations 2 and 6 h after reperfusion of the graft (RPF) were lower (P < 0.05) in the Celsior group than in the UW group. Hepatic tissue blood flow (HTBF) did not deteriorate as much (P < 0.05) in the Celsior group. The serum endothelin-1 level was lower (P < 0.05) in the Celsior group 2 h after RPF. Histopathology of liver specimens revealed portal congestion and hepatocyte necrosis with neutrophil infiltration in the UW group, while these findings were mild in the Celsior group. CONCLUSIONS: The Celsior solution improves vascular endothelial injury in livers from NHBDs and may have advantages in graft flush and preservation of grafts from NHBDs.     

In vitro function of porcine carotid arteries preserved in UW,HTK and Celsior solutions

We compared the efficacy of histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin (UW) solution with Celsior solution using hypothermically-preserved porcine carotid arteries and studied the importance of different components of these solutions by preserving carotid arteries in modified HTK solutions. Excised carotid arteries were stored at 4 degrees C in 0.9% (w/v) NaCl, UW, HTK, Celsior, or a modified HTK solution for up to 14 days. Preservation-induced changes in smooth muscle cell and endothelial cell function were determined using an organ bath for isometric tension recording. Short-term preservation (1-3 days) in UW, HTK and Celsior did not significantly alter contractile and relaxation responses of arterial segments when compared to freshly-excised segments, but significantly impaired these responses in arterial segments stored in 0.9% (w/v) NaCl solution. Long-term hypothermic preservation of arterial segments (7 and 14 days) in 0.9% (w/v) NaCl and HTK solution almost completely abolished all responses, but only slightly reduced the responses of arterial segments stored in UW solution. Intermediate results were obtained for Celsior. Modifying HTK by replacement of chloride for sulfate and phosphate resulted in improved contractile and relaxation responses after long-term preservation. With respect to smooth muscle and endothelial function, UW is superior to HTK and Celsior and the absence of chloride or presence of sulfate and phosphate plays a relevant role in this in vitro model of hypothermic preservation of porcine carotid arteries.     

In situ perfusion and UW solution used for storage did not decrease the incidence of ATN in kidneys harvested from hemodynamically unstable donors

Abstract The incidence of acute tubular necrosis ATN after cadaveric kidney transplantation in our centre has been in the range of 50%. A prospective study was carried out in 1991 and 1992 to assess the effect of in situ perfusion and hypothermic storage of kidneys harvested from brain-dead haemodynamically stable and unstable donors. Three litres of Ringer's solution were used for in situ perfusion. In 40 cases, the kidneys were stored in Euro-Collins (EC) solution and in the other 78 cases, in University of Wisconsin (UW) solution. Among the factors that could contribute to ATN, we analysed warm ischaemia time, anastomosis time and cold storage time. Function was considered to be delayed if the patient required posttransplantation dialysis. The donors were considered haemodynamically unstable when hypotension before harvesting was present (BP < 70 mm Hg over 2 h) despite high doses (> 15 μg/kg per minute) of dopamine or when cardiac arrest occurred at the time of harvesting and oliguria had been present for at least 2 h. Haemodynamically stable donors with a BP greater than 80 mm Hg had a normal diuresis. In all donors in this group the dose of dopamine was lower than 10 μg/kg per minute. The study showed that storage in UW solution did not influence the incidence of ATN in kidneys harvested from haemodynamically unstable donors. Differences observed in our study were due to haemodynamic status preceding donor nephrectomy and length of cold storage time.     

Optimal pH for simple cold storage or machine perfusion of dog kidneys with UW solution

Metabolic suppression by temperature is a key to successful organ preservation. Additional methods for inducing metabolic suppression may further improve organ preservation. Extracellular acidosis has been shown to suppress warm anoxic injury to various isolated cells. Acidosis may suppress enzymes with a pH optimum at the pH of the cytosol (pH 7.3). In this study, the combination of hypothermia and acidosis was used to determine if it would improve renal preservation. Dog kidneys were cold-stored (CS) for 48 h in University of Wisconsin (UW) solution with the pH adjusted to 6.4, 6.8, 7.4, or 7.8. Kidneys were also machine-perfused (MP) for 3 days with the gluconate perfusion solution (Belzer's machine perfusion solution, MPS) at pHs similar to those tested for CS. Renal function (serum creatinine, SCr) and survival were recorded in immediate contralateral nephrectomized recipients. On the basis of maximum SCr values, kidneys preserved by CS or MP were best preserved at pHs of 7.4 or 7.8. At a pH of 6.8, SCr values were elevated and returned to normal at a slower rate than in those preserved at higher pHs. This study shows that acidosis is not cytoprotective to cold-stored dog kidneys and causes preservation/reperfusion injury. Received: 25 August 1997 Received after revision: 18 November 1997 Accepted: 14 January 1998     

胰导管保护法提高胰岛获取量的实验研究

目的:探讨联合应用胰蛋白酶抑制剂和UW液的胰导管保护方法能否提高保存6 h和24 h大鼠胰腺中胰岛的获取量。方法:从雄性SD大鼠分离得到胰腺,分成5组:组1:新鲜的未用胰导管保护和UW液保存(n=8);组2:末用胰导管保护方法仅用UW液保存6 h(n=8);组3:应用胰导管保护方法并用UW液保存6 h(n=8);组4:未用胰导管保护方法仅用UW液保存24 h(n=8):组5:应用胰导管保护方法并用UW液保存24 h(n=8)。结果:分离纯化后每组的胰岛数量分别为1 820±639,585±151,1 496±676,567±182,1 442±857(胰岛当量±标准差);胰岛存活率分别为(93.8±4.9)%,(82.1±5.6)%,(89.6±4.9)%,(77.9±5.0)%,(86.7±7.1)%;本实验中未应用胰导管保护方法组和应用胰导管保护方法组间差异有显著性(P<0.05,组2和组3相比较,组4和组5相比较)结论:联合应用胰蛋白酶抑制剂和UW液的胰导管保护方法提高了保存6 h和保存24 h胰腺中的胰岛获取量。     

Effect of cardioplegic and organ preservation solutions and their components on coronary endothelium-derived relaxing factors

Cardioplegic (and organ preservation) solutions were initially designed to protect the myocardium (cardiac myocytes) during cardiac operation (and heart transplantation). Because of differences between cardiac myocytes and vascular (endothelial and smooth muscle) cells in structure and function, the solutions may have an adverse effect on coronary vascular cells. However, such effect is often complicated by many other factors such as ischemia-reperfusion injury, temperature, and perfusion pressure or duration. To evaluate the effect of a solution on the coronary endothelial function, a number of points should be taken into consideration. First, the overall effect on endothelium should be identified. Second, the effect of the solution on the individual endothelium-derived relaxing factors (nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor) must be distinguished. Third, the effect of each major component of the solution should be investigated. Lastly, the effect of a variety of new additives in the solution may be studied. Based on available literature these issues are reviewed to provide information for further development of cardioplegic or organ preservation solutions.