In Vivo Evaluation of Customized Aortic Repair Using a Novel Survival Model.

OBJECTIVE: The aim of the study was to evaluate the feasibility and mid-term biological behavior of the novel customized aortic repair (CAR) method for endovascular aneurysm treatment. METHODS: CAR consists of exclusion of an aneurysm from the circulation using a custom-designed dog bone-shaped balloon, followed by aneurysm sac filling with an in situ curing polymer. After curing and balloon deflation, the circulation is restored through a patent cast neolumen. A liquid two-component polymer was injected via a small bore catheter under X-ray control into a surgically created aneurysm. In 50% of the procedures, a self expanding bare metal stent was placed in the polymer cast lumen. A novel animal model was established involving creation of an aneurysm by anastomosing a venous interposition graft into the common carotid artery of eight adult sheep. Two animals were excluded because of non-device related complications. The remaining six animals were monitored for 20 weeks with duplex sonograms performed monthly to assess blood flow and polymer cast lumen patency. After the animals were sacrificed the polymer cast, common carotid artery, and the brain in the carotid outflow tract were removed for histological assessment. RESULTS: In four of the six animals, the aneurysm was successfully excluded using CAR with uneventful follow up. The aneurysm sac was filled incompletely in two animals, resulting in a stent malpositioning and cast lumen occlusion after 12 weeks in one case, and a type 1 endoleak in the other. All six animals survived for 20 weeks. Neither migration nor expansion of the polymer cast was observed and the polymer was demonstrated to be biocompatible and non-thrombogenic. Polymer emboli were not detected in the brain or meninges after sacrifice. CONCLUSIONS: The feasibility and mid-term biological safety of the CAR method for minimally invasive aneurysm repair was demonstrated using a simulated aneurysm survival model.

Promising future for the transgenic rat in transplantation research.

The rat is the most widely used animal species in surgical research and offers distinct advantages over the mouse in transplantation models due to its size and close genetic similarity to humans. Sequencing of the rat genome and successful application of transgenic technologies which had only been available for mice have since led to a resurgence of the use of rat models. Transplantation provides the possibility to deliver transgenes through a variety of routes which can potentially offer treatment modalities for post-transplant dysfunction and rejection. Moreover, the use of genetically encoded fluorescent light probes has enabled in vivo visualization of organs and tissue in living animals. In recent years, generation of gene knockout rats via the zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies has offered alternatives to the sophisticated embryonic stem cell based gene-targeting. In this review, we aim to provide an overview of transplantation studies involving transgenic techniques using rat models and recent advances in methods to modify the rat genome. Through novel gene modification techniques, precise, complete and conditional knockout and knockin rat models have become available which can provide promising new treatment options and opportunities for studying human transplant-related pathophysiology.

First clinical experience with polysol solution: pilot study in living kidney transplantation.

In this study, we assessed the safety of the new organ preservation solution polysol solution in the clinical setting of living kidney transplantation. We conducted a prospective pilot study in nine adult donor-recipient couples using polysol solution for washout and cold storage of kidney grafts. Adverse reactions possibly related to the use of polysol solution as well as renal function at 1, 6, and 12 months after transplantation were monitored. All living kidney transplantation performed in adults in our center within 2002 to 2008 using the University of Winconsin solution served as controls (n = 190). The use of polysol solution was associated with a higher acute rejection rate compared to University of Wisconsin solution at all time points. Also, antibody-mediated rejection occurred more frequently in the polysol group. Renal function at all time points was also comparable between the groups. This pilot study in living kidney transplantation is the first clinical study on the use of polysol solution. Although the study was not powered on the endpoint rejection, we observed a high number of acute rejection and antibody-mediated rejection episodes in recipients of polysol solution preserved grafts as compared to University of Wisconsin solution controls. As a consequence the study was terminated prematurely.

Hypothermic machine perfusion of kidney grafts: which pressure is preferred?

To assess the effect of the perfusion pressure (PP) during machine perfusion (MP) on the preservation quality of kidney grafts, we compared mean PPs of 25 and 30 mmHg using a porcine autotransplantation model. After assessment of the microcirculation, animals underwent left nephrectomy. Thereafter, kidneys were washed out followed by 20 h of MP at 25 mmHg (MP25, n = 7) or 30 mmHg (MP30, n = 7) using a novel MP system for hypothermic pulsatile perfusion. After MP preservation, the contralateral kidneys were removed and the preserved kidneys heterotopically autotransplanted. Ten minutes after reperfusion, the microcirculation was reassessed. Seven days posttransplant, animals were euthanized and the kidney grafts removed for histological analysis. MP using a mean PP of 25 mmHg resulted in higher capillary blood flow after reperfusion. In the MP30 group, 6 out of 7 animals survived, whereas in the MP25 group all animals survived. Overall, improvement in recovery of renal function and a better preservation of structural integrity were seen in the MP25 group compared to the MP30 group. Using a novel system for hypothermic MP, a mean PP of 25 mmHg is preferred over a mean PP of 30 mmHg.

Pulsatile perfusion preservation of warm ischaemia-damaged experimental kidney grafts.

BACKGROUND: Cold storage using histidine-tryptophan-ketoglutarate (HTK) solution is used widely in clinical practice for the preservation of warm ischaemia-damaged kidney grafts. This study assessed the efficacy of pulsatile machine perfusion in combination with Polysol for the preservation of warm ischaemia-damaged kidney grafts. METHODS: After induction of warm ischaemia by clamping of the left renal pedicle for 30 min, pigs were subjected to left nephrectomy. Thereafter, grafts were preserved for 20 h by cold storage with HTK (CS-HTK) or Polysol (CS-PS), or machine preservation with Polysol (MP-PS). Subsequently, contralateral kidneys were removed and preserved kidneys were transplanted. Control pigs underwent unilateral nephrectomy. Renal function was assessed daily for 1 week. Kidney biopsies were analysed for morphology and proliferative response. RESULTS: Renal function of warm ischaemia-damaged grafts preserved using MP-PS was comparable to that of non-ischaemic controls. MP-PS and CS-PS groups showed improved renal function compared with the CS-HTK group, with more favourable results for MP-PS than for CS-PS. The proliferative response of tubular cells in the CS-HTK group was higher than in all other groups. CONCLUSION: This study demonstrated that the function of warm ischaemia-damaged kidney grafts after pulsatile perfusion preservation was comparable to that of non-ischaemic controls.

Comparison of preservation solutions for washout of kidney grafts: an experimental study.

OBJECTIVE: The impact of different preservation solutions for washout of kidney grafts was evaluated regarding temperature, kidney weight, remaining red blood cells (RBCs) and histological evaluation after ex vivo washout using 500 mL cold preservation solution at 4 degrees C followed by 24 hours cold storage (CS). METHODS: Kidneys retrieved from Landrace pigs (20-30 kg) were immediately washed (warm ischemic time 0 min [WIT 0]), using 500 mL cold University of Wisconsin solution (UW), histidine-tryptophan-ketoglutarate (HTK), or Polysol (PS) followed by 24 hours, CS. Also, kidneys were retrieved after a WIT of 30 minutes followed by washout using HTK or PS. RESULTS: After washout, the weight of kidneys washed out with HTK had increased, whereas that of organs in the UW or PS group had decreased. After washout with UW, the core temperature of WIT 0 kidneys was lower than that with HTK. The time needed for washout using 500 mL solution was shorter using PS compared with HTK for both WIT 0 and WIT 30 groups. The amount of remaining RBCs was similar between all WIT 0 groups; whereas in the WIT 30 groups the amount was higher in kidneys washed out using HTK compared with PS. Histological evaluation showed less tissue injury among PS-washed kidneys compared with UW or HTK. CONCLUSION: Overall, kidneys washed-out with PS showed better preservation of structural integrity after 24 hours, CS compared with either UW or HTK. Washout of warm ischemically damaged kidneys was more effective using PS compared with HTK.

Improved renal function of warm ischemically damaged kidneys using Polysol.

OBJECTIVE: We sought to assess the efficacy of POLYSOL, a low-viscosity, colloid-based organ preservation solution, for the preservation of warm ischemically damaged kidney grafts compared with histidine-tryptophane-ketoglutarate (HTK) solution. METHODS: Pigs (25-30 kg) underwent a left nephrectomy after clamping the renal vessels for 30 minutes. Kidney grafts washed out with Polysol (n = 6) or HTK (n = 6) were cold stored (CS) for 20 hours at 4 degrees C. After the preservation period, the contralateral kidney was removed and the preserved kidney implanted heterotopically. Renal function was assessed daily for 7 days. Thereafter, animals were killed and the kidney grafts removed for histologic analysis. RESULTS: All animals survived for 7 days. All Polysol CS-preserved grafts showed immediate function, as demonstrated by urine production within 24 hours after reperfusion as compared with 3/6 grafts in the HTK CS group. Overall, the Polysol CS group showed improved renal function compared with HTK CS. Also, peak serum creatinine and blood urea values were lower in the Polysol CS group compared with HTK-preserved grafts. Histologic evaluation of warm ischemically damaged grafts showed less glomerular shrinking, less tubular damage, less edema, less inflammatory infiltration, and less necrosis in Polysol compared with HTK-preserved grafts. CONCLUSION: Application of Polysol solution for washout and CS preservation of warm ischemically damaged kidney grafts resulted in improved renal function and structural integrity when compared with HTK.

Machine perfusion preservation of the pig liver using a new preservation solution, polysol.

INTRODUCTION: The current gold standard for donor liver preservation is cold storage in a preservation solution (4 degrees C), such as Celsior or the University of Wisconsin solution (UW). Recent studies have suggested the benefits of machine perfusion (MP) over cold storage. To improve the results of MP, an enriched preservation solution (named Polysol) was developed, which in a rat liver preservation model proved to be superior to the UW-gluconate solution. The aim of this study was to assess Polysol in a pig liver preservation model. MATERIALS AND METHODS: Female pigs (35 to 40 kg) were used as liver donors. After heparinization, the liver was washed out using Ringer's lactate, followed by the preservation solution (4 degrees C). The liver was preserved for 24 hours by either cold storage using Celsior (n=5) or MP using Polysol (n=5). For analysis of liver damage and function, livers were reperfused for 60 minutes using oxygenated Krebs-Henseleit buffer. RESULTS: CS-Celsior caused significantly more damage compared with MP-Polysol (t=60, AST: 622+/-215 versus 222+/-55; ALT: 17+/-6 versus 5+/-1). Intravascular resistance during reperfusion was significantly higher after CS-Celsior compared with MP-Polysol (t=0, 0.20+/-0.01 and 0.11+/-0.02 mm Hg/mL/min, respectively). No differences were seen regarding ammonia clearance and urea production. In both groups, no bile was produced during reperfusion. CONCLUSIONS: In an ex vivo pig liver preservation model significantly less damage was observed after machine perfusion preservation using Polysol, in comparison to cold storage using Celsior.

The isolated perfused rat liver: standardization of a time-honoured model.

For many years, the isolated perfused rat liver (IPRL) model has been used to investigate the physiology and pathophysiology of the rat liver. This in vitro model provides the opportunity to assess cellular injury and liver function in an isolated setting. This review offers an update of recent developments regarding the IPRL set-up as well as the viability parameters that are used, with regards to liver preservation and ischaemia and reperfusion mechanisms.A review of the literature was performed into studies regarding liver preservation or liver ischaemia and reperfusion. An overview of the literature is given with particular emphasis on perfusate type and volume, reperfusion pressure, flow, temperature, duration of perfusion, oxygenation and on applicable viability parameters (liver damage and function). The choice of IPRL set-up depends on the question examined and on the parameters of interest. A standard technique is cannulation of the portal vein, bile duct and caval vein with pressure-controlled perfusion at 20 cm H2O (15 mmHg) to reach a perfusion flow of approximately 3 mL/min/g liver weight. The preferred perfusion solution is Krebs-Henseleit buffer, without albumin. The usual volume is 150-300 cm3, oxygenated to a pO2 of more than 500 mmHg. The temperature of the perfusate is maintained at 37 degrees C. Standardized markers should be used to allow comparison with other experiments.

Subnormothermic preservation maintains viability and function in a porcine hepatocyte culture model simulating bioreactor transport.

Bioartificial liver (BAL) systems have been developed to bridge patients with acute liver failure (ALF) to liver transplantation or liver regeneration. Clinical application of BAL systems is dependent on the supportive quality of cells used and direct availability of the whole system. Reliable transport of BAL systems from the laboratory to remote treatment centers is therefore inevitable. Subsequently, preservation conditions play a crucial role during transport of a BAL, with temperature being one of the most determining factors. In this study, we assessed the effect of subnormothermic preservation on freshly isolated porcine hepatocytes cultured in monolayer under oxygenation. Additionally, the effect of the University of Wisconsin (UW) preservation solution was compared with Williams' E (WE) culture medium at 4 degrees C. The control group was cultured for 3 days at 37 degrees C, whereas the transport groups were cultured at 4 degrees C, 15 degrees C, 21 degrees C, or 28 degrees C for 24 h at day 2. All groups were tested each day for cell damage and hepatic functions. Subnormothermic culture (i.e., 15 degrees C to 28 degrees C) for a period of 24 h did not reduce any hepatic function and did not increase cellular damage. In contrast, culture of hepatocytes in WE medium and preservation in UW solution at 4 degrees C significantly reduced hepatic function. In conclusion, freshly isolated porcine hepatocytes can be preserved for 24 h at subnormothermic temperatures as low as 15 degrees C. Future research will focus on the implementation of the AMC-BAL in an oxygenated culture medium perfusion system for transport between the laboratory and the hospital.

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

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

Optimization of a new preservation solution for machine perfusion of the liver: which is the preferred colloid?

AIMS: Machine perfusion (MP) has proven to be beneficial in experimental preservation of the liver. The modified University of Wisconsin solution (UW-Gluconate or UW-G) is used as the MP preservation solution of choice. We have developed Polysol, an enriched MP preservation solution based on a colloid. We sought to optimize Polysol by substituting the colloid hydroxyethylstarch (HES) with the colloids dextran and polyethylene glycol (PEG). METHODS: In an isolated perfused rat liver model, hepatocellular damage and liver function were assessed during reperfusion with Krebs-Henseleit buffer after 24 hours hypothermic MP using Polysol-HES, Polysol-dextran, or Polysol-PEG. Control livers were preserved by MP using UW-G. RESULTS: Compared to MP-UW-G, MP using Polysol resulted in significantly less damage and improved function during reperfusion. MP using Polysol-dextran or Polysol-PEG resulted in equal or less damage than Polysol-HES. Differences in ammonia clearance and bile production were not significant. Tissue edema was higher after MP using Polysol-HES as compared to Polysol-dextran and Polysol-PEG. CONCLUSIONS: MP of rat livers for 24 hours using UW-G results in more extensive damage and reduced liver function compared to MP using Polysol. MP using Polysol-dextran or Polysol-PEG results in equal or even better preservation compared to Polysol-HES.

Wash-out of the non-heart-beating donor liver: a comparison between ringer lactate, HTK, and polysol.

UNLABELLED: The solution of choice for wash-out of non-heart-beating donor (NHBD) livers is histidine tryptophan ketoglutarate (HTK). This solution has a lower viscosity, due to absence of a colloid, and is less expensive as compared to the University of Wisconsin (UW) solution. A new preservation solution for machine perfusion was developed, named Polysol. In order to apply Polysol clinically in NHBD organ retrieval, the efficacy as a wash-out solution was investigated. METHODS: After a warm ischemic time of 30 minutes, the rat liver was washed out via the portal vein with 50 mL of either ringer lactate (RL), HTK or Polysol. After wash-out and harvesting, the liver was reperfused with Krebs-Henseleit buffer. Samples were taken to assess hepatocellular injury and liver function. RESULTS: Liver damage parameters were elevated in the RL group as compared to the HTK and Polysol groups. Liver/rat weight ratios were significantly lower after wash-out with Polysol. Overall, no differences were seen in ammonia clearance and bile production. In conclusion, wash-out of the NHBD liver with Polysol results in equal to improved reperfusion results as compared to HTK. Polysol is feasible as a wash-out solution in combination with machine perfusion using Polysol.

Preservation of rat livers by cold storage: a comparison between the University of Wisconsin solution and Hypothermosol.

OBJECTIVES: The University of Wisconsin solution (UW) is the gold standard for cold storage (CS) of donor livers. However, UW contains the colloid Hydroxyethyl starch (HES), which may cause perfusion deficits due to its high viscosity. Recently, a new CS preservation solution, Hypothermosol (HTS), was introduced which contains the less viscous colloid Dextran. The aim of this study was to assess HTS as a cold storage solution for preservation of the liver. METHODS: In an isolated perfused rat liver model, hepatocellular damage was assessed after 24 hours of CS. Liver enzymes were measured during reperfusion with Krebs-Henseleit Buffer. Bile was collected during reperfusion as a parameter of liver function. RESULTS: CS using HTS showed a significant decrease of ALT and LDH levels (as compared to UW) at all time points during reperfusion. For LDH these results where most pronounced at t=10 min (84 +/- 7.09 vs 113 +/- 7.57: p < 0.05) and t=30 min (149.2 +/- 9.68 vs 194 +/-6.52: p< 0.05). Regarding liver function, more bile was produced after 24 hours CS in HTS, but this did not reach statistical significancy. CONCLUSIONS: Cold storage preservation of rat livers using Hypothermosol results in equal or even better preservation as compared to cold storage using UW.

The first disposable perfusion preservation system for kidney and liver grafts.

Machine perfusion systems for continuous hypothermic perfusion preservation are computer controlled and re-usable. The system of perfusion preservation proved beneficial for for storage of kidneys, particularly those harvested from NHBD donors. The first disposable continuous hypothermic perfusion system which can be used for storage of livers is presented.