Simple Cold Storage Research Articles

Oxygenated machine perfusion mitigates surface antigen expression and improves preservation of predamaged donor livers

The aim of the present study was to evaluate the potential benefit of machine preservation with the Belzer MPS or HTK solution, compared to standard cold storage, after procurement of marginal livers from non-heart beating donors in an experimental pilot study. Livers from male Wistar rats (250–300 g bw) were harvested after 60 min of cardiac arrest, flushed via the portal vein and cold stored submerged in HTK for 24 h at 4 °C while other organs were subjected to oxygenated machine perfusion with HTK or Belzer’s MPS at 5 ml/min at 4 °C. Cold perfusion of livers with the non-colloidal HTK was not compromised by the lack of oncotic agents and there was no rise in vascular resistance during the 24 h of machine preservation with HTK or the colloidal Belzer MPS. Viability of the livers was assessed after the cold preservation period by warm reperfusion in vitro. Oxygenated machine perfusion was found to significantly increase viability of the livers vs simple cold storage with respect to portal vascular resistance upon reperfusion, enzyme release as well as functional recovery of oxygen utilization or bile production. Moreover, tissue antigen expression of ICAM-1 or histocompatibility antigen class II could be markedly reduced by oxygenated perfusion preservation as compared to cold storage. It is concluded that predamaged organs should preferably be preserved by oxygenated machine perfusion thus minimizing functional alterations and immunogenicity of the graft. In this setup HTK appeared equally effective as Belzer’s MPS for machine preservation.

Extended preservation of non-heart-beating donor livers with normothermic machine perfusion.

Non-heart-beating donor (NHBD) livers represent an important organ pool, but are seldom utilized clinically and require rapid retrieval and implantation. Experimental work with oxygenated perfusion during preservation has shown promising results by recovering function in these livers. This study compared sanguinous perfusion with cold storage for extended preservation of the NHBD liver in a porcine model. Porcine livers were subjected to 60 min of in vivo total warm ischaemia before flushing, after which they were preserved by one of two methods: group 1 (n = 4), University of Wisconsin (UW) solution by standard cold storage for 24 h; group 2 (n = 4), oxygenated autologous blood perfusion on an extracorporeal circuit for 24 h. All livers were subsequently tested on the circuit during a 24-h reperfusion phase. Livers in group 1 showed no evidence of viability during the reperfusion phase with no bile production or glucose utilization; they also displayed massive necrosis. Livers in group 2 demonstrated recovery of function by synthetic function, substrate utilization and perfusion haemodynamics; these livers displayed less cellular injury by hepatocellular enzymes. All differences in parameters between the two groups were statistically significant (P < 0.05). These findings were supported by histological examination. Warm ischaemia for 1 h and simple cold storage (UW solution) for 24 h renders the liver non-viable. Oxygenated, sanguinous perfusion as a method of preservation recovers liver function to a viable level after 24 h of preservation.

Successful Six‐Day Kidney Preservation Using Trophic Factor Supplemented Media and Simple Cold Storage

This study examined the effect of trophic factor supplementation [TFS; bovine neutrophil peptide-1 (bactenecin), 1 mg/L; substance P, 2.5 mg/L; nerve growth factor, 20 microg/L; epidermal growth factor, 10 microg/L; insulin-like growth factor-1, 10 microg/L] during cold storage with UW lactobionate solution. Dogs transplanted with kidneys stored for 4days in TFS-UW had significantly lower peak serum creatinine values (mean 2.9 +/- 0.2mg/dL) and returned to normal values faster (6 days) than kidneys stored for 3days in unmodified UW solution (4.2 +/- 0.3 mg/dL and 14 days, respectively). Kidneys stored for 5days in TFS-UW (mean peak creatinine 3.7 +/- 0.3) functioned equivalently to kidneys stored for 3days and better than kidneys stored for 4 days in UW alone. Dogs with kidneys stored for 6days in TFS-UW had mean peak creatinines of 5.7 +/- 0.4 mg/dL. These returned to normal creatinine values in 14 days, equal to 3-day stored and significantly better than kidneys stored for 4days in UW alone (20days recovery time). This study shows trophic factor deprivation appears to be a critical mechanism of injury in organ preservation with current synthetic storage media, and marks the initial development of a synthetic biologically active preservation solution, the next generation of preservation media.

Ultrastructural analyses of pancreatic grafts preserved by the two-layer cold-storage method and by simple cold storage in University of Wisconsin solution

Ultrastructural analyses of pancreatic grafts preserved by the two-layer cold-storage method and by simple cold storage in University of Wisconsin solution

Hypothermic Perfusion of the Warm Ischemic Kidney: Is 32 °C Better Than 4 °C?

To the Editor: Brasile et al. (1) have developed an oxygen-carrying perfusion solution that can be used at 32°C to perfuse dog kidneys successfully after being exposed to 30 min of warm ischemia. This is a unique and meritorious achievement. The title of the article, ‘Hypothermia – a limiting factor in using warm ischemically damaged kidneys’, and their conclusion that hypothermia is the limiting factor, however, is unsupported by the evidence. In their experimental design they varied three parameters simultaneously (temperature, method of preservation, and solution). Thus, to conclude that the temperature was the limiting factor is premature and requires a better experimental design. Furthermore, 32°C is actually hypothermic compared with normal body temperature. Previous experimental and clinical experience has shown that hypothermia (0–4°C) can be safely used to preserve kidneys exposed to at least 30 min of warm ischemia. The development by Belzer in the late 1960s of a method to preserve kidneys for 3 d by continuous hypothermic perfusion (4–8°C) was used during the 1970s for human kidneys that often came from nonheart-beating donors. This was due to the lack of standard brain death criteria for organ donors and many donors were nonheart-beating. Thus, many kidneys had 25–40 min of warm ischemia. However, many of these organs were safely preserved for up to 24 h or more and transplanted after hypothermic perfusion. In our clinic at the University of Wisconsin Hospital, we continue to perfuse kidneys hypothermically (0–4°C), both from nonheart-beating (NHBD) and heart-beating cadavers (HBD). The delayed graft function rate for the NHBD kidneys that are machine-perfused is similar to the national average for HBD kidneys that are cold stored (about 25%). The warm ischemic period is variable and may range from 15 to 45 min. Early work by many authors showed that hypothermic perfusion (4–8°C) was superior to simple cold storage (0–4°C) for preservation of the dog kidney exposed to 30 min of warm ischemia. Our laboratory has successfully hypothermically preserved dog kidneys exposed to 45–60 min of warm ischemia by machine perfusion with the UW solution. Some transplant centers that have regularly used hypothermic (0–4°C) machine perfusion for kidney preservation often report significantly lower delayed graft function rates than centers that use simple cold storage. Hypothermic machine perfusion can lower delayed graft function rates of the ideal kidney to less than 10% and is suitable for the warm ischemically damaged kidney. Despite the benefits of machine perfusion, most kidneys continue to be preserved by simple cold storage. Matas and Delmonico (2) argue in the same issue of the journal that improvements in preservation (by shortening the cold ischemic time) could lower the discard rate for kidneys. One improvement in preservation that might achieve the same result as lowering cold ischemic time is the use of hypothermic (0–4°C) perfusion preservation. If more centers perfected this technology, an improvement in kidney transplant results might be achieved without the need for moderate (32°C) hypothermic preservation, both for HB and NHB cadaver kidneys. Anthony M. D'Alessandro and James H. Southard Department of Surgery, University of Wisconsin Medical School, Madison, WI, USA

Advantages of normothermic perfusion over cold storage in liver preservation.

To minimize the ischemia-reperfusion injury that occurs to the liver with the current method of preservation and transplantation, we have used an extracorporeal circuit to preserve the liver with normothermic, oxygenated, sanguineous perfusion. In this study, we directly compared preservation by the standard method of simple cold storage in University of Wisconsin (UW) solution with preservation by perfusion. Porcine livers were harvested from large white sows weighing between 30 and 50 kg by the standard procedure for human retrieval. The livers were preserved for 24 hr by either cold storage in UW solution (n=5) or by perfusion with oxygenated autologous blood at body temperature (n=5). The extracorporeal circuit used included a centrifugal pump, heat exchanger, and oxygenator. Both groups were then tested on the circuit for a 24 hr reperfusion phase, analyzing synthetic function, metabolic capacity, hemodynamics, markers of hepatocyte and reperfusion injury, and histology. Livers preserved with normothermic perfusion were significantly superior (P=0.05) to cold-stored livers in terms of bile production, factor V production, glucose metabolism, and galactose clearance. Cold-stored livers showed significantly higher levels of hepatocellular enzymes in the perfusate and were found to have significantly more damage by a blinded histological scoring system. Normothermic sanguineous oxygenated perfusion is a superior method of preservation compared with simple cold storage in UW solution. In addition, perfusion allows the possibility to assess viability of the graft before transplantation.

Effects of hypothermic machine perfusion on rat liver function depending on the route of perfusion.

The aim of this study was to evaluate the efficacy of hypothermic machine perfusion (HMP) to preserve rat livers according to the route of perfusion, i.e., via portal vein, hepatic veins (retrograde), or hepatic artery. Livers were preserved for 24 or 48 hr by simple cold storage (SCS) or by HMP. Preservation solution was supplemented with (HMP) or without (SCS) hydroxyethyl starch. After preservation, grafts were reperfused for 2 hr with an oxygenated Krebs-Henseleit bicarbonate buffer. After 24 hr of preservation, total glutathione concentrations in HMP livers were similar (1287+/-37, 1418+/-118, and 1471+/-62 nmol/g in hepatic artery, portal vein, and hepatic vein HMP livers, respectively) and higher than in the SCS (833+/-118 nmol/g, P<0.05) group. These higher total glutathione values were due to higher reduced glutathione concentrations. ATP concentrations in the liver tissue were similar in HMP groups (0.75+/-0.4, 0.64+/-0.1, and 0.77+/-0.1 micromol/g in hepatic artery, portal vein, and hepatic vein HMP livers, respectively) and higher than in SCS (0.32+/-0.06 micromol/g, P<0.05). After 2 hr of normothermic reperfusion, bile production in the HMP portal and HMP retrograde groups were similar (391+/-29 ml and 372+/-25 ml) and higher than in the HMP artery or SCS groups (275+/-25 ml and 277+/-32 ml, respectively; P<0.05). Aspartate transaminase, alanine transaminase, lactate dehydrogenase, and purine nucleoside phosphorylase release into the perfusate of HMP portal and HMP retrograde perfused livers was similar and significantly lower compared to the HMP artery and SCS groups. At the end of reperfusion, no statistical differences were found for glutathione concentration and energetic reserves in the livers of each group. After 48 hr of preservation, livers from the HMP portal and HMP retrograde groups did significantly better than livers from the HMP artery or SCS groups. This study confirms the superiority of HMP over SCS to preserve the liver graft. It shows that retrograde perfusion is similar to PV perfusion and that perfusion by HA is less beneficial.

Warm and cold ischemia result in different mechanisms of injury to the coronary vasculature during reperfusion of rat hearts

HEARTS for transplantation are preserved by flushing out blood from the vascular system with a preservation solution for storage at about 4°C. This method of cold ischemic storage provides safe preservation for about 4 to 6 hours. Longer preservation times would increase the distance hearts could be shipped, increase the sharing of hearts between hospitals, increase the number of hearts available for transplantation, provide time for better immunologic tissue matching between donor and recipient, and reduce the emergency nature of clinical heart transplantation. Although many laboratories report successful longerterm heart preservation in animal models, confidence by the transplant surgeons to extend preservation times beyond 4 to 6 hours has been very limited. Thus, better methods of heart preservation are needed and to accomplish this requires a better understanding of the nature of preservation-induced injury to the heart. Methods to improve heart preservation by simple cold storage have focused on the use of pharmacologic agents, either in the preservative solution or as a treatment of the recipient to suppress reperfusion injury created by the reestablishment of blood flow to the preserved heart. The choice of pharmacologic agents is often based on the vast literature describing the benefits of an agent to improve the recovery of hearts subjected to warm ischemic injury. Thus, the choice of agents is often based on the concept that the mechanisms of warm ischemic (WI) injury are similar to the mechanisms of cold ischemic (CI) injury. However, this concept may be misleading because there may be significant differences in the mechanisms of injury caused by WI versus CI. Differences in the mechanisms of injury caused by WI and CI have in fact been shown to exist in the liver. In this study, we used rat hearts and compared how WI and CI affected coronary flow and left ventricular function during reperfusion. Additionally, we compared the effects of some pharmacologic agents on the recovery of flow and function in the hearts. The study shows that there may be significant differences in how WI and CI injure the heart.

Prostaglandin E1 Influences Pulsatile Preservation Characteristics and Early Graft Function in Expanded Criteria Donor Kidneys

Introduction. Unlike simple cold storage, machine preservation allows dynamic assessment and manipulation of the donor organ prior to transplantation. We prospectively compared the effects of five pharmacological agents added to the perfusate during machine preservation of expanded criteria donor (ECD) kidneys in order to (1) describe their influence on perfusion parameters and (2) determine their influence on early graft outcome.Methods. Two hundred seventy-five consecutive ECD kidneys were preserved in our laboratory between 1/1/94 and 12/31/97 by either machine perfusion (MP) or cold storage (CS). ECD kidneys were defined as those requiring pretransplant biopsy. ECD kidneys were divided by method of preservation and MP kidneys were randomized to receive prostaglandin E1 (MP+PGE1), trifluoperazine (TFP), verapamil (VER), papaverine (PAP), mannitol (MAN), or no intervention during the period of machine perfusion. CS kidneys were randomized to receive PGE1 (CS+PGE1), TFP, VER, PAP, or no intervention. All MP kidneys were preserved by continuous hypothermic pulsatile perfusion using Belzer II solution and perfusion parameters were measured every hour during pulsatile perfusion. All CS kidneys were stored in 1.0 L of University of Wisconsin (UW) solution.Results. The addition of PGE1 to machine perfusate increased renal flow and decreased renal resistance. Moreover, the MP+PGE1-treated group was associated with improved early graft function compared to all other groups. The addition of VER, TFP, PAP, or MAN influenced neither the perfusion characteristics nor the incidence of early graft function in MP kidneys. Similarly, the addition of VER, TFP, or PAP did not influence early graft function in the CS kidneys. The CS+PGE1 group exhibited a significantly lower incidence of early graft function than did the MP+PGE1 group.Conclusions. PGE1 treatment during machine preservation improves hydrostatic perfusion parameters and reduces the incidence of delayed graft function in ECD kidneys. Moreover, the addition of PGE1, TFP, VER, or PAP to UW does not influence early graft function in the CS kidney.

USEFULNESS OF CONTINUOUS HYPOTERMIC PERFUSION PRESERVATION FOR CADAVERIC RENAL HIGH RISK GRAFTS

159 〈AIM〉 Because of the severe shortage of donors for cadaveric renal transplantation (Tx) in Japan, cadaveric renal grafts are being used for Tx even when they are high risk grefts (HRG). We always use continuous hypothermic perfusion preservation (CP) to evaluate whether HRG should be used for Tx and to improve the function of HRG. The aim of this study is to assess the usefulness of CP for Tx of cadaveric renal HRG, so-called non-heart-beating donors (NHBD). 〈METHODS〉 Of the 125 kidneys preserved by CP at our center from March 1981 to April 1998, 92 kidneys met the study criteria. 50 of 92 kidneys were in poor condition, the study criteria were defined as follows: Group 1:23 kidneys with a long warm ischemic time (WIT) of 60 minutes or more; Group 2:13 kidneys procured with donor serum creatinine levels of 3.0mg/dl or more; Group 3:14 kidneys preserved by CP following simple cold storage (CS) in Euro-Collins solution for at least 40 hours. And comparison of CP with CS of 16 pairs of kidneys procured from NHBD. 〈RESULTS〉 All of 85 grafts with a good perfusion flow rate of more than 0.4ml/min/g (1.10 ± 0.53 ml/min/g) and without a rise in perfusion pressure showed the good kidney functions after Tx. The remaining 7 grafts showed a poor perfusion flow rate of 0.4 ml/min/g or less (0.29 ± 0.04 ml/min/g, vs good, p<.001) and a rise in perfusion pressure. These grafts were determined as poor viability and discarded. They were histologically confirmed as unacceptable grafts for Tx. 4 of the contralateral kidneys procured from the same donors were transplanted at another center, all grafts resulted in primary non-function. When comparing preservation methods despite the longer total ischemic time in CS (7.8 hours in CS vs 11.7 hours in CP), CP achieved a higher incidence of immediate function (IF) 9 cases, a shorter period of postoperative hemodialysis days (HD) in 8 cases, and better graft survival in 3 cases when compared with CS from the same NHBD. In these study, CP achieved a higher incidence of IF, and a shorter period of HD. 〈CONCLUSION〉 We found CP is to be useful both for improving of HRG and for assessing HRG viability.

Investigation of Graft Function-Determining Factor in Cadaveric Renal Transplantations.

343 〈AIM〉 This study was conducted to identify the graft function -determining factors in cadaveric renal transplantations (RTx). 〈PATIENTS〉 One hundred fifty three patients treated by RTx in our center participated in this study. They were grouped by the type of donor and the mode of graft preservation as follows (HP: hypothermic continuous perfusion preservation, CS: simple cold storage): (Table)TableFor each group, multiple regression analysis was performed on donor age (D-Ag), recipient age (R-Ag), donor serum creatinine level (DS-Cr), warm ischemic time (WIT) and total ischemic time (TIT) against post-transplant graft function. 〈RESULTS〉 1) In the brain death group, even though D-Ag was significantly younger (p<0.01) and DS-Cr significantly lower (p<.01) for US kidney than for domestic kidney, ATN was significantly longer (p<.05) for US kidney, which may be attributed to a significantly longer TIT (p<.01). In domestic kidneys, ATN was significantly shorter for HP+CS group than for CS group (p<.05). 2) In the cardiac death group, even though D-Ag was significantly younger (p<.05) and DS-Cr significantly lower (p<.05) for NIS than for IS, ATN duration was significantly shorter (p<.01) for IS than for NIS. In IS cases, in spite of significantly longer WIT for HP+CS group than for CS group (p<.01), there was no difference in ATN between groups. 〈CONCLUSION〉 The function-determining factors were TIT for transplants from brain death donors and WIT for transplants from non-heart beating donors. In both the brain dead and cardiac dead groups, the presence or absence of HP was a factor for determining the graft function (whether or not the graft was treated by IS).

Coronary oxygen persufflation for long-term myocardial protection.

In human heart transplantation limited myocardial ischemia duration remains one of the most restricting factors. A new approach towards prolongation of this duration is the combination of cardioplegic arrest and continuous Coronary Oxygen Persufflation (COP) with gaseous oxygen. This technique, which is based on former experiments, was applied in pig hearts which we transplanted orthotopically after a hypothermic preservation time of 14 hours. For cardioplegic arrest we used either Euro-Flush glutathion solution (EFG; n=5), University of Wisconsin solution (UW; n=5), modified Bretschneider HTK cardioplegic solution (mHTK; n=6). In preliminary experiments all three solutions had shown equal cardioprotective qualities. Hearts of the mHTK group were submitted to continuous COP during storage (mHTK+COP). After 14 hours of preservation and orthotopic transplantation the mHTK+COP hearts showed significantly improved cardiac functional recovery compared to hearts preserved by simple cold storage techniques. Hemodynamics measured after 3 hours reperfusion were significantly better in the mHTK+COP group compared to EFG and UW: dp/dtmax in % of baseline+/-standard deviation (SD): 85+/-22, 65+/-26, 36+/-15, CO in % of baseline: 68+/-13, 35+/-8, 39+/-8. Postoperative preload recruitable stroke work in the mHTK+COP hearts was: 51.4+/-23.1 mmHg compared to preoperative: 57.3+/-17.2. ATP of left-ventricular myocardium in the mHTK+COP group: 14.7+2.1 micromol/g dry weight was significantly higher compared to EFG: 10.3+/-4.5 and UW: 5.9+/-3.2. CK-MB in percent of CK in all groups showed no increase during postoperative reperfusion. This study suggests that COP may present an effective complement to cold storage techniques currently used in heart transplantation. Prior to clinical application further investigations regarding long-term survival and endothelial function are required.

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.

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

Abstract 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/re-perfusion injury.

PROSTAGLANDIN E1 IMPROVES PERFUSION CHARACTERISTICS AND EARLY GRAFT FUNCTION IN EXTENDED CRITERIA DONOR KIDNEYS

334 Objective: Unlike simple cold storage, machine preservation allows dynamic assessment and manipulation of the donor organ prior to transplantation. We prospectively studied the effects of three vasoactive agents added to the perfusate during machine preservation of extended criteria donor kidneys (ECD) in order to (1) describe their impact on perfusion parameters and (2) determine their influence on early graft function. Methods: 100 consecutive ECD kidneys were preserved in our laboratory between 5/1/96 and 5/1/97. ECD kidneys were defined as those requiring pretransplant biopsy. ECD were randomized to receive prostaglandin E1 (PGE1), verapamil, trifluoperazine (TFP), or no intervention (control). All kidneys were preserved by continuous hypothermic pulsatile perfusion using Belzer II solution and perfusion parameters were measured every hour during pulsatile perfusion. Results (mean values): Table,Figure1)Comparison of selected variables (n=25 each)*:2. Incidence of DGF for treated groups vs. control (n=25 each):Conclusions: PGE1 treatment during machine preservation enhances perfusion characteristics (FL and RES) and reduces the incidence of DGF in ECD kidneys.

Nonfreezing cryopreservation--a possible means of improving long-term transplant function?

Improving organ preservation techniques for transplantation is one of the most important goals of transplantation research. We have established a new, nonfreezing cryopreservation method to optimize the viability of rat kidneys for transplantation with up to 4 M dimethylsulphoxide (DMSO) in EuroCollins solution (EC) at -5 degrees C to -15 degrees C. We have confirmed the occurrence of a tubular and glomerular defect pattern that mediates acute tubular necrosis (ATN) and that may be a cause of major histocompatibility complex (MHC) independent immunological components of chronic transplant rejection. The extent of this defect [transplant survival and function, 31P-NMR spectroscopy, histological defect index] in the nonfreezing cryopreserved groups (n = 22) is significantly (P = 0.017) lower than in the simple cold storage group (n = 12). Quality and localization of the lesions in kidney transplants can elucidate the context of organ preservation, progressive hyperfiltration defects, and the occurrence of graft failure without elevated frequency of acute rejection episodes. These results indicate that further efforts to provide higher pretransplant organ viability without using it to prolong cold storage intervals may provide better insight into MHC-independent factors of chronic transplant failure and may result in improved long-term transplant outcome.

Nonfreezing cryopreservation – a possible means of improving long-term transplant function?

Abstract Improving organ preservation techniques for transplantation is one of the most important goals of transplantation research. We have established a new, non-freezing cryopreservation method to optimize the viability of rat kidneys for transplantation with up to 4 M dimethylsulphoxide (DMSO) in EuroCollins solution (EC) at -5 °C to -15 °C. We have confirmed the occurrence of a tubular and glomerular defect pattern that mediates acute tubular necrosis (ATN) and that may be a cause of major histocompatibility complex (MHC) independent immunological components of chronic transplant rejection. The extent of this defect [transplant survival and function, 31P-NMR spectroscopy, histological defect index] in the nonfreezing cryopreserved groups (n= 22) is significantly (P= 0.017) lower than in the simple cold storage group (n= 12). Quality and localization of the lesions in kidney transplants can elucidate the context of organ preservation, progressive hyperfiltration defects, and the occurrence of graft failure without elevated frequency of acute rejection episodes. These results indicate that further efforts to provide higher pretransplant organ viability without using it to prolong cold storage intervals may provide better insight into MHC-independent factors of chronic transplant failure and may result in improved long-term transplant outcome.

Rat Liver Preservation by Hypothermic Oscillating Liver Perfusion Compared to Simple Cold Storage

Rat livers were preserved hypothermically for 10 or 24 hin vitroas if for transplantation. Two methods of preservation were compared using physiological and biochemical parameters: simple storage and oscillating perfusion. By measuring the nucleotides after preservation the calculated energy charge was significantly higher after 10 and 24 h of oscillating perfusion compared to the simple storage group. In addition, a significant energy charge loading was demonstrated by 10 h oscillating perfusion compared to the initial value prior to perfusion. The oscillating, computer-controlled perfusion permits continuous monitoring of perfusate temperature, O2consumption, pCO2, portal vein pressure, and pH and also automatic sample collection and pH compensation. In addition, the perfusate can be easily exchanged by using two different pumps or be rewarmed by a heat exchanger. For measuring of short-lived metabolites (interleukins, oxygen radicals, prostaglandins) sampling can be performed directly out of the vena cava outflow. pH and temperature stability was maintained by a data acquisition and controlling system. Because of a special designed liver chamber a combination of storage and perfusion with or without substrates was possible. The demonstrated standardized perfusion technique was achieved by a combination of special equipment and computer-aided monitoring and allows further experiments to improve understanding of ischemic and reperfusion injury.

Pulsatile preservation in renal transplantation

A subtle but important part of the success of cadaveric renal transplantation has been effective and prolonged organ preservation. During the early 1970s, pulsatile perfusion for preservation of kidneys was used by the majority of centres in the United States. This gradually changed later in the decade to a predominance of simple cold storage. There were a number of reasons for this change, including reports demonstrating few or no benefits of pulsatile perfusion over cold storage, as well as the perception that cold storage was significantly easier to accomplish from a technical standpoint and was less expensive. Interestingly, there now appears to be a trend towards pulsatile perfusion.

Excellence of the two-layer method (University of Wisconsin solution/perfluorochemical) in pancreas preservation before islet isolation

Background. In islet transplantation pancreatic preservation before islet isolation is an obstacle compromising islet yield and viability. We tested the feasibility of a two-layer method (University of Wisconsin solution [UW]/perfluorochemical) for pancreatic preservation before islet isolation. Methods. Dog pancreases were processed into pure islets by the method of Ricordi preceded by five different preservations (groups 1-a and 1-b, the two-layer method far 3 and 24 hours; groups 2-a and 2-b, simple cold storage in UW for 3 and 24 hours; group 3, without preservation). Islet yields and functional success after autotransplantation into the liver were compared among the groups. Results. Postpurification islet equivalents (IE)/gm pancreas and functional success rate were 5600 (mean), 83% in group 1-a; 4000, 56% in group 1-b; 4700, 33% in group 2-a; 1300, 0% in group 2-b; and 5000, 89% in group 3 (p < 0.05; 2b versus 1-a, 1-b, and 3), respectively. There was no statistical difference among groups 1-a, 1-b, and 3 in terms of islet yield and function (p > 0.2). Conclusions. The two-layer method is more effective than conventional simple cold storage in UW for pancreatic preservation before islet isolation. Clinical trials with the two-layer method are warranted.