Hours Of Perfusion Research Articles

Evaluation of an ex vivo model of the blood-perfused equine uterus

Uterine pathologies are the most common causes of infertility in mares. This study aimed to establish an ex vivo blood-perfused model for equine uteri and investigate the possible effects of different cycle stages (estrus, diestrus and anestrus) on the applicability of the model. Uteri (n = 13) were collected at an abattoir, flushed with preservation solution, transported to the laboratory on ice, and isolated perfused with autologous blood for 6 h (n = 12). For negative control, one uterus was handled as described but left without perfusion for 6 h. The cycle stage was determined by examination of the ovaries for the presence of Graafian follicles or corpora lutea and analysis of plasma progesterone concentration (estrus: n = 4; diestrus: n = 4; anestrus: n = 4). Sonomicrometry crystals were implanted into the myometrium to record spontaneous contractions and the response to 0.5 IU oxytocin after 6 h of perfusion. Analyses of the arterial and venous perfusate were performed every hour to determine glucose consumption, lactate production, pH, lactate dehydrogenase activity (LDH), and potassium concentration (K+). Biopsy samples were obtained directly after slaughter, after transportation, after equilibration, after 4, 5, and 6 h of perfusion, and immediately after removal from the perfusion system. The uteri's glucose consumption and lactate production increased over time (p < 0.05), but no differences among cycle stages were detected. pH (arterial and venous) increased over time (p < 0.05). No changes for LDH were observed. K+ increased after 4 h of perfusion (p < 0.05), but was unaffected by the cycle stage. Spontaneous contractions were present in all perfused uteri, but myometrial activity in the negative control was limited to the 2nd hour of perfusion. No effects of cycle stage on contraction amplitude and duration after oxytocin administration were detected. The cycle stage did not affect frequency (except after 5 h of perfusion), amplitude, duration of contractions, or edema formation. Histology revealed congestion of endometrial capillaries after 4, 5, and 6 h perfusion time. In conclusion, the ex vivo model was capable of supporting the functionality of equine uteri for 6 h. However, viability and histomorphology of the endometrium appeared to be impaired after 4 h of perfusion. Effects of the cycle stage on the applicability of the model were absent.

24-hour Perfusion of Porcine Myocutaneous Flaps Mitigates Reperfusion Injury: A 7-day Follow-up Study.

Background:Static cold storage is the gold standard of preservation in vascularized composite allotransplantation and allows a preservation time of 4–6 hours. Machine preservation is a promising technique for prolonged preservation; however, studies on extended preservation that compare different preservatives are scarce. This study aims to assess the feasibility of 24-hour acellular perfusion and compares different preservation solutions in a porcine myocutaneous flap replantation model.Methods:Six harvested bilateral myocutaneous flaps of three Dutch Landrace pigs were perfused hypothermically for 24 hours with University of Wisconsin machine perfusion solution (UW-MPS; n = 2) or histidine-tryptophan-ketoglutarate solution (HTK; n = 2) or preserved on ice for 4 hours (n = 2) before orthotopic replantation. Animals were observed for 7 days after replantation. Skeletal muscle injury was assessed by biochemical markers during perfusion, and muscle biopsies were analyzed for ischemia reperfusion injury directly after preservation and at 1, 3, and 7 days after replantation.Results:Markers of muscle damage varied during perfusion, but decreased overall in both perfusion groups. Flap weight increased 60% and 97% in the HTK-perfused flaps, compared with -6% and -7% in the UW-MPS-perfused flaps after 24 hours. Histopathologic evaluation demonstrated decreased muscle damage in flaps perfused with HTK compared with the UW-MPS-perfused flaps at 1 week after replantation.Conclusions:Machine perfusion of myocutaneous flaps for 24 hours with subsequent replantation is feasible, but warrants further research. Perfusion with HTK solution seemed to result in better histological outcomes 7 days after reperfusion compared with UW-MPS.

P-L14 Ex-vivo normothermic perfusion of an abattoir-derived porcine hepatic segment as a model for scientific research

Abstract Background The human immune response to bacterial and viral pathogens has been the focus of intense research, but details on the earliest phases of infection remain unclear. Better knowledge regarding the response of immune cells in the liver is important for the treatment of severe bacterial disease. Ex vivo perfusion which mimics physiological conditions of the liver may provide useful models for this research. An ex vivo model which perfuses hepatic segments would allow translational research on a physiological and reliable model in the scarce resource of human organs. Here we describe the extra-corporeal perfusion of a porcine hepatic segment. Methods Whole porcine livers were retrieved from animals slaughtered according to UK laws for food production and connected to a normothermic extracorporeal perfusion circuit. Constant perfusion via the hepatic artery and portal vein with heparinized autologous blood was established. Sodium bicarbonate, epoprostanol sodium and calcium chloride were also added to the perfusate to regulate acid-base status and reduce vasospasm. Functionality was assessed by monitoring blood-gases, perfusion pressures and flow rate. A segmental ex-vivo liver resection was performed to leave hepatic segment IV in circuit and isolated segment IV perfusion was maintained for one hour. Results Portal venous pressure was maintained between 8-16mmHg and hepatic arterial pressure between 80-90mmHg. Metabolic acidosis resolved with addition of sodium bicarbonate to the circuit with a pH of 7.42 after segmental perfusion for 1 hour. The lactate increased over the course of the perfusion to 20mmol/L after 1 hour of perfusion, however glucose levels were found to improve with the addition of sodium bicarbonate to the circuit. Conclusions Isolated segmental perfusion via ex-vivo resection of porcine hepatic segments is technically challenging. Ischaemic-reperfusion injury coupled with progressive metabolic acidosis may limit model viability. However, addition of sodium bicarbonate to the perfusate aids reduction of glucose levels and improves acidosis. Successful perfusion of a porcine hepatic segment provides the potential for segmental perfusion of human hepatic segments such as those resected during hemi-hepatectomies (HRA approved; REC 21/PR/0287). This is an important milestone leading to the creation of a model to study the early changes in human liver tissue for example during infection.

Mitochondrial respiratory chain and Krebs cycle enzyme function in human donor livers subjected to end-ischaemic hypothermic machine perfusion.

IntroductionMarginal human donor livers are highly susceptible to ischaemia reperfusion injury and mitochondrial dysfunction. Oxygenation during hypothermic machine perfusion (HMP) was proposed to protect the mitochondria but the mechanism is unclear. Additionally, the distribution and uptake of perfusate oxygen during HMP are unknown. This study aimed to examine the feasibility of mitochondrial function analysis during end-ischaemic HMP, assess potential mitochondrial viability biomarkers, and record oxygenation kinetics.MethodsThis was a randomised pilot study using human livers retrieved for transplant but not utilised. Livers (n = 38) were randomised at stage 1 into static cold storage (n = 6), hepatic artery HMP (n = 7), and non-oxygen supplemented portal vein HMP (n = 7) and at stage 2 into oxygen supplemented and non-oxygen supplemented portal vein HMP (n = 11 and 7, respectively). Mitochondrial parameters were compared between the groups and between low- and high-risk marginal livers based on donor history, organ steatosis and preservation period. The oxygen delivery efficiency was assessed in additional 6 livers using real-time measurements of perfusate and parenchymal oxygen.ResultsThe change in mitochondrial respiratory chain (complex I, II, III, IV) and Krebs cycle enzyme activity (aconitase, citrate synthase) before and after 4-hour preservation was not different between groups in both study stages (p > 0.05). Low-risk livers that could have been used clinically (n = 8) had lower complex II-III activities after 4-hour perfusion, compared with high-risk livers (73 nmol/mg/min vs. 113 nmol/mg/min, p = 0.01). Parenchymal pO2 was consistently lower than perfusate pO2 (p ≤ 0.001), stabilised in 28 minutes compared to 3 minutes in perfusate (p = 0.003), and decreased faster upon oxygen cessation (75 vs. 36 minutes, p = 0.003).ConclusionsActively oxygenated and air-equilibrated end-ischaemic HMP did not induce oxidative damage of aconitase, and respiratory chain complexes remained intact. Mitochondria likely respond to variable perfusate oxygen levels by adapting their respiratory function during end-ischaemic HMP. Complex II-III activities should be further investigated as viability biomarkers.

Prolonged ex-vivo normothermic kidney perfusion: The impact of perfusate composition.

Normothermic machine perfusion (NMP) of donor kidneys provides the opportunity for improved graft preservation and objective pre-transplant ex-vivo organ assessment. Currently, a multitude of perfusion solutions exist for renal NMP. This study aimed to evaluate four different perfusion solutions side-by-side and determine the influence of different perfusate compositions on measured renal perfusion parameters. Porcine kidneys and blood were obtained from a slaughterhouse. Kidneys underwent NMP at 37°C for 7 hours, with 4 different perfusion solutions (n = 5 per group). Group 1 consisted of red blood cells (RBCs) and a perfusion solution based on Williams’ Medium E. Group 2 consisted of RBCs, albumin and a balanced electrolyte composition. Group 3 contained RBCs and a medium based on a British clinical NMP solution. Group 4 contained RBCs and a medium used in 24-hour perfusion experiments. NMP flow patterns for solutions 1 and 2 were similar, solutions 3 and 4 showed lower but more stable flow rates. Thiobarbituric acid reactive substances were significantly higher in solution 1 and 4 compared to the other groups. Levels of injury marker N-acetyl-β-D glucosaminidase were significantly lower in solution 2 in comparison with solution 3 and 4. This study illustrates that the perfusate composition during NMP significantly impacts the measured perfusion and injury parameters and thus affects the interpretation of potential viability markers. Further research is required to investigate the individual influences of principal perfusate components to determine the most optimal conditions during NMP and eventually develop universal organ assessment criteria.

Transcriptomic Analysis during Normothermic Machine Perfusion Reveals Similar Responses in Steatotic and Lean Discarded Human Livers

Chronic liver disease is the 12th leading cause of death in the U.S., with end-stage liver disease alone accounting for 60,000 deaths in the U.S. each year.The only available treatment for liver failure is orthotopic liver transplantation.However, the severe shortage of viable donor organs results in less than 30% of patients on the wait list receiving transplants.The morbidity and mortality associated with liver disease could be dramatically reduced by expanding the donor organ pool by rendering marginal donor livers, especially steatotic grafts, transplantable.Steatotic livers are more susceptible to ischemia-reperfusion (I/R) injury, the major complication leading to graft dysfunction.Normothermic machine perfusion (NMP) combined with adjunct therapies may be able to rehabilitate discarded steatotic livers, thereby increasing the supply of organs for transplant.The goal of this study was to develop an understanding of the transcriptomic changes associated with NMP in steatotic compared to lean livers. Twelve discarded livers (6 lean, 6 steatotic), rejected by local transplant centers, were obtained and underwent 12 hours of NMP with oxygenated blood. Serial tissue and plasma samples were collected. Clinical viability criteria were used to determine if livers would have been transplantable (viable) or not (nonviable). Transcriptome sequencing was performed on biopsies taken at 0 (pre-), 3, and 6hr of perfusion. Comparative analysis between transcriptomes of individual conditions was performed using R. Ingenuity Pathway Analysis (IPA) was performed on significant genes to identify canonical pathways and upstream regulators. Three of the six livers from each group met viability criteria for transplant. IPA comparing viable livers at 0 and 3hr of perfusion demonstrated robust activation of innate immune responses, such as Toll-like receptor, IL-10, and IL-6 canonical pathways (P<10E-7) in both lean and steatotic livers, compared to nonviable livers. However, many of the same canonical pathways identified after 3 hours of perfusion in viable livers were not significant in the nonviable livers until 6hr of perfusion in both lean and steatotic livers, suggesting a delayed transcriptomic response in nonviable livers of both groups. There was significant overlap in upstream regulators identified in both viable and nonviable livers. However, these shared regulators demonstrated remarkably divergent downstream target activation.This is the first study to demonstrate activation of regenerative signaling in discarded lean and steatotic human livers during NMP. Comparison of gene expression profiles between lean and steatotic viable livers were similar as were the transcriptomic profiles of the nonviable livers from both groups.These data suggest that the response to NMP is similar in lean and steatotic liver.Our analysis provides numerous potential therapeutic targets for improving function and transplantability of nonviable lean and steatotic livers during ex vivo machine perfusion.

Lipopolysaccharide Levels in Ex Vivo Lung Perfusion are Associated with Lung Function and Suitability for Transplantation

Purpose Ex vivo lung perfusion (EVLP) has emerged as an important platform to assess marginal donor lungs prior to transplantation. Derived from the outer membrane of gram-negative bacteria, lipopolysaccharide (LPS) can stimulate strong immune responses. Studies have shown that donor lungs infected with bacteria may have worse outcomes, suggesting bacteria-induced donor lung injury. However, no large-scale studies have looked at the relationship between LPS levels and donor lung performance on EVLP. Methods LPS levels were measured after one and four hours of perfusion in n=100 clinical EVLP cases. To assess the effects of LPS on lung inflammation, IL-6, 8, 10, and 1β, sTNF-R1, sTREM-1 and ET-1 levels were measured with an ELISA-based platform. Biochemical and physiological assessments were compared to LPS levels. Finally, LPS levels and recipient intensive care unit length of stay after transplantation were investigated. Results There was a significant increase from 0.14 [0.09-0.21] to 0.23 [0.13-0.38] EU/mL (p Conclusion This study demonstrates the trends and importance of circulating LPS levels during EVLP assessment of human donor lung injury. We show that high LPS levels and increasing levels over time were associated with poor donor lung performance in EVLP. These results support the need for therapeutic strategies to target LPS during EVLP, which may improve lung function and rescue more donor lungs for transplantation.

Three-Dimensional Bioprinting for Tissue Engineering and Regenerative Medicine in Down Under: 2020 Australian Workshop Summary.

Three-Dimensional Bioprinting for Tissue Engineering and Regenerative Medicine in Down Under: 2020 Australian Workshop Summary.

Metabolic Profiling of Skeletal Muscle During Ex-Vivo Normothermic Limb Perfusion

Ex vivo normothermic limb perfusion (EVNLP) provides several advantages for the preservation of limbs following amputation: the ability to maintain oxygenation and temperature of the limb close to physiological values, a perfusion solution providing all necessary nutrients at optimal concentrations, and the ability to maintain physiological pH and electrolytes. However, EVNLP cannot preserve the organ viability infinitely. We identified evidence of mitochondrial injury (swelling, elongation, and membrane disruption) after 24 hours of EVNLP of human upper extremities. The goal of this study was to identify metabolic derangements in the skeletal muscle during EVNLP. Fourteen human upper extremities were procured from organ donors after family consent. Seven limbs underwent EVNLP for an average of 41.6 ± 9.4 hours, and seven contralateral limbs were preserved at 4°C for the same amount of time. Muscle biopsies were performed at 24 hours of perfusion, both from the EVNLP and control limbs. Perturbations in the metabolic profiles of the muscle during EVNLP were determined via untargeted liquid chromatography-mass spectrometry (MS) operated in positive and negative electrospray ionization modes, over a mass range of 50 to 750 Da. The data were deconvoluted using the XCMS software and further statistically analyzed using the in-house statistical package, MetaboLyzer. Putative identification of metabolites using exact mass within ±7 ppm mass error and MS/MS spectral matching to the mzCloud spectral library were performed via Compound Discoverer v.2.1 (Thermo Scientific, Fremont, CA, USA). We further validated the identity of candidate metabolites by matching the fragmentation pattern of these metabolites to those of their reference pure chemicals. A nonparametric Mann-Whitney U-test was used to compare EVNLP and control group spectral features. Differences were considered significantly different when P-value < 0.05. We detected over 13,000 spectral features of which 58 met the significance criteria with biologically relevant putative identifications. Furthermore we were able to confirm the identities of the ions taurine (P-value: 0.002) and tryptophan (P-value: 0.002), which were among the most significantly perturbed ions at 24 hours between the experimental and control groups. Metabolites belonging to the following pathways were the most perturbed at 24 hours: neuroactive ligand-receptor interaction (P-values: 0.031 and 0.036) and amino acid metabolism, including tyrosine and tryptophan metabolism (P-values: 0.015, 0.002, and 0.017). Taurine abundance decreased and tryptophan abundance increased at 24 hours. Other metabolites also identified at 24 hours included phenylalanine, xanthosine, and citric acid (P-values: 0.002, 0.002, and 0.0152). This study showed presence of active metabolism during EVNLP and metabolic derangement toward the end of perfusion, which correlated with detection of altered mitochondrial structure, swelling, and elongation.

A Multi-Mode System for Myocardial Functional and Physiological Assessment during Ex Situ Heart Perfusion

Ex situ heart perfusion (ESHP) has proven to be an important and valuable step toward better preservation of donor hearts for heart transplantation. Currently, few ESHP systems allow for a convenient functional and physiological evaluation of the heart. We sought to establish a simple system that provides functional and physiological assessment of the heart during ESHP. The ESHP circuit consists of an oxygenator, a heart–lung machine, a heater–cooler unit, an anesthesia gas blender, and a collection funnel. Female Yorkshire pig hearts (n = 10) had del Nido cardioplegia (4°C) administered, excised, and attached to the perfusion system. Hearts were perfused retrogradely into the aortic root for 2 hours before converting the system to an isovolumic mode or a working mode for further 2 hours. Blood samples were analyzed to measure metabolic parameters. During the isovolumic mode (n = 5), a balloon inserted in the left ventricular (LV) cavity was inflated so that an end-diastolic pressure of 6–8 mmHg was reached. During the working mode (n = 5), perfusion in the aortic root was redirected into left atrium (LA) using a compliance chamber which maintained an LA pressure of 6–8 mmHg. Another compliance chamber was used to provide an afterload of 40–50 mmHg. Hemodynamic and metabolic conditions remained stable and consistent for a period of 4 hours of ESHP in both isovolumic mode (LV developed pressure: 101.0 ± 3.5 vs. 99.7 ± 6.8 mmHg,p= .979, at 2 and 4 hours, respectively) and working mode (LV developed pressure: 91.0 ± 2.6 vs. 90.7 ± 2.5 mmHg,p= .942, at 2 and 4 hours, respectively). The present study proposed a novel ESHP system that enables comprehensive functional and metabolic assessment of large mammalian hearts. This system allowed for stable myocardial function for up to 4 hours of perfusion, which would offer great potential for the development of translational therapeutic protocols to improve dysfunctional donated hearts.

A Multi-Mode System for Myocardial Functional and Physiological Assessment during Ex Situ Heart Perfusion.

Ex situ heart perfusion (ESHP) has proven to be an important and valuable step toward better preservation of donor hearts for heart transplantation. Currently, few ESHP systems allow for a convenient functional and physiological evaluation of the heart. We sought to establish a simple system that provides functional and physiological assessment of the heart during ESHP. The ESHP circuit consists of an oxygenator, a heart-lung machine, a heater-cooler unit, an anesthesia gas blender, and a collection funnel. Female Yorkshire pig hearts (n = 10) had del Nido cardioplegia (4°C) administered, excised, and attached to the perfusion system. Hearts were perfused retrogradely into the aortic root for 2 hours before converting the system to an isovolumic mode or a working mode for further 2 hours. Blood samples were analyzed to measure metabolic parameters. During the isovolumic mode (n = 5), a balloon inserted in the left ventricular (LV) cavity was inflated so that an end-diastolic pressure of 6-8 mmHg was reached. During the working mode (n = 5), perfusion in the aortic root was redirected into left atrium (LA) using a compliance chamber which maintained an LA pressure of 6-8 mmHg. Another compliance chamber was used to provide an afterload of 40-50 mmHg. Hemodynamic and metabolic conditions remained stable and consistent for a period of 4 hours of ESHP in both isovolumic mode (LV developed pressure: 101.0 ± 3.5 vs. 99.7 ± 6.8 mmHg, p = .979, at 2 and 4 hours, respectively) and working mode (LV developed pressure: 91.0 ± 2.6 vs. 90.7 ± 2.5 mmHg, p = .942, at 2 and 4 hours, respectively). The present study proposed a novel ESHP system that enables comprehensive functional and metabolic assessment of large mammalian hearts. This system allowed for stable myocardial function for up to 4 hours of perfusion, which would offer great potential for the development of translational therapeutic protocols to improve dysfunctional donated hearts.

Association of Perfusion Characteristics and Posttransplant Liver Function in Ischemia-Free Liver Transplantation.

It has been shown that normothermic machine perfusion (NMP), a novel preservation method, is able to assess and resuscitate liver grafts with risk factors. However, there is no consistent criteria for the assessment of liver grafts with NMP. Ischemia-free liver transplantation (IFLT) includes innovative surgical techniques and NMP, which can protect liver grafts from ischemia throughout organ procurement, preservation, and implantation. In our center, 28 human livers from donation after brain death donors were subjected to IFLT between July 2017 and October 2018. The correlation between posttransplant liver function tests with the perfusion parameters, blood gas analysis of perfusate, and bile biochemistry were analyzed. During the preservation phase, the vascular flow was stable, and the lactate level decreased rapidly. The transaminase release in the perfusate was low but stable, whereas the glucose level remained high. The perfusate lactate and aspartate aminotransferase (AST) levels at 1 hour of perfusion were correlated with the posttransplant peak AST level. There were negative correlations between the portal vein and hepatic artery flows at the end of perfusion and the peak transaminase levels within 7 days after transplantation. In conclusion, during IFLT, NMP is able to bridge the liver grafts from donors to recipients and can allow the assessment of liver function by perfusion characteristics.

XENOGENEIC LUNG PERFUSION USING GALTKO.HCD46 LUNGS EXPRESSING HTFPI AND HCD47

Purpose: To address known xenogeneic rejection pathways, various genetic modifications have been introduced into donor pig lines. Here we evaluated whether tissue factor pathway inhibitor (TFPI), a protease inhibitor which blocks initial steps of the extrinsic coagulation cascade, and human CD47, a “don’t eat me” immunoregulatory protein, lessen inflammation and promote GalTKO.hCD46 lung xenograft survival. Methods: In a well-established ex vivo lung perfusion model, 8 GalTKO.hCD46 and 9 GalTKO.hCD46.hTFPI.hCD47 lungs were perfused with freshly collected heparinized human blood. All lungs were treated with an anti-GPIb Fab, 1-BIA, a thromboxane synthase inhibitor and Famotidine; and donor pigs were pre-treated with Desmopressin to reduce vWF expression. In both groups, 3 or 4 lungs in addition received selectin and integrin blockers. Organs were perfused for up to 8 hours or until the lung failed. Results: All except two GalTKO.hCD46 organs (183, 440 min) ‘survived’ until elective termination after 8 hours of perfusion. While the pulmonary vascular resistance (PVR) remained low with both genotypes, additional selectin and integrin blockade lowered PVRs in both groups. Neutrophil but not platelet sequestration and activation were reduced during the first 2 hours in association with hTFPI.hCD47 expression; this trend was more pronounced in the absence of selectin and integrin blockade, reaching significance at the 1 and 2 hour time point. Further biochemical analyses are underway. Conclusion: While the expression of hTFPI and hCD47 showed a trend toward protective effects (improved survival, delayed neutrophil sequestration), overall lung physiology and hematological analyses did not demonstrate significant improvements. Lack of improvement of parameters other than PVR with addition of selectin and integrin blockade suggests that other injury pathways (e.g. non-gal antibody; cytokines; coagulation pathway dysregulation; GPIIb/IIIa-driven platelet activation) contribute to xenolung injury. Identifying and addressing these factors may be necessary to reveal effects attributable to hTFPI and hCD47.NIH U19 AI090959. Unrestricted educational gifts from United Therapeutics/ Lung Biotechnology LLC.

Strategies to prolong homeostasis of ex vivo perfused lungs

ObjectivesEx vivo lung perfusion provides an innovative method to assess and repair donor lungs. The current Toronto ex vivo lung perfusion protocol can reliably and reproducibly preserve lungs for 12 hours. A longer ex vivo lung perfusion preservation time could enable the application of more advanced repair therapies and the rescue of more donor lungs for lung transplant. Our objective was to achieve stable 24-hour normothermic ex vivo lung perfusion. MethodsWe systematically examined 3 modifications of ex vivo lung perfusion perfusate administration in a large animal 24-hour ex vivo lung perfusion model. Pig lungs were assigned to 4 groups (n = 5 per group): (1) control; (2) continuous replacement of ex vivo lung perfusion perfusate; (3) modified feed, which used a modified solution to maintain perfusate osmolality by adjusting glucose and sodium levels; and (4) total parenteral nutrition, in which we added parenteral nutrition to the perfusate. ResultsOnly 1 lung in the control group completed 24-hour ex vivo lung perfusion. However, 24-hour perfusion was achieved in 4 lungs in the continuous replacement group, 3 lungs in the modified feed group, and 4 lungs in the total parenteral nutrition group. The total parenteral nutrition group achieved significantly longer stable perfusion time compared with control (P = .03). Lung function was significantly improved and inflammatory cytokine production was reduced in the continuous replacement and total parenteral nutrition groups compared with control. ConclusionsModifications of ex vivo lung perfusion perfusate toward achieving a stable homeostatic state can extend perfusion time for up to 24 hours. Although these modifications allow for prolonged ex vivo lung perfusion, further research will be required to develop stable lung support beyond 24 hours.

Cell-free DNA in human ex vivo lung perfusate as a potential biomarker to predict the risk of primary graft dysfunction in lung transplantation

BackgroundCell-free DNA (cfDNA), such as mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA), are known to be released from injured cells and as such have been explored as biomarkers for tissue injury in different clinical settings. Ex vivo lung perfusion (EVLP) has been developed as an effective technique for marginal donor lung functional assessment. We hypothesized that the level of cfDNA in EVLP perfusate may reflect tissue injury and thus can be developed as a biomarker to quantify the degree of donor lung injury or predict the development of primary graft dysfunction (PGD) after lung transplantation (LTx). MethodsThe perfusate from 62 donor lungs transplanted at our institution between May 2010 and December 2015 was sampled for cfDNA at 1 and 4 hours of perfusion. Sequences of genes encoding nicotinamide adenine dinucleotide dehydrogenase 1 (NADH-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to represent mtDNA and nuDNA, respectively. Levels were quantified by real-time polymerase chain reaction and correlated with clinical outcome after LTx. ResultsIn our entire cohort, 14 patients developed PGD grade 3 (PGD3) within 72 hours after LTx. The non-PGD group included 48 patients (PGD0-1). Concentrations of mtDNA in the perfusate of the PGD3 group were significantly higher than those in non-PGD group at 1 hour of EVLP (1874 ± 844 vs 1259 ± 885 copies/μL; P = .011). The perfusate of the PGD3 group had significantly higher levels of nuDNA compared with the non-PGD group at both 1 hour (1498 ± 1895 vs 675 ± 391 copies/μL; P = .008) and 4 hours (4521 ± 5810 vs 1764 ± 1494 copies/μL; P = .001). In donation after cardiac death (DCD) cases, mtDNA levels were significantly higher in the PGD3 group compared with the non-PGD group at 1 hour of EVLP (2060 ± 997 vs 1184 ± 782 copies/μL; P = .040), and the levels of nuDNA were significantly higher in the PGD3 group compared with the non-PGD group at both 1 hour (1021 ± 495 vs 606 ± 305 copies/μL; P = .041) and 4 hours (2358 ± 1028 vs 1185 ± 967 copies/μL; P = .006). In donation after brain death (DBD) cases, cfDNA scores did not show a significant difference. ConclusionsWe found that the amount of cfDNA, especially nuDNA, in EVLP perfusate was higher in the severe PGD group (PGD3) compared with the non-PGD group. This proof-of-concept study supports the concept that the analysis of cfDNA levels in EVLP perfusate can help estimate the damage to donor lungs before implantation. Larger studies are needed to validate this concept.

Dual versus single vessel normothermic ex vivo perfusion of rat liver grafts using metamizole for vasodilatation.

Normothermic ex vivo liver perfusion (NEVLP) is a promising strategy to increase the donor pool in liver transplantation. Small animal models are essential to further investigate questions regarding organ preservation and reconditioning by NEVLP. A dual vessel small animal NEVLP (dNEVLP) model was developed using metamizole as a vasodilator and compared to conventional portovenous single vessel NEVLP (sNEVLP). Livers of male Wistar rats were perfused with erythrocyte-supplemented culture medium for six hours by either dNEVLP via hepatic artery and portal vein or portovenous sNEVLP. dNEVLP was performed either with or without metamizole treatment. Perfusion pressure and flow rates were constantly monitored. Transaminase levels were determined in the perfusate at the start and after three and six hours of perfusion. Bile secretion was monitored and bile LDH and GGT levels were measured hourly. Histopathological analysis was performed using liver and bile duct tissue samples after perfusion. Hepatic artery pressure was significantly lower in dNEVLP with metamizole administration. Compared to sNEVLP, dNEVLP with metamizole treatment showed higher bile production, lower levels of transaminases during and after perfusion as well as significantly lower necrosis in liver and bile duct tissue. Biochemical markers of bile duct injury showed the same trend. Our miniaturized dNEVLP system enables normothermic dual vessel rat liver perfusion. The administration of metamizole effectively ameliorates arterial vasospasm allowing for six hours of dNEVLP, with superior outcome compared to sNEVLP.

Improvement of steatotic rat liver function with a defatting cocktail during ex situ normothermic machine perfusion is not directly related to liver fat content.

There is a significant organ shortage in the field of liver transplantation, partly due to a high discard rate of steatotic livers from donors. These organs are known to function poorly if transplanted but make up a significant portion of the available pool of donated livers. This study demonstrates the ability to improve the function of steatotic rat livers using a combination of ex situ machine perfusion and a “defatting” drug cocktail. After 6 hours of perfusion, defatted livers demonstrated lower perfusate lactate levels and improved bile quality as demonstrated by higher bile bicarbonate and lower bile lactate. Furthermore, defatting was associated with decreased gene expression of pro-inflammatory cytokines and increased expression of enzymes involved in mitochondrial fatty acid oxidation. Rehabilitation of marginal or discarded steatotic livers using machine perfusion and tailored drug therapy can significantly increase the supply of donor livers for transplantation.

Urine Recirculation Improves Hemodynamics and Enhances Function in Normothermic Kidney Perfusion.

Kidney pairs were recovered uninjured (as live-donor nephrectomy) and perfused consecutively. Pig kidneys (n = 10) were allocated to either NMP with URC (n = 5) or NMP with volume replacement (n = 5). Cold ischemia time was either 2 or 27 hours for the first or second perfusion (URC or UR) of a kidney pair. An autologous blood-based perfusate, leukocyte-filtered, was used and NMP performed up to 24 hours. Perfusion parameters, biochemistry/metabolic parameters were monitored and samples collected. Physiological mean arterial pressures and flows were achieved in both groups but were sustainable only with URC. Significantly higher arterial flow was observed with URC (326.7 ± 1.8 versus 242.5 ± 14.3 mL/min, P = 0.001). Perfusate sodium levels were lower with URC, 129.6 ± 0.7 versus 170.3±2.7 mmol/L, P < 0.001). Stable physiological pH levels were only observed with URC. Perfusate lactate levels were lower with URC (2.2 ± 0.1 versus 7.2 ± 0.5 mmol/L, P < 0.001). Furthermore, the hourly rate of urine output was lower with URC and closer to physiological levels (150 versus 548 mL/h, P = 0.008). Normothermic kidney perfusion with URC was associated with longer achievable durations of perfusion: the objective in all experiments was a 24-hour perfusion, but this was not achieved in every case. The mean perfusions were 17.3 ± 9.2 hours with URC versus 5.3 ± 1.3 hours NMP with UR; P = 0.02. There appeared to be no differences in baseline tubular condition with and without URC. URC facilitates long-term kidney NMP in a porcine model. Perfusate homeostasis and stability of renal arterial flow throughout the perfusion period was only achievable with URC, independent of cold ischemia time duration.

Minimizing Cardiac Oedema during Ex Vivo Perfusion in a Juvenile Porcine Model - How Much Does Coronary Flow Matter?

Transmedic Organ Care System (OCS) allows preservation of the donor heart by perfusing the organ at 34°C in a beating state and providing additional assessment options. The perfusion protocols as established by the manufacturer is valid for adult human hearts, using coronary flows in the 8-900ml/min range guided by lactate measurements. Higher flow can facilitate higher degrees of cardiac oedema which can be associated with diastolic dysfunction, and generel impairment of contractility of the heart. The aim of the study is to establish the optimal coronary flow in a juvenile porcine heart, similar in size to the adult human heart, that minimizes oedema and still meets the metabolic requirements. 18 pigs in the range of 60-70kg was used for the trial and randomized into three groups with a coronary flow of 6-, 7- or 800ml/min for 180 minuts. After intubation and propofol/fentanyl anesthesia was induced, median sternotomy was performed and the heart prepared for harvest. Immediately after crossclamp 1000ml of HTK Custodiol cardioplegia was administered, and the heart prepared as per protocol for the human heart. The heart was connected a OCS-based system and randomized to one of three regimens: a coronary flow of 6-, 7- or 800ml/min for 180 minutes at 34 degree celsius, with the pressure autoregulated to 65mmHg. The pump flow was adjusted to give the desired coronary flow. Haemodynamic parameteres were continously recorded, arterial and venous lactates sampled every 30 minutes. Biopsies taken form the apex of the left ventricle and the heart was weighed after 180 minuts of perfusion. Our primary endpoint was met, as the weight gain in the 800ml/min group was significantly higher than the 600ml/min group (103g +/-20g vs 72g +/- 15g, p<0.05). There were no significant difference in the final lactate (relative to the initial levels) after three hours of perfusion. All hearts had end arterial lactate under 5.0 mmol/l. At the end of the perfusion, Troponin T levels in the blood and pathohistological examination of the biopsies showed no significant differences between the groups. We found a lower degree of oedema formation in the 600ml/min perfusion group, but similar metabolic response as the group with higher flows. . We suggest using this slightly reduced flow when investigating juvenile porcine hearts in an ex-vivo perfusion model.

Ex Vivo Normothermic Perfusion, a Novel Method to Assess Pancreases after Preservation

Julien Branchereau1,2* and Etohan Ogbemudia1*, Kaithlyn Rozenberg1, Fungai Dengu1, Gabriella Hakim1, Flavia Neri1, Georg Ebeling1, Letizia Lo Faro1, James Hunter1, Rutger Ploeg1* and Peter Friend1*&#x0D; * "Authors contributed equally to this work"&#x0D; 1-Nuffield Department of Surgical Sciences - University of Oxford&#x0D; 2-Nantes Hospital University - France&#x0D; Introduction&#x0D; Static cold storage (SCS) of pancreases remains the current standard method for preservation prior to transplantation. Pulsatile hypothermic machine perfusion (HMP) is an emerging method that could potentially improve the preservation of pancreases to enhance graft function. This is based on personal extensive experience with HMP of pancreases in human, porcine allotransplantation and large non-human primate models. All models consistently showed preservation of pancreatic parenchyma on histological assessment for up to 24hours. To provide reassurance of organ viability prior to transplantation further investigations are necessary. We propose that normothermic perfusion (NMP) of pancreases after cold preservation allows necessary functional and physiological assessments.&#x0D; Method&#x0D; Porcine pancreases (3) were retrieved from the slaughterhouse after 30 minutes of warm ischaemia and were flushed. After 3 hours of cold ischaemia two pancreases were perfused by HMP (Wave machine; Waters Medical Systems) for 6 hours followed by 1 hour of NMP. One pancreas after 3 hours of CIT was placed on NMP for 2.5hours, this was the first pancreas NMP to assess feasibility of this technique.NMP was achieved by modification of the kidney assist device. Oxygenation was with 21% oxygen. The pancreases were cannulat ed via the aorta with free drainage of perfusate from the portal vein. NMP parameters for all pancreases were a pressure of 40mmHg and temperature of 37C. Perfusate was composed of red blood cells, plasma to provide a haematocrit of 25%, with additives of co-amoxiclav and 25,000IU of heparin. During the perfusions we collected serial perfusate samples for blood gas analysis and for insulin enzyme-linked immunosorbent assay (ELISA).&#x0D; Results&#x0D; The macroscopic appearance of the pancreases and the attached duodenum at the end of NMP appeared viable. Average resistance index during NMP was 0.62 ru (range 0.30 to 0.90 ru). Average flow rate was 77mls/min (range 53 to 100 mls/min). Throughout, the duration of NMP in the two pancreases perfused for one-hour lactate remain stable with no increase from baseline. In the one pancreas perfused for 2.5hours lactate was also stable in the first hour then increased by 50% during the last 1.5hours of perfusion. ELISA confirmed the presence of insulin in the perfusate for all three perfusions.&#x0D; Conclusion&#x0D; Normothermic perfusion is a feasible method to allow physiological and functional assessment of pancreases after cold preservation techniques encouraging us to further develop this model.&#x0D;