Cold Organ Preservation Research Articles

(554) - Lung Transplant Outcomes After Implementation of a Hospital-Based 10OC Refrigeration for Cold Organ Preservation

(554) - Lung Transplant Outcomes After Implementation of a Hospital-Based 10OC Refrigeration for Cold Organ Preservation

Retroperitoneal fibrosis as a postoperative complication following renal transplantation in cats.

The aim of this report was to describe the clinical signs, diagnostic imaging findings, surgical management, histopathological findings, outcome and possible risk factors for cats that developed retroperitoneal fibrosis (RPF) following renal transplantation. Medical records of cats that underwent renal transplantation and developed clinically significant RPF between 1995 and 2019 were reviewed. Eighty-one cats underwent 83 renal transplantations. Of these 81 cats, six developed clinically significant RPF. For all six cats, renal transplantation was performed using cold organ preservation solution and ureteral papilla implantation. Immunosuppression protocol included ciclosporin and prednisolone. All cats had at least one subtherapeutic trough ciclosporin level (<250 ng/ml) in the postoperative period. Cats presented with moderate-to-severe azotemia 39-210 days following renal transplantation. Abdominal ultrasonography and contrast pyelography revealed various degrees of hydroureter and hydronephrosis of the transplanted kidney. Surgical examination revealed a layer of dense fibrous tissue surrounding the transplanted kidney, ureter and bladder resulting in ureteral obstruction. Ureteral obstruction was managed by reimplantation of the proximal ureter or renal pelvis to the bladder. Histopathologic examination of the fibrous tissue and affected portion of the distal ureter revealed fibrous connective tissue with lymphoplasmacytic infiltration and perivascular inflammation suggestive of an autoimmune type reaction. Of the six cats, two died within 5 days after revision surgery, two developed signs consistent with recurrent partial ureteral obstruction (40 and 41 days after revision), one was euthanized 6 years later for an unrelated disease and one was lost to follow-up. The incidence of RPF in this population of cats was relatively low (7%), but still represents a significant cause of morbidity and mortality. The cause of RPF remains unknown, although investigation into suboptimal immunosuppression as a potential cause for local rejection reaction is warranted.

Reconstruction surgery case report: ex vivo surgery and auto lung transplantation for pulmonary artery sarcoma

This is a report on the lung autotransplant techniques for the treatment of intimal sarcoma of pulmonary artery (PA). A 58-year-old man was diagnosed with PA sarcoma occluding the right main PA and peripherally extending into the right interlobar PA. First, the tumor was completely removed by pulmonary endarterectomy combined with right pneumonectomy under cardiopulmonary bypass. A following ex vivo evaluation of the explanted right lung revealed that the right lower PA was intraluminally intact, so the right lower lobe was used for reimplantation after being flushed with cold organ preservation solution on a back table. The patient was safely weaned from cardiopulmonary bypass following transplantation. His early post-transplant course was uneventful. He died of tumor recurrence at the distal left PA and liver and bone metastasis 14 months after surgery.

A Hibernation-Like State for Transplantable Organs: Is Hydrogen Sulfide Therapy the Future of Organ Preservation?

Renal transplantation is the treatment of choice for end-stage renal disease, during which renal grafts from deceased donors are routinely cold stored to suppress metabolic demand and thereby limit ischemic injury. However, prolonged cold storage, followed by reperfusion, induces extensive tissue damage termed cold ischemia/reperfusion injury (IRI) and puts the graft at risk of both early and late rejection. Recent Advances: Deep hibernators constitute a natural model of coping with cold IRI as they regularly alternate between 4°C and 37°C. Recently, endogenous hydrogen sulfide (H2S), a gas with a characteristic rotten egg smell, has been implicated in organ protection in hibernation. In renal transplantation, H2S also seems to confer cytoprotection by lowering metabolism, thereby creating a hibernation-like environment, and increasing preservation time while allowing cellular processes of preservation of homeostasis and tissue remodeling to take place, thus increasing renal graft survival. Although the underlying cellular and molecular mechanisms of organ protection during hibernation have not been fully explored, mammalian hibernation may offer a great clinical promise to safely cold store and reperfuse donor organs. In this review, we first discuss mammalian hibernation as a natural model of cold organ preservation with reference to the kidney and highlight the involvement of H2S during hibernation. Next, we present recent developments on the protective effects and mechanisms of exogenous and endogenous H2S in preclinical models of transplant IRI and evaluate the potential of H2S therapy in organ preservation as great promise for renal transplant recipients in the future. Antioxid. Redox Signal. 28, 1503-1515.

A safety evaluation of profound hypothermia-induced suspended animation for delayed resuscitation at 90 or 120\xa0min

BackgroundThe successful treatment of military combat casualties with penetrating injuries is significantly dependent on the time needed to get the patient to an adequate treatment facility. Profound hypothermia-induced suspended animation for delayed resuscitation (SADR) is a novel approach for inducing cardiac arrest and buying additional time for such injuries. However, the time used to safely administer circulatory arrest (CA) is controversial. The goal of this study was to evaluate the safety of hypothermia-induced SADR over 90 and 120 min time intervals.MethodsSixteen male BAMA minipigs were randomized into two groups: CA90 group (90 min, n = 8) and CA120 group (120 min, n = 8). Cannulation of the right common carotid arteries and internal jugular veins was performed to establish cardiopulmonary bypass for each animal. Through the perfusion of cold organ preservation solution (OPS), cardioplegia and profound hypothermia (15 °C) were induced. After CA, cardiopumonary bypass (CPB) was restarted, and the animals were gradually re-warmed and resuscitated. The animals were assisted with ventilators until spontaneous breathing was achieved. The index of hemodynamic perioperative serum chemistry values [alanine transaminase (ALT), aspartate aminotransferase (AST), creatinine (CR), lactic dehydrogenase (LDH) and troponin T (TnT)] and survival were observed from pre-operation to 7 days post-operation.ResultsFifteen animals were enrolled in the experiment, while 1 animal in CA120 group died from surgical error. All 8 animals in CA90 group recovered, with only 1 animal displaying mild disability. However, in CA120 group, only 2 animals survived with severe disability, and the other 5 animals died after 2 days post-operation. In CA90 group, the perioperative serum chemistry values increased at 1 day post-operation (ALT 84.43 ± 18.65 U/L; AST 88.99 ± 23.19 U/L; Cr 87.90 ± 24.49 μmol/L; LDH 1894.13 ± 322.26 U/L; TnT 0.849 ± 0.135 ng/ml) but decreased to normal or almost normal levels at 7 days post-operation (ALT 52.48 ± 9.04 U/L; AST 75.23 ± 21.46 U/L; Cr 82.69 ± 18.41 μmol/L; LDH 944.67 ± 834.32 U/L; TnT 0.336 ± 0.076 ng/ml).ConclusionsProfound hypothermia-induced SADR is an effective method for inducing cardiac arrest. Our results indicate that inducing CA for 90 min (at 15 °C) is safer than doing so for 120 min. Our results indicate that 120 min of CA at 15 °C is dangerous and can result in high mortality and severe neurological complications. Further experimentation is needed to determine whether 120 min of CA at temperatures lower than 15 °C can lead to safe recovery.

Hyperbranched polyglycerol as a colloid in cold organ preservation solutions.

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.

Antibiotic Prophylaxis in (Sub)Normothermic Organ Preservation

Bacterial contamination during cold organ preservation occurs without major complications. However, with organ preservation steering toward (sub)normothermic temperatures, bacterial contamination may be detrimental with limited evidence to support the choice of antibiotic. This study aimed to determine the effective antibiotic prophylaxis for (sub)normothermic preservation by investigating whether Staphylococcus epidermidis was capable of growing in a subnormothermia-compatible preservation solution Polysol (PS) and in solutions designed for cold preservation (University of Wisconsin solution, histidine-tryptophan-ketoglutarate solution, and Belzer-machine perfusion solution). Various S. epidermidis and Staphylococcus aureus strains were exposed to ceftriaxone and cefazolin at concentrations from 0 to 1000 μg/mL under subnormothermic and normothermic conditions in PS. To mimic procedural conditions, the effect of cefazolin was determined after exposure of bacteria to 20-hr incubation at 28°C in the presence of cefazolin and subsequent incubation at 37°C in the absence of cefazolin. The toxicity of cefazolin was assessed by cell viability and caspase activation assays in porcine kidney endothelial cells. Without antibiotics, PS sustained bacterial growth under sub(normothermic) conditions, whereas growth was absent in cold preservation solutions. Cefazolin exhibited greater bactericidal effect on S. epidermidis than ceftriaxone. However, after inoculating PS with 10 colony-forming units/mL, only a cefazolin concentration of 1000 μg/mL was able to exert a complete bactericidal effect on S. epidermidis and S. aureus strains and maintain sterility after removal of cefazolin. Finally, 1000 μg/mL cefazolin showed no adverse effects on porcine kidney endothelial cells. Based on these findings, we recommend that high-dose cefazolin be used for prophylaxis in (sub)normothermic organ preservation with PS.

Beneficial Effects of Ischemic Preconditioning on Pancreas Cold Preservation

Ischemic preconditioning (IPC) confers tissue resistance to subsequent ischemia in several organs. The protective effects are obtained by applying short periods of warm ischemia followed by reperfusion prior to extended ischemic insults to the organs. In the present study, we evaluated whether IPC can reduce pancreatic tissue injury following cold ischemic preservation. Rat pancreata were exposed to IPC (10 min of warm ischemia followed by 10 min of reperfusion) prior to ~18 h of cold preservation before assessment of organ injury or islet isolation. Pancreas IPC improved islet yields (964 ± 336 vs. 711 ± 204 IEQ/pancreas; p = 0.004) and lowered islet loss after culture (33 ± 10% vs. 51 ± 14%; p = 0.0005). Islet potency in vivo was well preserved with diabetes reversal and improved glucose clearance. Pancreas IPC reduced levels of NADPH-dependent oxidase, a source of reactive oxygen species, in pancreas homogenates versus controls (78.4 ± 45.9 vs. 216.2 ± 53.8 RLU/μg; p = 0.002). Microarray genomic analysis of pancreata revealed upregulation of 81 genes and downregulation of 454 genes (greater than twofold change) when comparing IPC-treated glands to controls, respectively, and showing a decrease in markers of apoptosis and oxidative stress. Collectively, our study demonstrates beneficial effects of IPC of the pancreas prior to cold organ preservation and provides evidence of the key role of IPC-mediated modulation of oxidative stress pathways. The use of IPC of the pancreas may contribute to increasing the quality of donor pancreas for transplantation and to improving organ utilization.

Islet Isolation From Human Pancreas With Extended Cold Ischemia Time

The general consensus among transplant centers is that a cold ischemia time (CIT) beyond 8 hours results in reduced yields and quality of human islets. We sought to optimize the isolation process and enzymes for pancreata with extended CIT. We processed 16 extended CIT pancreata (13.2 ± 0.7 hours). Donors averaged 50.8 ± 2.6 (standard error of the mean) years old with a body mass index of 28.6 ± 1.5. Glands were shipped in cold organ preservation solution without oxygenated perfluorocarbon. Isolations were performed under a protocol optimized for digestion with the new cGMP collagenase from Roche. Purification used continuous Euroficoll/University of Wisconsin gradients. Islets were cultured in two types of Prodo cGMP islet culture media and/or in Miami 1A media. Glucose-stimulated insulin secretion assays were performed after 8 to 16 days of culture. Prepurification yield averaged 415 ± 41 KIEQ postpurification, 359 ± 29 KIEQ (purification loss 13.5%); and postculture 317 ± 27 KIEQ (culture loss 11.7%). Our process liberated an average of 4278 IEQ/g of pancreas (97 ± 5 g). Most islets were recovered in the purest fraction (purity 79.7% ± 1.9%). Culture loss in our enhanced culture media was 11.7%. After 2 to 3 days in culture, viability was 92% ± 1%. Islets exhibited compactness and dithizone staining. Glucose-stimulated insulin secretion assays performed after 3 to 23 days in our PIM(R) media resulted in a stimulation index of 6.8 ± 1.7 (G50 to G350). We concluded that our human islet isolation process permitted the recovery of large numbers of high-quality human islets from extended CIT pancreata and that our cGMP islet culture media was superior to the current standard CMRL-based media.

Putting Life On Hold—For How Long? Profound Hypothermic Cardiopulmonary Bypass in a Swine Model of Complex Vascular Injuries

Rapid induction of profound hypothermia for emergency preservation and resuscitation can improve survival from uncontrolled lethal hemorrhage in large animal models. We have previously demonstrated that profound hypothermia (10 degrees C) must be induced rapidly (2 degrees C/min) and reversed gradually (0.5 degrees C/min) for best results. However, the maximum duration of hypothermic arrest in a clinically relevant trauma model remains unknown. Uncontrolled lethal hemorrhage was induced in 22 swine by creating an iliac artery and vein injury, followed 30 minutes later (simulating transport time) by laceration of the descending thoracic aorta. Through a thoracotomy approach, a catheter was placed in the aorta, and cold organ preservation solution was infused using a roller pump to rapidly induce profound hypothermia (10 degrees C) which was maintained with low-flow cardiopulmonary bypass. Vascular injuries were repaired during the asanguinous hypothermic low flow period. Profound hypothermia was maintained (n = 10-12 per group) for either 60 minutes or 120 minutes. After repair of injuries, animals were rewarmed (0.5 degrees C/min) and resuscitated on cardiopulmonary bypass, and whole blood was infused during this period. Animals were monitored for 4 weeks for neurologic deficits, organ dysfunction, and postoperative complications. The 4-week survival rates in 60- and 120-minute groups were 92% and 50%, respectively (p < 0.05). The surviving animals were neurologically intact and had no long-term organ dysfunction, except for one animal in the 120-minute group. The animals subjected to 120 minutes of hypothermia had significantly worse lactic acidosis, displayed markedly slower recovery, and had significantly higher rates of postoperative complications, including late deaths because of infections. In a model of lethal injuries, rapid induction of profound hypothermia can prevent death. Profound hypothermia decreases but does not abolish metabolism. With current methods, the upper limit of hypothermic arrest in the setting of uncontrolled hemorrhage is 60 minutes.

Profound Hypothermic Cardiopulmonary Bypass Facilitates Survival Without a High Complication Rate in a Swine Model of Complex Vascular, Splenic, and Colon Injuries

Induction of a profound hypothermia for emergency preservation and resuscitation in severe hemorrhagic shock can improve survival from lethal injuries, but the impact of hypothermia on bleeding and infectious complications has not been completely determined. Uncontrolled hemorrhage was induced in 26 swine (95 to 135 lbs) by creating an iliac artery and vein injury, and 30 minutes later, by lacerating the descending thoracic aorta. Through a left thoracotomy approach, profound total body hypothermia (10 degrees C) was induced (2 degrees C/min) by infusing cold organ preservation solution into the aorta. The experimental groups were: vascular injuries alone (group 1, n=10), vascular and colon injuries (group 2, n=8), and vascular, colon, and splenic injuries (group 3, n=8). All injuries were repaired during 60 minutes of low-flow cardiopulmonary bypass (CPB) with hemodilution and profound hypothermia; then the animals were slowly rewarmed (0.5 degrees C/min) back to normothermia. Survivors were monitored for 6 weeks for postoperative bleeding, neurologic deficits, cognitive function (learning new skills), organ dysfunction, and septic complications. Six-week survival rates were 90% in group 1, 87.5% in group 2, and 75% in group 3 (p > 0.05). One animal in each group died from acute cardiac failure during the early postoperative phase. Splenic salvage was possible in all animals, and none required complete splenectomy for hemorrhage control. All surviving animals were neurologically intact, displayed normal learning capacity, and had no longterm organ dysfunction. None of the animals had postoperative hemorrhage or experienced septic complications. One animal in group 3 died on the ninth postoperative day because of bowel obstruction (volvulus). Induction of profound hypothermia can preserve the viability of key organs during repair of lethal injuries. This strategy can be used even in the presence of solid organ and bowel injuries to improve survival, without any considerable increase in postoperative complication rates.

Development and Testing of Portable Pump for the Induction of Profound Hypothermia in a Swine Model of Lethal Vascular Injuries

Rapid induction of a profound hypothermic state (suspended animation) can maintain viability of key organs during repair of lethal injuries. Conventional cardiopulmonary bypass equipment (roller pump) used to induce and reverse hypothermia is expensive, bulky, requires standard electricity, and is not transportable. Development of a small, portable, battery operated, disposable, pump can logistically facilitate induction and maintenance of hypothermia. In this experiment, a portable prototype pump was tested and its performance was compared with the regular roller pump in a swine model of lethal vascular injuries. Uncontrolled hemorrhage was induced in 16 swine (80-120 lbs) by creating an iliac artery and vein injury (nonlethal). After 30 minutes of pulseless shock, the descending thoracic aorta was lacerated (lethal injury). Through a left thoracotomy approach, a catheter was placed in the aorta and cold organ preservation solution was infused to rapidly (2 degrees C/min) induce hypothermia (10 degrees C) for 60 minutes. The performance of the prototype pump was initially tested in a nonsurvival experiment (four animals). Then, 12 animals were cooled either with (n = 6/group) (1) conventional roller pump or (2) small prototype pump. The injuries were repaired during hypothermic arrest and the animals were re-warmed (0.5 degrees C/min). Whole blood was infused during resuscitation on cardiopulmonary bypass. Surviving animals were closely monitored for 3 weeks for postoperative complications, neurologic deficits, and organ dysfunction. The flow rates and the time needed to induce and reverse profound hypothermia were no different between the prototype and the conventional roller pumps. Three-week survival rates were 83% in both groups. Only a transient increase in liver enzymes, and markers of cellular injury (creatine kinase, lactate dehydrogenase) was noted (no meaningful difference between groups), with no long-term organ dysfunction. In this large animal model of lethal vascular injuries, a portable, battery operated, disposable, rotary pump performed as well as the conventional roller pump. The logistical advantages of this system make it an attractive choice for inducing hypothermia in emergency departments and austere settings, and for maintaining hypothermia during transport.

Profound hypothermia is superior to ultraprofound hypothermia in improving survival in a swine model of lethal injuries

Rapid induction of profound hypothermia can improve survival from uncontrolled lethal hemorrhage. However, the optimal depth of hypothermia in this setting remains unknown. This experiment was designed to compare the impact of deep (15 degrees C), profound (10 degrees C), and ultraprofound (5 degrees C) hypothermia on survival and organ functions. Uncontrolled lethal hemorrhage was induced in 32 swine (80-120 lb) by creating an iliac artery and vein injury, followed 30 minutes later by laceration of the descending thoracic aorta. Hypothermia was induced rapidly (2 degrees C/min) by infusing cold organ preservation solution into the aorta through a thoracotomy. The experimental groups were (n = 8 per group): a normothermic control, and 3 hypothermic groups in which the core temperature was reduced to 15 degrees C, 10 degrees C, and 5 degrees C. Vascular injuries were repaired during 60 minutes of hypothermia. Animals were then rewarmed (0.5 degrees C/min) and resuscitated on cardiopulmonary bypass, and monitored for 6 weeks for neurologic deficits, cognitive function, and organ dysfunction. All normothermic animals died, whereas 6-week survival rates for the 15 degrees C, 10 degrees C, and 5 degrees C groups were 62.5%, 87.5%, and 25%, respectively (P < .05: normothermic vs 15 degrees C and 10 degrees C; 10 degrees C vs 5 degrees C). The surviving animals from the 15 degrees C and 10 degrees C groups were neurologically intact, displayed normal learning capacity, and had no long-term organ dysfunction. The survivors from the 5 degrees C group displayed slower recovery and impaired cognitive functions. In a model of lethal injuries, rapid induction of profound hypothermia can prevent death. The depth of hypothermia influences survival, with a better outcome associated with a core temperature of 10 degrees C compared with 5 degrees C.

Organ preservation solutions increase endothelial permeability and promote loss of junctional proteins.

To investigate the effects of the organ preservation solutions UW and Plegisol on endothelial permeability; occludin and vascular endothelial (VE)-cadherin content in human umbilical vein endothelial cells (HUVEC); and junctional localization of these proteins after exposure to these solutions. Organ preservation for transplantation is limited by several challenges, including loss of tissue function, tissue injury, and tissue edema. Occludin and VE-cadherin are responsible for maintaining and regulating the endothelial solute barrier. Several studies have noted organ edema and dysfunction with preservation, as well as gaps between endothelial cells suggesting that disorganization of junctional proteins (e.g., occludin and VE-cadherin) is responsible for interstitial edema. HUVEC monolayers were treated with 4 degrees C UW and Plegisol for 3 and 6 hours and then reperfused with normal buffer. Permeability was examined using FITC-dextran tracer during the reperfusion phase. Occludin and VE-cadherin content at different time points was measured by Western blotting. Treated groups were also examined by immunofluorescence for occludin, VE-cadherin, and F-actin. Compared with untreated controls, cold preservation for 3 and 6 hours increased endothelial permeability after rewarming, which appears to depend on the duration of cold exposure. Monolayers exposed to 3 hours of cold preservation did not have increased permeability in the first hour after rewarming but had significantly increased permeability after the first hour and all subsequent time points. Monolayers exposed to 6 hours of cold preservation had increased permeability after the first hour and at all later time points. Western blotting demonstrated that occludin content was decreased to a similar extent with all solutions after 3 hours of cold preservation. Six hours of cold preservation in Plegisol reduced the occludin content significantly compared with UW and control. VE-cadherin content was unchanged after 3 hours of cold preservation but was dramatically reduced in all groups at 6 hours. Immunofluorescent staining demonstrated junctional gap formation and discontinuous staining of occludin and VE-cadherin with all cold preservation protocols; changes in F-actin organization were observed at 3 and 6 hours after cold preservation. The changes in occludin, VE-cadherin, and F-actin content and organization and increased permeability associated with cold storage demonstrate that alterations of the tight and adherens junctions may underlie organ edema associated with cold organ preservation. These data also suggest that novel strategies to maintain the content and integrity of endothelial junctional proteins may provide an important therapeutic avenue for organ preservation.

A new technique for isolating and culturing human hepatocytes from whole or split livers not used for transplantation.

Large numbers of human hepatocytes were obtained from split and whole livers by using an adaptive form of the collagenase perfusion technique employed in rodent and human biopsies. In order to guarantee a homogenous distribution of the perfusate within the whole specimen, major hepatic veins were cannulated with large bore catheters. This technique allowed for the isolation of human hepatocytes on a large scale (up to 18.5 x 10(9) in one case) from normal and diseased liver specimens. The yield of isolated normal viable hepatocytes is inversely proportional to the donor age. In addition, it was noted that a short time between declared death and organ harvest (cross clamp time) results in higher viability of hepatocytes. In contrast, the time of cold organ preservation did not correlate with the viability or the yield of isolated hepatocytes. We conclude that the technique presented here allows isolation of large numbers of human hepatocytes from specimens unsuitable for transplantation but very valuable for biomedical research.

Viability and primary culture of rat hepatocytes after hypothermic preservation: The superiority of the leibovitz medium over the university of wisconsin solution for cold storage

Hepatocytes isolated from adult rat livers were hypothermically preserved for 24 or 48 hr before being plated under conventional culture conditions. They were stored either in the Leibovitz medium, a cell culture medium with and without polyethylene glycol (PEG), a compound known to suppress ischemia-induced cell swelling, or in the University of Wisconsin solution, the most effective solution for cold organ preservation. After 24 or 48 hr of storage at 4.5 degrees C in Leibovitz medium, cell viability and adherence efficiency to plastic dish, were only slightly reduced, whereas University of Wisconsin hepatocytes had a decreased viability and (especially after 48-hr storage) lost their adhesion ability; they did not survive in vitro. The metabolic competence of hepatocytes maintained in Leibovitz medium was retained over the 3 days of culture, as shown by low extracellular levels of the membrane-bound and cytosolic hepatic enzymes, as well as by intracellular glutathione content, albumin secretion rate and several phase I and phase II drug metabolic reactions very close to those found with fresh hepatocytes maintained under similar culture conditions. Addition of polyethylene glycol to the Leibovitz medium resulted in slightly higher viability and function of hepatocytes after cold storage. These results clearly demonstrate that viability of a transplanted liver does not correlate with long-term in vitro viability of isolated hepatocytes after hypothermic preservation in University of Wisconsin solution. They also suggest that nutritional and energy substrates as found in the Leibovitz medium are probably required to define a suitable solution for cold preservation of isolated parenchymal cells. The findings with Leibovitz medium favor the conclusion that hypothermically preserved hepatocytes could be used for various metabolic studies and for the treatment of liver insufficiency.