An Examination of Packing Methods for Grafts to Prevent Freezing Injury during Transportation for Liver Transplantation.

BACKGROUND: University of Wisconsin solution (UW) may freeze at temperatures below -0.7 °C, damaging the graft. The present study assessed the effectiveness of the liver graft package protocol, which recommends filling a package with sufficient liquid to prevent grafts from sustaining freezing injury. METHODS: We filled ice cubes at two temperatures (-80 and -20 °C) around packages and performed a comparative study with four groups based on the temperature and filling of the second layer with lactated Ringer's solution (LR) (A: -80 °C, LR-; B: -80 °C, LR+; C: -20 °C, LR-; D: -20 °C, LR+). The bovine liver was used as a graft and preserved for 6 h in the first isolation bag filled with UW. RESULTS: While temperatures dropped below -0.7 °C at some points for 6 h in groups A, B, C, they never dropped to -0.7 °C in group D. The macroscopic findings in groups A, B, C showed freezing of the UW and grafts, but no such results in group D. A pathological study including electron microscopy showed freezing injury in groups A, B, and C but no significant changes in group D. CONCLUSIONS: The graft package protocol prevents freezing of the UW and liver grafts.

Excellent Islet Yields after 18-h Porcine Pancreas Preservation by Ductal Injection, Pancreas Preservation with MK Solution, Bottle Purification, and Islet Purification Using Iodixanol with UW Solution and Iodixanol with MK Solution.

Successful islet isolation is the key to successful islet transplantation. Our group recently modified the islet isolation protocol to include pancreatic ductal injection of the preservation solution, pancreas storage in modified extracellular-type trehalose-containing Kyoto (MK) solution, and use of an iodixanol-based purification solution and bottle purification. In this study, we applied these methods to porcine islet isolation after 18-h pancreas preservation and compared two solutions with different compositions in bottle purification. Islet yield before purification was 651,661 ± 157,719 islet equivalents (IE) and 5576 ± 1538 IE/g pancreas weight. An IU solution was made by adding iodixanol to University of Wisconsin solution and an IK solution was made by adding iodixanol to MK solution. The efficacy of the two solutions for islet isolation was compared. There were no significant differences between the two purification methods with regard to islet yield, survival rate, purity, score, or stimulation index. These results indicate that our isolation protocol produces efficient islet yields from prolonged cold-stored pancreas and that IU and IK solutions are equally useful for islet purification.

Preservation of lower extremity spare parts using the University of Wisconsin solution.

The management of a mangled limb is a challenging endeavor. With the advancement in microsurgery, spare parts surgery (fillet flaps) has gained recent interest. In the context of lower extremity amputation secondary to trauma, viable spare parts can provide stump soft tissue coverage, potentially preserving critical length and obviating above-knee amputations. Commonly, spare parts surgery is performed in the acute setting but tissue preservation is sometimes necessary. The authors report their experience preserving a fillet flap of a mangled lower extremity for 48 h using the University of Wisconsin solution. A sole fillet flap and a split-thickness skin graft were harvested and preserved from the amputated lower extremity (based on the posterior tibial artery and vein). Stump coverage was achieved by anastomosing the fillet flap to the proximal posterior tibial artery and vein. This solution has not been previously described for preservation of fillet flaps.

University of Wisconsin vs normal saline solutions for preservation of blood vessels of brain dead donors: A histopathological study.

OBJECTIVE: To compare the cellular changes of harvested arteries which were preserved in normal saline (NS) and the standard and routinely used University of Wisconsin (UW) solution. METHODS: This experimental study was conducted on 20 brain dead patients. The femoral and iliac arteries were bilaterally removed and were placed in NS and UW solutions. The vascular change indices including endothelial detachment (ED), medial detachment (MD), and internal elastic membrane disruption (IEMD) were surveyed for each preserver in the first, 5th, 10th, and 21st day. RESULTS: The mean age of the included patients was 32.28 ± 8.88 years, and there were 13 (65.0%) men and 7 (35.0%) women among the patients. The NS and UW preservation solutions were comparable regarding the indices of vascular changes at first, 5th, and 10th day of the study. Only in 21st day of the study, there was a significant difference between 2 group regarding MD changes (P = .049). CONCLUSION: The results of this in vitro study demonstrated that NS can be used as a worthy preserver for harvested vessels for up to 21 days, especially in resource-limited transplantation centers.

Storage effect and transplant outcomes of University of Wisconsin preservation solution and histidine-tryptophan-ketoglutarate preservation solution on liver allografts: a Meta-analysis / 中华消化外科杂志

Objective To systematically evaluate the storage effect and transplant outcomes of University of Wisconsin (UW) preservation solution and histidine-tryptophan-ketoglutarate (HTK) preservation solution on liver allografts.Methods Literatures were researched using PubMed,Embase (1980-),Ovid Medline (1948-),The Cochrane Library,Wanfang database,VIP database from the database establishement to October 2015 with the key words including organ preservation,storage solutions,Histidine-tryptophan-ketoglutarate or HTK,custodial,bretschneider,University of Wisconsin,UW solution,viaspan,cardiosol,belzer solution,hepatic transplantation,liver transplantation,viscera transplantation,liver grafts,hepatic grafts,liver allografts,hepatic allografts,器官移植,器官保存液,UW,HTK,肝移植and比较.Two reviewers independently screened literatures,extracted data and assessed the risk of bias.All the patients using UW and HTK preservation solutions were respectively allocated into the UW group and HTK group.Count data were represented as the odds ratio (OR) and measurement data were represented as the standardized mean difference (SMD) and 95％ confidence interval (CI).The heterogeneity of the studies was analyzed using the I2 test.Results Eleven literatures were retrieved,and the total sample size were 34 475 patients including 25 248 in the UW group and 9 227 in the HTK group.The results of Meta analysis showed that there were no statistically significant differences in the primary transplants nonfunction,retransplant rate and 1-year grafts overall survival rate between the 2 groups (OR =1.18,0.84,0.97,1.02,95％ CI:0.55-2.57,0.47-1.50,0.66-1.42,0.66-1.58,P ＞0.05).There were also no statistically significant differences in the levels of alanine transaminase (ALT),aspartate transaminase (AST),total bilirubin (TBil) at postoperative day 1 between the 2 groups (SMD =-0.19,-O.30,0.30,95％ CI:-0.62-0.23,-0.70-0.10,-0.01-0.61,P ＞0.05).There were no statistically significant differences in the postoperative prothrombin time(PT) and alkaline phosphatase(ALP) between the 2 groups (P ＞0.05) and in the incidence of postoperative biliary complications between the 2 groups (OR =1.49,95％ CI:0.97-2.30,P ＞ 0.05).Conclusion There is similar storage effect between UW and HTK preservation solutions on liver allografts,and no difference in the transplant outcomes.

Polyethylene glycol protects primary hepatocytes during supercooling preservation.

Cold storage (at 4°C) offers a compromise between the benefits and disadvantages of cooling. It allows storage of organs or cells for later use that would otherwise quickly succumb to warm ischemia, but comprises cold ischemia that, when not controlled properly, can result in severe damage as well by both similar and unique mechanisms. We hypothesized that polyethylene glycol (PEG) 35 kDa would ameliorate these injury pathways and improve cold primary hepatocyte preservation. We show that reduction of the storage temperature to below zero by means of supercooling, or subzero non-freezing, together with PEG supplementation increases the viable storage time of primary rat hepatocytes in University of Wisconsin (UW) solution from 1 day to 4 days. We find that the addition of 5% PEG 35 kDa to the storage medium prevents cold-induced lipid peroxidation and maintains hepatocyte viability and functionality during storage. These results suggest that PEG supplementation in combination with supercooling may enable a more optimized cell and organ preservation.

Tetramethylpyrazine protects Schwann cells from ischemia-like injury and increases cell survival in cold ischemic rat nerves

Tetramethylpyrazine (TMP), a major active ingredient of Ligusticum wallichi Franchat extract (a Chinese herb), exhibits neuroprotective properties in ischemia. In this study, we assessed its protective effects on Schwann cells (SCs) by culturing them in the presence of oxygen glucose deprivation (OGD) conditions and measuring cell survival in cold ischemic rat nerves. In the OGD-induced ischemic injury model of SCs, we demonstrated that TMP treatment not only reduced OGD-induced cell viability losses, cell death, and apoptosis of SCs in a dose-dependent manner, and inhibited LDH release, but also suppressed OGD-induced downregulation of Bcl-2 and upregulation of Bax and caspase-3, as well as inhibited the consequent activation of caspase-3. In the cold ischemic nerve model, we found that prolonged cold ischemic exposure for four weeks was markedly associated with the absence of SCs, a decrease in cell viability, and apoptosis in preserved nerve segments incubated in University of Wisconsin solution (UWS) alone. However, TMP attenuated nerve segment damage by preserving SCs and antagonizing the decrease in nerve fiber viability and increase in TUNEL-positive cells in a dose-dependent manner. Collectively, our results indicate that TMP not only provides protective effects in an ischemia-like injury model of cultured rat SCs by regulating Bcl-2, Bax, and caspase-3, but also increases cell survival and suppresses apoptosis in the cold ischemic nerve model after prolonged ischemic exposure for four weeks. Therefore, TMP may be a novel and effective therapeutic strategy for preventing peripheral nervous system ischemic diseases and improving peripheral nerve storage.

Tetrametilpirazina (TMP), o principal componente do extrato de Ligusticum wallichi Franchat (erva chinesa), apresenta propriedades neuroprotetoras na isquemia. Nesse estudo, avaliamos seus efeitos protetores nas células de Schwann (SC), cultivando-as na presença de condições de depleção de oxigênio da glicose (OGD) e medindo a sobrevivência dos nervos de ratos isquêmicos pelo resfriamento. No modelo de lesão isquêmica em SC induzida por OGD, demonstramos que o tratamento com TMP não somente reduziu as perdas de viabilidade celular induzida por OGD, a morte celular, a apoptose de SC dose-dependente e inibiu a liberação de LDH, mas, também, suprimiu a infra-regulação do Vcl-2 e a supra-regulação de Bax e caspase-3, e inibiu a consequente ativação da caspase-3. No modelo de nervo isquêmico por resfriamento, observamos que a exposição prolongada ao resfriamento por quatro semanas estava, marcadamente, associada com a ausência de SC, com o decréscimo da viabilidade celular e a apoptose em segmentos de nervo incubados na solução da Universidade de Wisconsin apenas. Entretanto, a TMP atenuou o dano no segmento do nervo preservando SC e antagonizando a diminuição da viabilidade da fibra nervosa e o aumento das células TUNEL-positiva de modo dose-dependente. De forma conjunta, nossos resultados indicam que o TMP não só fornece efeitos protetores em um modelo de dano semelhante à isquemia de SC de ratos cultivados pela regulação de BCl-2, Bax e caspase 3, mas, também, aumenta a sobrevivência celular e suprime a apoptose no modelo de isquemia por resfriamento por exposição prolongada por quatro semanas. Então, TMP pode ser uma estratégia terapêutica eficaz para prevenir doenças isquêmicas do sistema nervoso periférico e melhora a armazenagem do nervo periférico.

Comparative study of effect of mechanical perfusion and simple cold preservation on DCD pancreas in pigs / 器官移植

Objective To compare the effect of mechanical perfusion and simple cold preservation on donation after cardiac death (DCD)pancreas in pigs.Methods Ten healthy pigs were randomized into simple cold preservation group and the mechanical perfusion group (5 pigs in each group ).DCD model was established in pigs.The pancreas was cut and stored in University of Wisconsin solution (UW solution).The pancreas of the simple cold preservation group was preserved with simple UW solution and that of the mechanical perfusion group was preserved by mechanical perfusion.Specimen was collected from pancreatic tail at 1,2,3,4,6 and 24 h to prepare tissue section.Then,the tissue section was stained with hematoxylin-eosin (HE).Histopathological examination was conducted and pathological score of the two groups was compared.Results Microthrombus in DCD pancreas of pig was removed at 180 min of mechanical perfusion and injury to pancreas islet caused by excessive perfusion was avoided.The pathological score of the mechanical perfusion group was (4.2 ±0.8)and that of the simple cold preservation group was (8.4 ±1.1),and the difference had statistical significance (P <0.05).Conclusions Mechanical perfusion may effectively remove thrombus in pancreatic vessels.Compared with simple cold preservation,mechanical perfusion may maintain the integrity of pancreas islet better after the preservation of the same period of time.

The application of polymerized porcine hemoglobin (pPolyHb) in the rat small bowel preservation.

Small bowel transplantation (SBTx) has become a standard clinical treatment for short bowel syndrome or irreversible intestinal function failure. Optimum preservation of the organ is essential for the success of transplantation. In this study, pPolyHb was used as an additive to hypertonic citrate adenine solution (HCA) to provide oxygen for rat small bowel transplant. Rat small bowels were preserved in HCA, HCA with pPolyHb, and University of Wisconsin solution (UW) for 12, 24, and 36 h, respectively. The results suggested that the preservation effect of HCA with pPolyHb was comparable with the UW solution, and more effective than the HCA solution.

Effect of melatonin on kidney cold ischemic preservation injury.

Melatonin is a potent free radical scavenger of reactive oxygen species, nitric oxide synthase inhibitor and a well-known antioxidant secreted from pineal gland. This hormone has been reported to protect tissue from oxidative damage. In this study, we aim to investigate the effect of melatonin on kidney cold ischemia time when added to preservation solution. Thirty male Wistar albino rats were divided equally into three groups; Ringer Lactate (RL) solution, University of Wisconsin (UW) solution with and without melatonin. The serum Lactate Dehydrogenase (LDH) activities of the preservation solutions at 2(nd), 24(th), 36(th), and 48(th) hours were determined. Tissue malondialdehyde (MDA) levels were also measured and a histological examination was performed at 48(th) hour. Melatonin that added to preservation solution prevented enzyme elevation and decreased lipid peroxidation in preservation solution when compared to the control group (p<0.05). The histological examination revealed that UW solution containing melatonin significantly prevented the kidney from pathological injury (p<0.05). Melatonin added to preservation solutions such as UW solution seemed to protect the tissue preserved effectively from cold ischemic injury for up to 48 hour.

MicroRNA profiles in graft preservation solution are predictive of ischemic-type biliary lesions after liver transplantation.

BACKGROUND & AIMS: Ischemic-type biliary lesions (ITBL) are the second most common cause of graft loss after liver transplantation. Though the exact pathophysiology of ITBL is unknown, bile duct injury during graft preservation is considered to be a major cause. Here we investigated whether the release of cholangiocyte-derived microRNAs (CDmiRs) during graft preservation is predictive of the development of ITBL after liver transplantation. METHODS: Graft preservation solutions (perfusates) and paired liver biopsies collected at the end of cold ischemia were analysed by RT-qPCR for CDmiR-30e, CDmiR-222, and CDmiR-296 and hepatocyte-derived miRNAs (HDmiRs) HDmiR-122 and HDmiR-148a. MicroRNAs in perfusates were evaluated on their stability by incubation and fractionation experiments. MicroRNA profiles in perfusates from grafts that developed ITBL (n=20) and grafts without biliary strictures (n=37) were compared. RESULTS: MicroRNAs in perfusates were proven to be stable and protected against degradation by interacting proteins. Ratios between HDmiRs/CDmiRs were significantly higher in perfusates obtained from grafts that developed ITBL (p<0.01) and were identified as an independent risk factor by multivariate analysis (p<0.01, HR: 6.89). The discriminative power of HDmiRs/CDmiRs in perfusates was validated by analysis of separate brain death- (DBD) and cardiac death donors (DBD; p ≤ 0.016) and was superior to expression in liver biopsies (C=0.77 in perfusates vs. C<0.50 in biopsies). CONCLUSIONS: This study demonstrates that differential release of CDmiRs during graft preservation is predictive of the development of ITBL after liver transplantation. This provides new evidence for the link between graft-related bile duct injury and the risk for later development of ITBL.

Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions.

INTRODUCTION: Endothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed endothelial barrier integrity and viability as function of preservation temperature and perfusion solution, and hypothesized that endothelial cell preservation at subnormothermic conditions using metabolism-supporting solutions constitute optimal preservation conditions. METHODS: Human umbilical vein endothelial cells (HUVEC) were preserved at 4-37°C for up to 20 h using Ringer's lactate, histidine-tryptophan-ketoglutarate solution, University of Wisconsin (UW) solution, Polysol, or endothelial cell growth medium (ECGM). Following preservation, the monolayer integrity, metabolic capacity, and ATP content were determined as positive parameters of endothelial cell viability. As negative parameters, apoptosis, necrosis, and cell activation were assayed. A viability index was devised on the basis of these parameters. RESULTS: HUVEC viability and barrier integrity was compromised at 4°C regardless of the preservation solution. At temperatures above 20°C, the cells' metabolic demands outweighed the preservation solutions' supporting capacity. Only UW maintained HUVEC viability up to 20°C. Despite high intracellular ATP content, none of the solutions were capable of sufficiently preserving HUVEC above 20°C except for ECGM. CONCLUSION: Optimal HUVEC preservation is achieved with UW up to 20°C. Only ECGM maintains HUVEC viability at temperatures above 20°C.

Improved hypothermic short-term storage of isolated mouse islets by adding serum to preservation solutions.

Preserving isolated islets at low temperature appears attractive because it can keep islet quantity comparable to freshly isolated islets. In this study, we evaluated the effect of serum as an additive to preservation solutions on islet quality after short-term hypothermic storage. Isolated mouse islets were preserved at 4°C in University of Wisconsin solution (UW) alone, UW with serum, M-Kyoto solution (MK) alone or MK with serum. We then assessed islet quantity, morphology, viability and function in vitro as well as in vivo. Islet quantity after storage in all four solutions was well maintained for up to 120 h. However, islets functioned for different duration; glucose-stimulated insulin release assay revealed that the duration was 72 h when islets were stored in UW with serum and MK with serum, but only 24 h in UW alone, and the islet function disappeared immediately in MK alone. Viability assay confirmed that more than 70% islet cells survived for up to 48 h when islets are preserved in UW with serum and MK with serum, but the viability decreased rapidly in UW alone and MK alone. In in vivo bioassays using 48-h preserved isogeneic islets, all recipient mice restored normal blood glucose concentrations by transplants preserved in UW with serum or MK with serum, whereas 33.3% recipients and no recipient restored diabetes by transplants preserved in UW alone and in MK alone respectively. Adding serum to both UW and MK improves their capability to store isolated islets by maintaining islet functional viability.