Variability in perfusion conditions and set-up parameters used in ex vivo human placenta models: A literature review.

The ex vivo human placenta perfusion model has proven to be clinically relevant to study transfer- and fetal exposure of various drugs. Although the method has existed for a long period, the setup of the perfusion model has not been generalized yet. This review aims to summarize the setups of ex vivo placental perfusion models used to examine drug transfer across the placenta to identify generalized properties and differences across setups. A literature search was carried out in PubMed September 26, 2022. Studies were labeled as relevant when information was reported, between 2000 and 2022, on the setups of ex vivo placental perfusion models used to study drug transfer across the placenta. The placenta perfusion process, and the data extraction, was divided into phases of preparation, control, drug, and experimental reflecting the chronological timeline of the different phases during the entire placental perfusion process. 135 studies describing an ex vivo human placental perfusion experiment were included. Among included studies, the majority (78.5%) analyzed drug perfusion in maternal to fetal direction, 18% evaluated bi-directional drug perfusion, 3% under equilibrium conditions, and one study investigated drug perfusion in fetal to maternal direction. This literature review facilitates the comparison of studies that employ similar placenta perfusion protocols for drug transfer studies and reveals significant disparities in the setup of these ex vivo placental perfusion models. Due to interlaboratory variability, perfusion studies are not readily comparable or interchangeable. Therefore, a stepwise protocol with multiple checkpoints for validating placental perfusion is needed.

Pancreas Transplantation from Donors after Circulatory Death: an Irrational Reluctance?

PURPOSE OF REVIEW: Beta-cell replacement is the best therapeutic option for patients with type 1 diabetes. Because of donor scarcity, more extended criteria donors are used for transplantation. Donation after circulatory death donors (DCD) are not commonly used for pancreas transplantation, because of the supposed higher risk of complications. This review gives an overview on the pathophysiology, risk factors, and outcome in DCD transplantation and discusses different preservation methods. RECENT FINDINGS: Studies on outcomes of DCD pancreata show similar results compared with those of donation after brain death (DBD), when accumulation of other risk factors is avoided. Hypothermic machine perfusion is shown to be a safe method to improve graft viability in experimental settings. DCD should not be the sole reason to decline a pancreas for transplantation. Adequate donor selection and improved preservation techniques can lead to enhanced pancreas utilization and outcome.

Chronic pain after hand-assisted laparoscopic donor nephrectomy.

BACKGROUND: Data on chronic pain after kidney donation are sparse. The aim of this study was to assess the incidence of chronic pain after hand-assisted laparoscopic nephrectomy. METHODS: Living kidney donors who donated between 2011 and 2017 at the University Medical Centre Groningen were included. All patients underwent hand-assisted laparoscopic donor nephrectomy. Postdonation pain and movement disabilities were assessed using the Carolinas Comfort Scale (CCS) and a visual analogue scale (VAS). The prevalence, severity of pain and the need for analgesics were reported. RESULTS: Some 333 living kidney donors with a mean age of 56 years were included. At a median of 19 (i.q.r. 10-33) months after donation, 82 donors (24·6 per cent) had a CCS score above 0, of which 58 (71 per cent) had a CCS score of at least 2 and 57 (70 per cent) reported movement limitations. Some 110 donors (33·0 per cent) had a VAS score of more than 0. Complaints mainly occurred during bending over (12·3 per cent) and exercising (12·4 per cent). Thirty-two donors (9·7 per cent) required analgesics during follow-up between donation and the time of measurement, and six of 82 (7 per cent) reported chronic inguinal pain. In multivariable analysis, donor age (odds ratio (OR) 0·97, 95 per cent c.i. 0·95 to 0·99; P = 0·020) and length of hospital stay (OR 1·21, 1·01 to 1·51; P = 0·041) were independently associated with chronic pain. CONCLUSION: One-quarter of donors experienced chronic postdonation pain or discomfort, most of which was bothersome. Younger donors and those with a longer postoperative hospital stay had more symptoms.

Organ-specific responses during brain death: increased aerobic metabolism in the liver and anaerobic metabolism with decreased perfusion in the kidneys.

Hepatic and renal energy status prior to transplantation correlates with graft survival. However, effects of brain death (BD) on organ-specific energy status are largely unknown. We studied metabolism, perfusion, oxygen consumption, and mitochondrial function in the liver and kidneys following BD. BD was induced in mechanically-ventilated rats, inflating an epidurally-placed Fogarty-catheter, with sham-operated rats as controls. A 9.4T-preclinical MRI system measured hourly oxygen availability (BOLD-related R2*) and perfusion (T1-weighted). After 4 hrs, tissue was collected, mitochondria isolated and assessed with high-resolution respirometry. Quantitative proteomics, qPCR, and biochemistry was performed on stored tissue/plasma. Following BD, the liver increased glycolytic gene expression (Pfk-1) with decreased glycogen stores, while the kidneys increased anaerobic- (Ldha) and decreased gluconeogenic-related gene expression (Pck-1). Hepatic oxygen consumption increased, while renal perfusion decreased. ATP levels dropped in both organs while mitochondrial respiration and complex I/ATP synthase activity were unaffected. In conclusion, the liver responds to increased metabolic demands during BD, enhancing aerobic metabolism with functional mitochondria. The kidneys shift towards anaerobic energy production while renal perfusion decreases. Our findings highlight the need for an organ-specific approach to assess and optimise graft quality prior to transplantation, to optimise hepatic metabolic conditions and improve renal perfusion while supporting cellular detoxification.

Propofol-based anaesthesia versus sevoflurane-based anaesthesia for living donor kidney transplantation: results of the VAPOR-1 randomized controlled trial.

BACKGROUND: Kidney transplantation is associated with harmful processes affecting the viability of the graft. One of these processes is associated with the phenomenon of ischaemia-reperfusion injury. Anaesthetic conditioning is a widely described strategy to attenuate ischaemia-reperfusion injury. We therefore conducted the Volatile Anaesthetic Protection of Renal Transplants-1 trial, a pilot project evaluating the influence of two anaesthetic regimens, propofol- vs sevoflurane-based anaesthesia, on biochemical and clinical outcomes in living donor kidney transplantation. METHODS: Sixty couples were randomly assigned to the following three groups: PROP (donor and recipient propofol), SEVO (donor and recipient sevoflurane), and PROSE (donor propofol and recipient sevoflurane). The primary outcome was renal injury reflected by urinary biomarkers. The follow-up period was 2 yr. RESULTS: Three couples were excluded, leaving 57 couples for analysis. Concentrations of kidney injury molecule-1 (KIM-1), N -acetyl-ß- d -glucosaminidase (NAG), and heart-type fatty acid binding protein (H-FABP) in the first urine upon reperfusion showed no differences. On day 2, KIM-1 concentrations were higher in SEVO [952.8 (interquartile range 311.8-1893.0) pg mmol -1 ] compared with PROP [301.2 (202.0-504.7) pg mmol -1 ]. This was the same for NAG: SEVO, 1.835 (1.162-2.457) IU mmol -1 vs PROP, 1.078 (0.819-1.713) IU mmol -1 . Concentrations of H-FABP showed no differences. Measured glomerular filtration rate at 3, 6, and 12 months showed no difference. After 2 yr, there was a difference in the acute rejection rate ( P =0.039). Post hoc testing revealed a difference between PROP (35%) and PROSE (5%; P =0.020). The difference between PROP and SEVO (11%) was not significant ( P =0.110). CONCLUSIONS: The SEVO group showed higher urinary KIM-1 and NAG concentrations in living donor kidney transplantation on the second day after transplantation. This was not reflected in inferior graft outcome. CLINICAL TRIAL REGISTRATION: NCT01248871.

Exogenous administration of thiosulfate, a donor of hydrogen sulfide, attenuates angiotensin II-induced hypertensive heart disease in rats.

BACKGROUND AND PURPOSE: Hypertension is an important mediator of cardiac damage and remodelling. Hydrogen sulfide (H2S) is an endogenously produced gasotransmitter with cardioprotective properties. However, it is not yet in clinical use. We, therefore, investigated the protective effects of sodium thiosulfate (STS), a clinically applicable H2 S donor substance, in angiotensin II (Ang II)-induced hypertensive cardiac disease in rats. EXPERIMENTAL APPROACH: Male Sprague Dawley rats were infused with Ang II (435 ng kg min(-1)) or saline (control) for 3 weeks via s.c. placed osmotic minipumps. During these 3 weeks, rats received i.p. injections of either STS, NaHS or vehicle (0.9% NaCl). KEY RESULTS: Compared with controls, Ang II infusion caused an increase in systolic and diastolic BP with associated cardiac damage as evidenced by cardiac hypertrophy, an increase in atrial natriuretic peptide (ANP) mRNA, cardiac fibrosis and increased oxidative stress. Treatment with NaHS and STS prevented the development of hypertension and the increase in ANP mRNA levels. Furthermore, the degree of cardiac hypertrophy, the extent of histological fibrosis in combination with the expression of profibrotic genes and the levels of oxidative stress were all significantly decreased. CONCLUSIONS AND IMPLICATIONS: Ang II-induced hypertensive cardiac disease can be attenuated by treatment with STS and NaHS. Although BP regulation is the most plausible mechanism of cardiac protection, the antifibrotic and antioxidant properties of released sulfide may also contribute to their effects. Our data show that H2 S might be a valuable addition to the already existing antihypertensive and cardioprotective therapies.

Emerging role of gasotransmitters in renal transplantation.

Once patients with kidney disease progress to end-stage renal failure, transplantation is the preferred option of treatment resulting in improved quality of life and reduced mortality compared to dialysis. Although 1-year survival has improved considerably, graft and patient survival in the long term have not been concurrent, and therefore new tools to improve long-term graft and patient survival are warranted. Over the past decades, the gasotransmitters nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) have emerged as potent cytoprotective mediators in various diseases. All three gasotransmitters are endogenously produced messenger molecules that possess vasodilatory, anti-apoptotic, anti-inflammatory and anti-oxidant properties by influencing an array of intracellular signaling processes. Although many regulatory functions of gasotransmitters have overlapping actions, differences have also been reported. In addition, crosstalk between NO, CO and H2S results in synergistic regulatory effects. Endogenous and exogenous manipulation of gasotransmitter levels modulates several processes involved in renal transplantation. This review focuses on mechanisms of gas-mediated cytoprotection and complex interactions between gasotransmitters in renal transplantation.

Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.

In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. We have studied the feasibility of normothermic machine perfusion in four discarded human donor livers. Normothermic machine perfusion consisted of pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion for 6 h. Two hollow fiber membrane oxygenators provided oxygenation of the perfusion fluid. Biochemical markers in the perfusion fluid reflected minimal hepatic injury and improving function. Lactate levels decreased to normal values, reflecting active metabolism by the liver (mean lactate 10.0 ± 2.3 mmol/L at 30 min to 2.3 ± 1.2 mmol/L at 6 h). Bile production was observed throughout the 6 h perfusion period (mean rate 8.16 ± 0.65 g/h after the first hour). Histological examination before and after 6 h of perfusion showed well-preserved liver morphology without signs of additional hepatocellular ischemia, biliary injury or sinusoidal damage. In conclusion, this study shows that normothermic machine perfusion of human donor livers is technically feasible. It allows assessment of graft viability before transplantation, which opens new avenues for organ selection, therapeutic interventions and preconditioning.

The prognostic value of renal resistance during hypothermic machine perfusion of deceased donor kidneys.

Vascular renal resistance (RR) during hypothermic machine perfusion (HMP) is frequently used in kidney graft quality assessment. However, the association between RR and outcome has never been prospectively validated. Prospectively collected RR values of 302 machine-perfused deceased donor kidneys of all types (standard and extended criteria donor kidneys and kidneys donated after cardiac death), transplanted without prior knowledge of these RR values, were studied. In this cohort, we determined the association between RR and delayed graft function (DGF) and 1-year graft survival. The RR (mmHg/mL/min) at the end of HMP was an independent risk factor for DGF (odds ratio 38.1 [1.56-934]; p = 0.026) [corrected] but the predictive value of RR was low, reflected by a c-statistic of the receiver operator characteristic curve of 0.58. The RR was also found to be an independent risk factor for 1-year graft failure (hazard ratio 12.33 [1.11-136.85]; p = 0.004). Determinants of transplant outcome are multifactorial in nature and this study identifies RR as an additional parameter to take into account when evaluating graft quality and estimating the likelihood of successful outcome. However, RR as a stand-alone quality assessment tool cannot be used to predict outcome with sufficient precision.

Activation of hemostasis in brain dead organ donors: an observational study.

BACKGROUND: Brain death is associated with a systemic inflammatory response resulting in diminished organ function in individuals transplanted with organs from brain dead donors. As inflammation is accompanied by activation of coagulation, we hypothesized that activation of hemostasis occurs in brain dead organ donors. OBJECTIVES: To assess the hemostatic status in brain dead organ donors. PATIENTS AND METHODS: In this study, we systematically assessed the hemostatic system in samples taken from 30 brain dead donors. As controls, blood samples from 30 living kidney donors were included. RESULTS AND CONCLUSIONS: Compared with the living donors, brain dead donors showed significant platelet activation (assessed by glycocalicin plasma levels), and a profound dysbalance in the von Willebrand factor/ADAMTS13 axis, which is key in platelet attachment to damaged vasculature. Furthermore, compared with the living donors, brain dead donors showed a significantly increased activation of secondary hemostasis with formation of fibrin (assessed by plasma levels of prothrombin fragment 1 + 2, fibrinopeptide A and D-dimer). Finally, brain dead donors showed profound hypofibrinolysis as assessed by a global clot lysis assay, which was attributed to substantially elevated plasma levels of plasminogen activator inhibitor type 1. Collectively, our results show activation of hemostasis and dysregulated fibrinolysis in brain dead organ donors. This prothrombotic state may contribute to formation of microthrombi in transplantable organs, which potentially contributes to deterioration of organ function.

Kidney injury molecule-1 is an early noninvasive indicator for donor brain death-induced injury prior to kidney transplantation.

With more marginal deceased donors affecting graft viability, there is a need for specific parameters to assess kidney graft quality at the time of organ procurement in the deceased donor. Recently, kidney injury molecule-1 (Kim-1) was described as an early biomarker of renal proximal tubular damage. We assessed Kim-1 in a small animal brain death model as an early and noninvasive marker for donor-derived injury related to brain death and its sequelae, with subsequent confirmation in human donors. In rat kidney, real-time PCR revealed a 46-fold Kim-1 gene upregulation after 4 h of brain death. In situ hybridization showed proximal tubular Kim-1 localization, which was confirmed by immunohistochemistry. Also, Luminex assay showed a 6.6-fold Kim-1 rise in urine after 4 h of brain death. In human donors, 2.5-fold kidney injury molecule-1 (KIM-1) gene upregulation and 2-fold higher urine levels were found in donation after brain death (DBD) donors compared to living kidney donors. Multiple regression analysis showed that urinary KIM-1 at brain death diagnosis was a positive predictor of recipient serum creatinine, 14 days (p < 0.001) and 1 year (p < 0.05) after kidney transplantation. In conclusion, we think that Kim-1 is a promising novel marker for the early, organ specific and noninvasive detection of brain death-induced donor kidney damage.

[A successful transplant begins with organ preservation]. / Het succes van transplantatie begint met orgaanpreservatie.

Organ preservation is a critical link in the chain of donation and transplantation and has a significant effect on post-transplant graft function and graft survival. Clinically, the most widely used form of preservation is static cold storage, which is based on the reduction of cellular metabolism by hypothermia. Although static cold storage is simple and effective, it is questionable whether it still meets present day requirements. Due to the persistent shortage of donors, increasing numbers of organs are being accepted from older and non-heart-beating donors. Organs from such donors may benefit from a more dynamic method of preservation: hypothermic machine perfusion.

High mobility group box 1 and adenosine are both released by endothelial cells during hypothermic preservation.

Hypothermic preservation of solid allografts causes profound damage of vascular endothelial cells. This, in turn, might activate innate immunity. In the present study we employed an in vitro model to study to what extent supernatants of damaged endothelial cells are able to activate innate immunity and to study the nature of these signals. The expression of high mobility group box 1 (HMGB1) and adhesion molecules on human umbilical vein endothelial cell was studied by immunofluorescence, fluorescence activated cell sorter and Western blotting. Cytokine production was performed by enzyme-linked immunosorbent assay. HMGB1 expression was lost completely in endothelial cells after hypothermic preservation. This was associated with cell damage as it occurred only in untreated endothelial cell but not in cells rendered resistant to hypothermia-mediated damage by dopamine treatment. Only supernatants from hypothermia susceptible cells up-regulated the expression of interleukin (IL)-8 and adhesion molecules in cultured endothelial cells in an HMGB1-dependent manner. In whole blood assays, both supernatants of hypothermia susceptible and resistant cells inhibited tumour necrosis factor (TNF)-alpha production concomitantly with an increased IL-10 secretion. The activity of the supernatants was already found after 6 h of hypothermic preservation, and paralleled the decrease in intracellular adenosine triphosphate (ATP) levels. Modulation of TNF-alpha and IL-10 production by these supernatants was abrogated completely by prior treatment with adenosine deaminase and was similar to the response of an A2R agonist. Our study demonstrates that both HMGB1 and adenosine are released during hypothermic preservation. While release of HMGB1 is caused by cell damage, release of adenosine seems to be related to ATP hydrolysis, occurring in both susceptible and resistant cells.

Kidney grafts from brain dead donors: Inferior quality or opportunity for improvement?

Major improvements in immunosuppressive treatment, surgical techniques, and treatment of post-transplant complications have contributed considerably to improved outcome in renal transplantation over the past decades. Yet, these accomplishments have not led to similar improvements in transplant outcome when the results of living and deceased donors are compared. The enormous demand for donor kidneys has allowed for the increase in acceptance of suboptimal donors. The use of brain dead patients as organ donors has had a tremendous positive influence on the number of renal transplants. Unfortunately, the physiologically abnormal state of brain death has a negative effect on transplant outcome. The fact that transplanted kidneys derived from brain dead donors have a decreased viability indicates that potential grafts are already damaged before retrieval and preservation. In this review, we present an overview of the current knowledge of (patho)-physiological effects of brain death and its relevance for renal transplant outcome. In addition, several options for therapeutic intervention during brain death in the donor with the goal to improve organ viability and transplant outcome are discussed.

The Groningen hypothermic liver perfusion pump: functional evaluation of a new machine perfusion system.

To improve preservation of donor livers, we have developed a portable hypothermic machine perfusion (HMP) system as an alternative for static cold storage. A prototype of the system was built and evaluated on functionality. Evaluation criteria included 24 h of adequate pressure controlled perfusion, sufficient oxygenation, a maintained 0-4 degrees C temperature and sterile conditions. Porcine livers were perfused with pump pressures that were set at 4 mmHg (continuous, portal vein) and 30/20 mmHg, at 60 BPM (pulsatile, hepatic artery). Control livers were preserved using the clinical golden standard: static cold storage. In the HMP group, pressure, flow and temperature were continuously monitored for 24 h. At time-points t = 0, 2, 4, 8, 12, and 24 h samples of University of Wisconsin machine preservation solution were taken for measurement of partial oxygen pressure (pO(2)) and lacto-dehydrogenase. Biopsies in every lobe were taken for histology and electron microscopy; samples of ice, preservation solution, liver surface, and bile were taken and cultured to determine sterility. Results showed that temperature was maintained at 0-4 degrees C; perfusion pressure was maintained at 4 mmHg and 30/20 mmHg for portal vein and hepatic artery, respectively. Flow was approximately 350 and 80 ml/min, respectively, but decreased in the portal vein, probably due to edema formation. Arterial pO(2) was kept at 100 kPa. Histology showed complete perfusion of the liver with no major damage to hepatocytes, bile ducts, and non-parenchymal cells compared to control livers. The machine perfusion system complied to the design criteria and will have to demonstrate the superiority of machine perfusion over cold storage in transplant experiments.

Time-dependent changes in donor brain death related processes.

Donor brain death (BD) affects kidney function and survival after transplantation. Studies on brain dead kidney donors indicate that, besides inflammation and coagulation, cytoprotective gene expression is activated as well. Here, we evaluated in a time-course experiment progression of these renal BD-related processes. Animals were sacrificed 0.5, 1, 2 or 4 h after BD and compared to sham-operated controls. Proinflammatory genes (E-selectin, MCP-1, II-6) were massively up-regulated (p < 0.05) already 0.5 h after BD. Inducers of proinflammatory gene expression were either activated (NF-kappaB) or induced in expression (Egr-1) after 0.5 h of BD. Increased numbers of infiltrating granulocytes were seen in the interstitium from 0.5 h on. Also, expression of protective genes HO-1 and HSP70 were increased within 0.5 h. Remarkably, reactive oxygen species formation was detectable only in the later phase of BD. Among 14 measured serum cytokines, MCP-1 and KC-protein were significantly elevated from 0.5 h on. In conclusion, a fast induction of proinflammatory and stress-induced protective processes in brain dead donor kidneys was demonstrated, probably triggered by changes occurring during BD induction. Importantly, hypoxia appeared not to be one of the initial triggers, and early increased systemic levels of chemokines MCP-1 and KC may be regarded as the starting point for the inflammatory cascade in brain dead donor kidneys.

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.

Development of the isolated dual perfused rat liver model as an improved reperfusion model for transplantation research.

The Isolated Perfused Liver (IPL) model is a widely used and appreciated in vitro method to demonstrate liver viability and metabolism. Reperfusion is performed in a controlled setting, however, via the portal vein only. To study transplant related questions concerning bile and transport of bile, the in vitro Isolated dual Perfused Liver model is revisited. The IdPL is an in vitro reperfusion model, using both portal vein and hepatic artery. Livers from 12 Wistar rats were flushed with University of Wisconsin-organ preservation solution, procured and reperfused in either the conventional IPL-model (n = 6) or the new IdPL-model (n = 6). Liver injury, assessed by the release of aspartate amino transferase and lactate dehydrogenase, showed similar levels during both IPL and I dPL reperfusion, only alanine amino transferase showed an improvement. Cumulative bile production showed an improvement: 176.3 +/- 8.4 in the IdPL compared to 126.1 +/- 12.2 microg/g-liver in the IPL (p < 0.05). Clearance of phenol red (PR) and taurocholic acid (TC) remained similar. At 90 minutes reperfusion the PR clearance showed 0.11 +/- 0.01 and 0.11 +/- 0.02 mg/30min/g-liver and the TC clearance 1.01 +/- 0.10 and 1.01 +/- 0.07 micromol/ml/30min/g-liver in the IPL and IdPL, respectively. Increasing the reperfusion time beyond the normally used 90 minutes resulted in a significant increase in transaminases and LDH and a decrease in bile production, liver morphology remained intact and glycogen content was appropriate. In conclusion, the IdPL-model showed similar or better results than the IPL-model, but the liver could not endure an extended reperfusion time using the IdPL.

Hypothermic machine perfusion of the liver and the critical balance between perfusion pressures and endothelial injury.

Hypothermic machine perfusion (HMP) provides better protection against cold ischemic injury than cold storage in marginal donor kidneys. Also, in liver transplantation a switch from static cold storage to HMP could be beneficial as it would allow longer preservation times and the use of marginal donors. A critical question concerning application of HMP in liver preservation is the crucial balance between perfusion pressure and occurrence of endothelial injury. Rat livers were cold-perfused for 24 hours to study perfusion pressures for both hepatic artery and portal vein. Cold storage served as control and was compared to HMP-preserved livers using a mean arterial perfusion pressure of 25 mm Hg and a portal perfusion pressure of 4 mm Hg (25% of normothermic liver circulation) and to HMP at 50 mm Hg and 8 mm Hg perfusion, respectively (50% of normothermic liver circulation). UW solution was enriched with 14.9 micromol/L propidium iodide (PI) to stain for dead cells and with an additional 13.5 micromol/L acridine orange to stain for viable hepatocytes. A low PI-positive cell count was found using HMP at 25% of normal circulation compared to cold storage. The PI count was high for the HMP group perfused at just 50% of normal circulation compared to HMP at 25% and compared to cold storage. In summary, for liver HMP, perfusion at 25% showed complete perfusion with minimal cellular injury. HMP using perfusion pressures of 25 mm Hg for the hepatic artery and 4 mm Hg for the portal vein is feasible without induction of endothelial injury.