Cold Preservation Research Articles

VEGF-C attenuates ischemia reperfusion injury of liver graft in rats

BackgroundVascular endothelial growth factor receptor-3 (VEGFR-3) / vascular endothelial growth factor -c (VEGF-C) signaling is reported to negatively regulate TLR4-triggered inflammation of macrophages. This study aims to clarify whether the VEGFR-3/VEGF-C signaling can suppress Kupffer cells (KCs) activation and attenuate hepatic ischemia-reperfusion injury (IRI) after liver transplantation. MethodsA rat model of liver transplantation was performed. Donor livers were perfused with VEGF-C injection via portal vein during cold preservation, and controls were perfused with UW solution. Serum levels of alanine transaminase (ALT), total bilirubin (TBIL) and inflammatory cytokines, as well as histology were analyzed after 24 h. KCs were isolated from grafts, RT-PCR and immunofluorescence were used to evaluate polarization-specific marker genes, western bolt was employed to assess the expression of suppressor of cytokine signaling 1(SOCS1) and phosphorylated glycogen synthase kinase 3β (p-GSK3β), and EMSA was utilized to quantify the NF-κB transcriptional activity. ResultsCompared with controls, VEGF-C perfusion reduced ALT and TBIL levels and alleviated liver damage. Furthermore, VEGF-C perfusion suppressed serum proinflammatory cytokines secretion and increased IL-10.In addition, the VEGFR-3 mRNA of KCs was increased after reperfusion. VEGF-C perfusion suppressed NF-κB activity and up-regulated the expression of SOCS1 and p-GSK3β in KCs, and shifted the M1/M2 balance toward an anti-inflammatory profile. ConclusionExogenous VEGF-C protects liver graft from IRI by regulating the inflammatory response and modifying polarization of KCs.

Oxygenated UW Solution Decreases ATP Decay and Improves Survival After Transplantation of DCD Liver Grafts

Donation after circulatory death (DCD) liver grafts are known to be predisposed to primary nonfunction and ischemic cholangiopathy. Many DCD grafts are discarded because of older donor age or long warm ischemia times. Thus, it is critical to improve the quality of DCD liver grafts. Here, we have tested whether an enriched oxygen carrier added to the preservation solution can prolong graft survival and reduce biliary damage. We assessed the adenosine triphosphate (ATP) content decay of mouse liver grafts after cold ischemia, warm ischemia, and combined warm+cold ischemia. In addition, we used a rat model of liver transplantation to compare survival of DCD grafts preserved in high-oxygen solution (preoxygenated perfluorocarbon [PFC] + University of Wisconsin [UW] solution) versus lower oxygen solution (preoxygenated UW solution). Adenosine triphosphate levels under UW preservation fall to less than 10% after 30 minutes of warm ischemia. Preoxygenated UW solution with PFC reached a significantly higher PaO2. After 45 minutes of warm ischemia in oxygenated UW + PFC solution, grafts showed 63% higher levels of ATP (P = 0.011). In addition, this was associated with better preservation of morphology when compared to grafts stored in standard UW solution. Animals that received DCD grafts preserved in higher oxygenation solution showed improved survival: 4 out of 6 animals survived long-term whereas all control group animals died within 24 hours. The additional oxygen provided by PFC during static cold preservation of DCD livers can better sustain ATP levels, and thereby reduce the severity of ischemic tissue damage. PFC-based preservation solution extends the tolerance to warm ischemia, and may reduce the rate of ischemic cholangiopathy.

Uncontrolled deceased cardiac donation: An unutilized source for organ transplantation in the United States.

The practice of uncontrolled donation after cardiac death (uDCD) has been met with tepid interest within the United States transplant community. Hesitancy stems largely from fears of eroding public trust due to complex ethical issues involving consent. Beyond ethical concerns, uDCD creates unique logistic challenges to obtain and to preserve organs within a short time frame. This mandates that organ recovery centers be able to rapidly mobilize, and that traditional cold preservation techniques may be inadequate. Proof of effective uDCD organ recovery comes from several European nations, and the frequency of its use is increasing due to early promising results. These scarce resources provide life-saving organs to desperate transplant candidates who otherwise experience high morbidity and mortality on a transplant waitlist. The objective of this review will be to provide an overview of the European experience with uDCD and discuss the unique ethical and logistic challenges associated with its implementation in the United States. Given existing models for it successful use, uDCD remains a poorly utilized source of donors in the United States at this time.

α-ketoglutarate attenuates ischemia-reperfusion injury of liver graft in rats

ObjectiveThe α-ketoglutarate (αKG), a metabolite of glutaminolysis, is reported to orchestrate macrophages activation. This study aims to clarify whether the αKG / glutaminolysis metabolism can suppress Kupffer cells (KCs) activation during liver transplantation and attenuate hepatic ischemia-reperfusion injury (IRI). MethodsDonor livers were perfused with DM-αKG (a cell-permeable analog of αKG) or BPTES (an inhibitor of glutaminase 1) via portal vein during cold preservation, and controls were perfused with UW solution. Then, a rat model of liver transplantation was performed. Serum levels of alanine transaminase (ALT), total bilirubin (TBIL) and inflammatory cytokines, as well as histology, were analyzed after 24 h. KCs were isolated from grafts. RT-PCR and immunofluorescence were used to evaluate polarization-specific marker genes. Western bolt was employed to assess the expression of phosphorylation of glycogen synthase kinase 3β (p-GSK3β) and suppressor of cytokine signaling 1 (SOCS1). EMSA was utilized to quantify the NF-κB transcriptional activity. ResultsCompared with controls, DM-αKG perfusion decreased ALT and TBIL levels, alleviated liver damage, and reduced apoptosis, while BPTES group showed higher ALT and TBIL levels, severe damage and more apoptosis. Furthermore, DM-αKG perfusion suppressed NF-κB activity, up-regulated the expression of p-GSK3β and SOCS1 in KCs, and shifted the M1/M2 balance toward an anti-inflammatory profile. Besides, DM-αKG suppressed serum pro-inflammatory cytokines secretion and increased IL-10. ConclusionsαKG produced by glutaminolysis protects liver graft from IRI by regulating the inflammatory response and modifying the polarization of KCs.

Normothermic Machine Perfusion Protects Against Liver Ischemia-Reperfusion Injury During Reduced-Size Liver Transplantation in Pigs.

BackgroundNormothermic machine perfusion (NMP) preservation is superior to cold preservation during reduced-size liver transplantation (RSLT) in pigs. However, the mechanism of this protective effect has not been explained. We aimed to compare the effects of NMP preservation with that of cold preservation (CS) in protecting against ischemia-reperfusion injury (IRI) during RSLT in pigs.Material/MethodsTwenty-four healthy Bama miniature pigs were randomized into 2 groups: 1) the NMP group in which donor livers harvested without warm ischemia time and cardiac activity were connected to the NMP system to reduce liver size under normothermic conditions, and 2) the CS group in which donor livers harvested without warm ischemia time and cardiac activity were perfused using the University of Wisconsin (UW) solution and then preserved in the 0–4°C UW solution to reduce liver size under cold conditions. Livers were then transplanted without veno-venous bypass. Amounts of bile secretion for the NMP groups were recorded hourly. The serological indices were measured. Expressions of cytochrome C, caspase 3, and NF-κB p65 in liver tissue were observed.ResultsThe levels of bile secretions were gradually diminished from 16.50±2.66 mL/h before splitting to 6.35±1.24 mL/h after splitting. With the exception of TNF-α on postoperative day 2, overall, levels of TNF-α, IL-1, IL-6, and MDA were significantly lower in the NMP group versus CS group for all 5 days postoperatively. Finally, cytochrome C, caspase 3, and NF-κB p65 expressions were all significantly suppressed in the NMP group as compared with the CS group.ConclusionsMP preservation is superior to cold preservation in protecting against liver IRI during RSLT in pigs.

“Non-Touch” Vena Cava Technique as an Improvement in Combined Lung and Liver Procurement in Controlled Donation After Circulatory Death

The number of organs retrieved from donation after circulatory death (DCD) donors has continued to rise in recent years. The functional superiority of DCD organs is achieved when the lungs are perfused with cold perfusion and livers with normothermic regional perfusion (NRP). Thus, a precise surgical technique is required to combine thoracic and abdominal organ procurement.The technique used at our center consists of a rapid laparotomy and middle sternotomy, then the abdominal aorta (Ao) and abdominal inferior vena cava (VC) are cannulated and the descending thoracic Ao is cross-clamped. NRP is started at that point. As a variation of previously described techniques, the thoracic vena cava is not initially clamped in order to improve the return of blood volume to the NRP circuit. The pulmonary artery is cannulated to flush the lungs and the left atrial appendage is opened for drainage. After 120 minutes, NRP perfusion is stopped and the organs are flushed with cold preservation solution.In 2016, 3 livers and 6 lungs were harvested at our center using the technique described. After a minimum follow-up of 1 year, no evidence of biliary complications was observed.The combined procurement of lungs after room temperature perfusion and liver after NRP without initial clamping of the thoracic VC is feasible, with excellent function post-transplantation.

Gene Silencing With siRNA (RNA Interference): A New Therapeutic Option During Ex Vivo Machine Liver Perfusion Preservation

RNA interference (RNAi) is a natural process of posttranscriptional gene regulation that has raised a lot of attention culminating with the Nobel Prize in Medicine in 2006. RNAi-based therapeutics have been tested in experimental transplantation to reduce ischemia/reperfusion injury (IRI) with success. Modulation of genes of the innate immune system, as well as apoptotic genes, and those involved in the nuclear factor kappa B pathways can reduce liver injury in rodent liver pedicle clamping and transplantation models of IRI. However, in vivo use of RNAi faces limitations regarding the method of administration, uptake, selectivity, and stability. Machine perfusion preservation, a more recent alternative approach for liver preservation showing superior results to static cold preservation, could be used as a platform for gene interference therapeutics. Our group was the first to demonstrate uptake of small interfering RNA (siRNA) during liver machine preservation under both normothermic and hypothermic perfusion. Administering siRNA in the perfusion solution during ex vivo machine preservation has several advantages, including more efficient delivery, lower doses and cost-saving, and none/fewer side effects to other organs. Recently, the first RNAi drug was approved by the US Food and Drug Administration for clinical use, opening a new avenue for new drugs with different clinical applications. RNAi has the potential to have transformational therapeutic applications in several areas of medicine including transplantation. We believe that machine preservation offers great potential to be the ideal delivery method of siRNA to the liver graft, and future studies should be initiated to improve the clinical applicability of RNAi in solid organ transplantation.

Will the machine perfusion of the liver increase the number of donor organs suitable for transplantation?

Worldwide, there is a trend towards an increase in the number of patients waiting for liver transplantation, despite an increase in the total number of liver transplantation operations. Solving the problem of donor organ shortage is possible through the use of organs from marginal donors: organs removed after cardiac arrest, organs with a high percentage of steatosis, organs from donors over the age of 60 years. The main reason for refusing to use them is the risk of severe complications and an unfavorable outcome of the operation. Static cold preservation today is the main method of protecting donor organs from thermal damage, which possesses rather effective protective properties. At the same time, the duration of cold preservation has a limited time interval. There is always uncertainty about the viability of the organ. Modern methods for assessing donor organs such as donor history, laboratory data, visual examination and morphology, do not reliably predict liver function after transplantation. In this regard, the development of methods for preserving the organ after removing, which do not degrade the quality of the organ or even are capable of restoring the lost functions, is relevant. The machine perfusion of the liver is one of the new concepts aimed at solving this problem. The article highlights the international experience of using the machine perfusion of the donor liver over the past 15 years. Machine perfusion is a promising trend of transplantation development, which allows reducing the shortage of donor organs and improving their quality.

Direct comparison of the infarct-limiting and hemodynamic effects of various necroptosis inhibitors in the model of prolonged static cold storage of the donor rat heart

Objective . To investigate the effects of various necroptosis inhibitors on the morphological and functional characteristics of the myocardium in the model of prolonged cold preservation of the donor rat heart. Design and methods. Thirty eight Wistar rats were divided into the following groups: 1) control (n = 7), 2) dimethylsulfoxide (DMSO) (n = 7), 3) necrostatin-1s (n = 6), 4) necrosulfonamide (n = 6), 5) necrostatin-5 (n = 6), 6) necrostatin-1 (n = 6). Necroptosis inhibitors were administered by intraperitoneal injection 1 hour prior to the start of the experiment, previously dissolved in DMSO. HTK solution cooled to 4 °C was used as a preservative solution. The functional state of the myocardium was assessed by continuous recording of intraventricular pressure, heart rate and coronary flow velocity. Morphological changes in the myocardium were assessed by the size of myocardial necrosis, measured planimetrically after the end of 8-hour ischemia and 2-hour reperfusion. Results. All investigated necroptosis inhibitors significantly improved the morphological and functional state of the myocardium, which manifested itself in a significantly smaller amount of myocardial necrosis in the groups of necroptotic inhibitors in comparison with control groups and DMSO. Thus, in the groups of necrosulfonamide and necrostatin-1s, myocardial necrosis size was 31 ± 8,7 % and 27 ± 8,2 %, respectively; in necrostatin-5 and necrostatin-1 groups — 37 ± 9,2 % and 34 ± 7,8, respectively, which is lower than in control groups and DMSO (58 ± 8,6 % and 60 ± 7,6 %, respectively, p < 0,05). Also, diastolic intraventricular pressure was lower and pulse intraventricular pressure and coronary flow velocity were higher during the reperfusion period in the groups of necroptosis inhibitors compared to control group and DMSO group (p < 0,05). We did not find any significant differences between necroptosis inhibitors regarding cardioprotective activity in the model of prolonged cold preservation of the donor heart. Conclusions. All inhibitors of necroptosis equally improved the morphological and functional state of the myocardium in the model of prolonged static cold storage of the donor heart.

Cardiotrophin-1 Improves Kidney Preservation, Graft Function, and Survival in Transplanted Rats.

Cold ischemia-reperfusion injury is unavoidable during organ transplantation, and prolonged preservation is associated with poorer function recovery. Cardiotrophin-1 (CT-1) is an IL-6 family cytokine with cytoprotective properties. This preclinical study in rats tested whether CT-1 mitigates cold renal ischemia-reperfusion injury in the context of the transplantation of long-time preserved kidneys. Kidneys were flushed with cold (4°C) University of Wisconsin solution containing 0.2 μg/mL CT-1 and stored for several periods of time at 4°C in the same solution. In a second approach, kidneys were first cold-preserved for 6 hours and then were perfused with University of Wisconsin solution containing CT-1 (0, 16, 32, or 64 μg/mL) and further cold-preserved. Organ damage markers were measured in the kidneys at the end of the storage period. For renal transplantation, recipient consanguineous Fischer rats underwent bilateral nephrectomy and received a previously cold-preserved (24 hours) kidney as described above. Survival and creatinine clearance were monitored over 30 days. Cardiotrophin-1 in perfusion and preservation fluids reduced oxidative stress markers (superoxide anion and inducible nitric oxide synthase), inflammation markers (NF-κB and tumor necrosis factor-α), and vascular damage (vascular cell adhesion molecule-1) and activated leukemia inhibitory factor receptor and STAT-3 survival signaling. Transplantation of kidneys cold-preserved with CT-1 increased rat survival and renal function (ie, lower plasma creatinine and higher creatinine clearance) and improved kidney damage markers after transplantation (ie, lower superoxide anion, tumor necrosis factor-α, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 and higher NF-κB). Cardiotrophin-1 represents a novel therapeutic strategy to reduce ischemia-reperfusion and cold preservation injury to rescue suboptimal kidneys and, consequently, to improve the clinical outcomes of renal transplantation.

Advances in machine perfusion, organ preservation, and cryobiology: potential impact on vascularized composite allotransplantation.

In this review, we discuss novel strategies that allow for extended preservation of vascularized composite allografts and their potential future clinical implications for the field of vascularized composite allotransplantation (VCA). The current gold standard in tissue preservation - static cold preservation on ice - is insufficient to preserve VCA grafts for more than a few hours. Advancements in the field of VCA regarding matching and allocation, desensitization, and potential tolerance induction are all within reasonable reach to achieve; these are, however, constrained by limited preservation time of VCA grafts. Although machine perfusion holds many advantages over static cold preservation, it currently does not elongate the preservation time. More extreme preservation techniques, such as cryopreservation approaches, are, however, specifically difficult to apply to composite tissues as the susceptibility to ischemia and cryoprotectant agents varies greatly by tissue type. In the current scope of extended preservation protocols, high subzero approaches of VCA grafts will be particularly critical enabling technologies for the implementation of tolerance protocols clinically. Ultimately, advances in both preservation techniques and tolerance induction have the potential to transform the field of VCA and eventually lead to broad applications in reconstructive transplantation.

Defining the optimal duration for normothermic regional perfusion in the kidney donor: A porcine preclinical study.

Kidneys from donation after circulatory death (DCD) are highly sensitive to ischemia-reperfusion injury and thus require careful reconditioning, such as normothermic regional perfusion (NRP). However, the optimal NRP protocol remains to be characterized. NRP was modeled in a DCD porcine model (30minutes of cardiac arrest) for 2, 4, or 6hours compared to a control group (No-NRP); kidneys were machine-preserved and allotransplanted. NRP appeared to permit recovery from warm ischemia, possibly due to an increased expression of HIF1α-dependent survival pathway. At 2hours, blood levels of ischemic injury biomarkers increased: creatinine, lactate/pyruvate ratio, LDH, AST, NGAL, KIM-1, CD40 ligand, and soluble-tissue-factor. All these markers then decreased with time; however, AST, NGAL, and KIM-1 increased again at 6hours. Hemoglobin and platelets decreased at 6hours, after which the procedure became difficult to maintain. Regarding inflammation, active tissue-factor, cleaved PAR-2 and MCP-1 increased by 4-6hours, but not TNF-α and iNOS. Compared to No-NRP, NRP kidneys showed lower resistance during hypothermic machine perfusion (HMP), likely associated with pe-NRP eNOS activation. Kidneys transplanted after 4 and 6hours of NRP showed better function and outcome, compared to No-NRP. In conclusion, our results confirm the mechanistic benefits of NRP and highlight 4hours as its optimal duration, after which injury markers appear.

Changes in graft gene expression contribute to coagulopathy in uDCD liver transplantation

Uncontrolled DCD donors suffer cardiac arrest followed by unsuccessful resuscitation. Their livers have been successfully transplanted, though strict maintenance and selection criteria are necessary to avoid adverse outcomes, namely ischemic type biliary lesions. We demonstrated that uDCD liver recipients develop severe coagulopathy and fibrinolysis (Blasi 2016). Given the liver is the major source of clotting and fibrinolytic factors, we hypothesized that changes in gene expression may be detected in uDCD livers and provide a basis for abnormalities observed. Methods: 32 samples were taken from 16 uDCD livers: “A” before cold preservation and “B” after reperfusion. Total RNA was extracted and analyzed using microarrays; results were confirmed using RT-PCR. Finally, 61 biopsies from 38 uDCD livers were evaluated by light microscopy to rule out fibrin microthrombi. Results: Both before preservation and after reperfusion, uDCD livers had significantly upregulated expression of genes that provoke fibrinolysis and inhibit coagulation, including TPA, urokinase plasminogen activator, urokinase plasminogen activator receptor, and thrombomodulin. There was also decreased expression of genes implicated in hemostasis (vWF) and inhibition of fibrinolysis (alpha-2-macroglobulin):TableGene"A"FCFDR"B"FCFDRA2M−2.20.006−2.60.0009PLAT1.90.0022.70.0002PLAU2.00.0052.20.0008PLAUR2.50.00023.22E-5TM----1.5<0.01VWF−2.40.0004−2.10.001 Open table in a new tab On histology, no fibrin microthrombi were detected, even in cases that developed ITBL (13%). Conclusion: Changes in gene expression in uDCD livers help explain severe coagulopathy and fibrinolysis in recipients. Endogenous fibrinolysis is upregulated in these livers most at risk for ITBL, and clinical DCD protocols using TPA to help prevent ITBL should undergo critical appraisal.

Aldehyde Dehydrogenase 2 (ALDH2) in Rat Fatty Liver Cold Ischemia Injury.

Institut George Lopez-1 (IGL-1) and Histidine-tryptophan-ketoglutarate (HTK) solutions are proposed as alternatives to UW (gold standard) in liver preservation. Their composition differs in terms of the presence/absence of oncotic agents such as HES or PEG, and is decisive for graft conservation before transplantation. This is especially so when fatty (steatotic) livers are used since these grafts are more vulnerable to ischemia insult during conservation. Their composition determines the extent of the subsequent reperfusion injury after transplantation. Aldehyde dehydrogenase-2 (ALDH2), a mitochondrial enzyme, has been reported to play a protective role in warm ischemia-reperfusion injury (IRI), but its potential in fatty liver cold ischemic injury has not yet been investigated. We evaluated the relevance of ALDH2 activity in cold ischemia injury when fatty liver grafts from Zucker Obese rats were preserved in UW, HTK, and IGL-1 solutions, in order to study the mechanisms involved. ALDH2 upregulation was highest in livers preserved in IGL-1. It was accompanied by a decrease in transaminases, apoptosis (Caspase 3 and TUNEL assay), and lipoperoxidation, which was concomitant with the effective clearance of toxic aldehydes such as 4-hydroxy-nonenal. Variations in ATP levels were also determined. The results were consistent with levels of NF-E2 p45-related factor 2 (Nrf2), an antioxidant factor. Here we report for the first time the relevance of mitochondrial ALDH2 in fatty liver cold preservation and suggest that ALDH2 could be considered a potential therapeutic target or regulator in clinical transplantation.

A Hyperbaric Warm Perfusion System Preserves Tissue Composites Ex vivo and Delays the Onset of Acute Rejection.

Ischemia-reperfusion injury (IRI) precipitates acute rejection of vascularized composite allografts (VCA). Hyperbaric preservation of tissues ex vivo, between harvest and revascularization, may reduce IRI and mitigate acute rejection of VCA. A porcine heterotopic musculocutaneous gracilis flap model was used. In phase 1, control autografts (n = 5) were infused with University of Wisconsin Solution (UWS) and stored at 4°C for 3 hours. Intervention autografts (n = 5) were placed in a hyperbaric oxygen organ preservation system for 5 hours and infused with hyperoxygenated UWS at 20°C and 3 atm. Grafts were replanted into the animals' necks. In phase 2, similarly treated control (n = 8) and intervention grafts (n = 8) were allotransplanted into the necks of animals separated by a typed and standardized genetic mismatch. No systemic immunosuppression was given. Systemic markers of IRI, and clinical and histopathological assessments of necrosis and rejection were performed. Autotransplanted tissue composites preserved in the hyperbaric chamber showed histopathological evidence of less muscle necrosis at 3 hours (p = 0.05). Despite a longer period of ischemia, no evidence was found of a difference in systemic markers of IRI following revascularization in these groups. Allotransplanted tissues supported ex vivo within the hyperbaric perfusion device experienced acute rejection significantly later than corresponding controls. Hyperbaric warm perfusion preserves musculocutaneous tissue composites ex vivo for longer than standard cold preservation in this model. This translates into a delay in acute rejection of allotransplanted tissue composites.

Impact of In Situ Cold Perfusion of Kidney Grafts during Vascular Anastomosis

This study investigated simple in situ perfusion of a kidney to reduce its susceptibility to warm ischemia in rodent and porcine ischemic kidney models. Organ temperature was managed via simple continuous perfusion using newly developed thermal barrier sheet (TBS). ATP retention following cold perfusion with extracellular-trehalose-Kyoto (ETK) solution was investigated in a rat model. Materials and Methods Kidneys from adult male genetically modified luciferase transgenic (Luc Tg) rats were removed. The renal artery/vein underwent simple cold preservation at 4°C for 5 to 18 hours using ETK solution. After cold storage, the kidney was transplanted into a dummy living body that was heated to 37°C and perfused with ETK solution using a drip. The surface temperature (Ts) was measured with an infrared camera and ATP luminescence in organs was observed for 60 minutes. In porcine, the renal artery and vein were clamped within the abdominal cavity and the left kidney was prepared as a donor organ after 25 minutes of exposure to warm ischemia. The kidney was perfused via the renal artery at 4°C using ETK solution and subsequently underwent cold preservation (Fig1.a). We continued the surgery, while the kidney continued to undergo cold perfusion (Fig2.b) using a TBS. Continuous suturing was performed and the cannula was removed immediately after reflux. Blood flow then resumed. Control kidneys were sutured without undergoing perfusion. The time required for anastomosis was 1 hour, during which time the Ts of the kidney was measured. After resuming blood flow, the right kidney was removed. Following transplantation, blood and tissue were collected. Results Using a TBS and cold reflux in the dummy body, the rodent kidney Ts was maintained at 15°C or less for more than 30 minutes. During perfusion with the conventional method, the kidney Ts was at least 28°C immediately following removal. In the case of cold perfusion and TBS, the ATP level was elevated by approximately 4-fold that of pre-removal levels during preservation. Meanwhile, while using the conventional method, the ATP level was 0.9-fold of the pre-removal level during cold preservation. The porcine kidney Ts in the control group increased to 32°C during transplantation. Whereas, in the cold perfusion group, transplantation was performed while the kidney Ts was maintained at 24°C. The primary non-function rate after transplantation was 60% in the control group and 25% in the cold perfusion group. Discussion The cold perfusion method using a TBS can maintain the temperature and ischemic time in all organs obtained after cardiac death. In addition, the preclinical porcine model is a useful renal transplantation graft model that does not exhibit rejection and can be used to identify appropriate constant temperature perfusion solutions. Conclusions By developing this perfusion system, we believe that we can promote research toward practical application of machine perfusion.

The Effects of Short-Term Subnormothermic Perfusion after Cold Preservation on Liver Grafts from Donors after Cardiac Death

Background We previously reported that short oxygenated warm perfusion before cold storage (CS) had improved the graft viability of rat livers from donors after cardiac death (DCD). In this study, we investigated the effectiveness of short-term oxygenated subnormothermic perfusion for different durations after CS in a rat DCD model, and investigated the effects of 30-min subnormothermic perfusion for DCD livers in pigs. Methods In Study 1: We used an isolated rat liver perfusion system. The grafts were retrieved from Wistar rats 30min after cardiac arrest (thoracotomy), preserved in CS for 6h, and perfused with oxygenated subnormothermic (23-26°C) Krebs-Henseleit buffer for different durations (0, 30, 60, and 90min). After that, 15min incubation at 23-26°C, the grafts were reperfused under normothermic condition for 60min as a model of liver transplantation (0, 30, 60, and 90min groups; n=5 in each). In Study 2, landrace pigs (25-30kg) were randomly allocated into three types of groups (n = 5 per group): heart-beating (HB) graft, transplanted after a 4-h period of cold storage (CS); DCD graft, retrieved 20min after apnea induced cardiac arrest (respiratory withdrawal) and transplanted after a 4-h period of CS; and subnormothermic ex vivo liver perfusion (SELP) graft, retrieved in the same manner as the DCD graft but perfused with a subnormothermic oxygenated Krebs-Henseleit buffer (21-25°C, 10-15cmH2O) for 30min in the simplified dripping manner, without a machine perfusion system, after the 4-h period of CS, and subsequently transplanted. Results In Study 1, performing SELP groups significantly improved portal flow volume (P <0.05), and bile production (P <0.05), decreased liver enzymes (P <0.05), and increased tissue ATP (P <0.01) compared to DCD group. In the histological examinations, additional SELP ameliorated tissue deterioration, preserved the parenchymal structure, and decreased apoptosis compared to DCD group (P <0.01). In Study 2, the survival rate in the SELP group was significantly better than that in the DCD group (P = 0.0016). In the histological examination, preserved structure of the parenchyma was observed in the SELP group, and SELP significantly decreased apoptosis compared to DCD group (P <0.0001). Conclusion Even 30min of subnormothermic perfusion after CS recovered DCD livers from ischemia-reperfusion injury and might improve the viability of the grafts. Although 30min SELP was not sufficient to improve the survival rate as a level of HB group and to prevent primary graft non-function completely, this simple technique has the potential to expand the donor liver pool.

Cold Preservation with a Hyperbranched Polyglycerol-Based Solution Improves Functional Recovery of Kidney Transplants in a Pig Model

Cold Preservation with a Hyperbranched Polyglycerol-Based Solution Improves Functional Recovery of Kidney Transplants in a Pig Model

A Correlation Model of Thermal Entrainment Factor for Single Air Curtain without Back Panel Airflow

Thermal entrainment factor (TEF) is an important parameter to describe the cold preservation performance of a display cabinet. In order to calculate the TEF of a display cabinet rapidly and accurately, the method of establishing a correlation model based on computational fluid dynamics (CFD) simulation results is presented and the correlation model of TEF for typical single air curtain is developed in this paper. The TEF for single air curtain without back panel airflow can be regressed by an exponential function of Reynolds number and deflection modulus.

Effect of Cold-Preservation on the Vessels of Vascularized Composite Allografts in Rats

Introduction We evaluated the effect of cold ischemia on the development of chronic rejection in vascularized composite allotransplantation. Methods Thirty rat hindlimbs were transplanted from LBN to WL rats and divided into two experimental groups: immediate transplantation and transplantation after 7 hours of cold preservation ischemia. The animals received daily low-dose immunosuppression with cyclosporine A for 2 months. Intimal proliferation, arterial permeability rate, leukocyte infiltration and tissue fibrosis were assessed. To elucidate the mechanism of rejection immunohistochemical and RT-PCR cytokine analysis (Roche LightCycler) were performed. Results Significant differences were found in the intimal proliferation and arterial permeability rate between the two groups (p = 0.004). The arterial permeability rate worsened in the most distal and small vessels (p = 0.047). The numbers of CD3+, CD8+, CD20+, and CD68+ were also statistically higher in the cold ischemia group (p < 0.05, all levels). A trend toward significance was observed with C4d deposition (p = 0.059). No differences were found in the RNA of cytokines. Conclusions An association between cold ischemia and chronic rejection was observed in rat hindlimb allografts in a sub-optimal immunosuppression setting. Chronic rejection intensity and distal progression were significantly related with cold ischemia. The leukocyte infiltrates were a rejection marker, but their exact implication in monitoring and their relation with cold ischemia are yet to be clarified. This work was granted by the Fundación Investigación IdiPaz, Madrid.J.B. presented this work and won the Residents Award of the Spanish Society of Plastic & Reconstructive Surgery, during the National Meeting held in Tenerife (Spain) in 2014.