Exploring the Role of Mesenchymal Stem Cells During Normothermic Organ Perfusion: A New Paradigm to Enhance Outcome Following Allograft Transplantation

Normothermic Acellular Machine Perfusion and Bile Duct Injury in Pig Livers Retrieved After Cardiac Death

Strategies in Organ Preservation-A New Golden Age.

Lohmann 1/M. Pool 2, K. Rozenberg 3, M. Eijken 4, U. Møldrup 5, B.K. Møller 6, J.M. Sierra Parraga 7, M. Hoogduijn 7, L. Lo Faro 3, C. Moers 2, J. Hunter 3, A.K. Keller 1, H. Leuvenink 2, C.C. Baan 7, R.J. Ploeg 3, B. Jespersen 1 Department of Clinical Medicine, Aarhus University, Aarhus, Denmark Department of Surgery, University Medical Center Groningen, Groningen, The Netherlands Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom Department of Renal Medicine, Aarhus University Hospital, Aarhus, Denmark Department of Urology, Aarhus University Hospital, Aarhus, Denmark Department of Clinical Immunology, Aarhus University Hospital, Aarhus, Denmark Department of Internal Medicine, Nephrology and Transplantation, Erasmus MC, University Medical Center, Rotterdam, the Netherlands Introduction Marginal kidneys are increasingly being accepted to decrease waiting time for a transplant. Normothermic machine perfusion (NMP) is a technique that allows delivery of therapies that may help condition or repair the organ prior to transplantation. Mesenchymal stromal cells (MSC) may be able to ameliorate ischaemia reperfusion injury as they possess potent anti-inflammatory and regenerative properties. We investigated the safety and effect of MSCs administered during ex vivo NMP prior to transplantation in a pig auto-transplant model of donation after circulatory death. Methods Porcine kidneys subjected to 75 min warm ischaemia were retrieved and preserved for 14h by oxygenated HMP (oxHMP) and 4h NMP and then auto-transplantation. Kidneys were randomised to three different intervention strategies (n=7 per group): following 1h NMP, either a vehicle (NMP), 10 million pig MSC (NMP+pMSC) or 10 million human MSC (NMP+hMSC) were intra-arterially infused. The NMP groups were all compared to a control group, where kidneys were only preserved with oxHMP. The pig was re-anaesthetised, the contralateral kidney was removed and the treated kidney was auto-transplanted and the animals were recovered for 14 days. Results Renal blood flow during NMP was no different between the groups (p=0.0685). Post-transplant plasma creatinine increased in all groups but there were no significant differences between the groups (p=0.517). Plasma kidney injury biomarker NGAL was significantly higher in the NMP+pMSC group compared to the NMP (p=0.003) and NMP+hMSC (p=0.017) groups at day 14. On day 14, mGFR significantly improved in the NMP group compared to the control (55 ± 3 vs 42 ± 12 ml/min, p=0.025). No differences in GFR were observed on day 14 in the other groups (NMP+pMSC, p=0.090 and NMP+hMSC, p=0.387). MSC were detectable in biopsies of MSC treated kidney after NMP and post-transplantation. Conclusion NMP alone improved renal graft function compared to oxHMP of DCD kidneys post-transplant. The method of MSC administration during NMP proved to be safe, however in this model MSC treatment did not improve renal function. Nevertheless viable MSC remained detectable in the transplanted kidney at postoperative day 14 which may have an effect on longer term outcomes.

Solid Renal Masses in Transplanted Allograft Kidneys: A Closer Look at the Epidemiology and Management.

Transplantation of declined livers after normothermic perfusion

The shortage of donor kidneys is a worldwide problem. One possibility to increase the donor organ pool is to improve the quality of suboptimal donor kidneys. For this reason, an increasing amount of research is being performed on the subject of machine perfusion, a technique during which an organ is connected to a perfusion circuit. Apart from the fact that this technique can be used to bridge the period between donation and actual transplantation, it also provides the opportunity for active interventions to an isolated organ. The first part of my thesis focuses on a cellular intervention during normothermic (37 degrees) machine perfusion with mesenchymal stromal cells. The aim was to determine if it was technically feasible to administer mesenchymal stromal cells, cells with regenerative capacities, during normothermic machine perfusion and what effect these cells had on the donor kidney. It proved to be feasible to administer these cells in such way that a proportion remained detectable after machine perfusion. In a porcine autotransplantation model, mesenchymal stromal cells did not affect early graft function, but further research is necessary to determine if these cells could have a beneficial effect on longer term graft function of the donor kidney. The second part of my thesis focuses on the use of different perfusion solutions and oxygen carriers during normothermic machine perfusion. From these experiments it can be concluded that the composition of the perfusion solution, as well as the choice for a specific oxygen carrier, has a significant impact on kidney function and injury markers during normothermic machine perfusion.

K Rozenberg 1, L Knijff 1, J M Sierra Parraga 2, L Lo Faro 1, M J Hoogduijn 2, C 2, J Hunter 1, R Ploeg 1 Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands BackgroundDonation after circulatory death kidneys are widely used to increase the donor pool, despite their inferior quality. Normothermic machine perfusion (NMP) could provide a platform to assess organ viability prior to transplantation and offers the unique opportunity for active interventions to an isolated organ. Mesenchymal stromal cells (MSC) have been shown to possess potent anti-inflammatory and regenerative properties ameliorating ischaemia reperfusion injury. However, the most common delivery route of MSC is intravenous infusion, which is associated with off-target distribution. We aimed to determine the dose of MSC needed to allow successful homing in donor kidneys during NMP without adversely affecting renal perfusion dynamics. MethodsPorcine slaughterhouse kidneys with 20 min warm ischaemia were retrieved and underwent 3h hypothermic machine perfusion followed by NMP for 7h. An oxygenated, autologous blood-based solution containing albumin, electrolytes and nutrients was used as perfusate. Following 1h of NMP, either a vehicle, 2 x 106, 10 x 106 or 50 x 106 labelled (Qdots) porcine adipose derived MSC (n=3 per group) were injected into the cannulated renal artery. Physiological recordings were taken regularly. Perfusate, urine and biopsies were obtained for biomarker analysis and to locate MSC. ResultsNo difference was found in average renal blood flow (p=0.668), renal resistance (p=0.828) and urine production (p=0.307) between all MSC groups compared to control. Damage markers LDH and AST increased during NMP and remained similar between groups (p=0.816 and p=0.312). Confocal microscopy demonstrated mainly glomerular localization of MSC (Figure 1), but they were also observed in the capillary network around the tubules. Additionally, a significant percentage of localised MSC was observed in the glomeruli in the 10 and 50 million MSC groups compared to 2 million MSC (p=0.002 and p<0.0001). No differences were observed between the 2 and 10 million MSCs group (p>0.05). There is a significant increase in secreted inflammatory mediator PGE2 with time in all groups (P<0.05). DiscussionAdministration of different doses of MSC to the donor kidneys during NMP is safe and feasible. MSC successfully homed in the glomeruli. This model provides us with the opportunity of getting a better insight in the interaction between MSC and injured donor kidney: how do MSC or their released secretome affect tissue integrity and renal function.

Normothermic machine perfusion (NMP) of kidneys offers the opportunity to perform active interventions, such as the addition of mesenchymal stromal cells (MSCs), to an isolated organ prior to transplantation. The purpose of this study was to determine whether administering MSCs to kidneys during NMP is feasible, what the effect of NMP is on MSCs and whether intact MSCs are retained in the kidney and to which structures they home. Viable porcine kidneys were obtained from a slaughterhouse. Kidneys were machine perfused during 7 h at 37 °C. After 1 h of perfusion either 0, 105, 106 or 107 human adipose tissue derived MSCs were added. Additional ex vivo perfusions were conducted with fluorescent pre-labelled bone-marrow derived MSCs to assess localisation and survival of MSCs during NMP. After NMP, intact MSCs were detected by immunohistochemistry in the lumen of glomerular capillaries, but only in the 107 MSC group. The experiments with fluorescent pre-labelled MSCs showed that only a minority of glomeruli were positive for infused MSCs and most of these glomeruli contained multiple MSCs. Flow cytometry showed that the number of infused MSCs in the perfusion circuit steeply declined during NMP to approximately 10%. In conclusion, the number of circulating MSCs in the perfusate decreases rapidly in time and after NMP only a small portion of the MSCs are intact and these appear to be clustered in a minority of glomeruli.

Machine Perfusion as "Comfort Zone": What Are Key Challenges of Liver Viability Assessment Today?

Background/Objectives: The rising use of liver grafts from donation after circulatory death (DCD) has been enabled by advances in normothermic regional perfusion (NRP) and machine perfusion (MP) technologies. We aimed to identify predictive biomarkers in DCD grafts subjected to NRP, followed by randomization to either normothermic machine perfusion (NMP) or dual hypothermic oxygenated perfusion (D-HOPE). Methods: Among 57 DCD donors, 32 liver grafts were transplanted, and recipients were monitored for one week post-transplant. Biomarkers linked with oxidative stress, hepatic injury, mitochondrial dysfunction, inflammation, regeneration, and autophagy were measured during NRP, end-ischemic MP, and one week post-transplant. Results: Arginase-1 (ARG-1) levels were consistently higher in discarded grafts and in recipients who later developed early allograft dysfunction (EAD). Specifically, ARG-1 levels at the end of MP correlated with markers of hepatic injury. Receiver operating characteristic analysis indicated that ARG-1 at the end of MP had a good predictive accuracy for EAD (AUC = 0.713; p = 0.02). Lipid peroxidation (TBARS) elevated at the start of NRP, declined over time, with higher levels in D-HOPE than in NMP, suggesting a more oxidative environment in D-HOPE. Metabolites like flavin mononucleotide (FMN) and NADH exhibited significant disparities between perfusion types, due to differences in perfusate compositions. Inflammatory biomarkers rose during NRP and NMP but normalized post-transplantation. Regenerative markers, including osteopontin and hepatocyte growth factor, increased during NRP and NMP and normalized post-transplant. Conclusions: ARG-1 demonstrates strong potential as an early biomarker for assessing liver graft viability during perfusion, supporting timely and effective decision-making in transplantation.

Normothermic machine perfusion (NMP) has emerged as technology for organ preservation and assessment. While NMP has been widely adopted for liver, lung, and heart transplantation, kidney NMP has faced slower clinical integration. Normothermic perfusion may potentially improve kidney transplant through improved preservation, graft viability assessment, mitigation of ischemia reperfusion injury, and treatment prior to transplant. The purpose of this review is to highlight the applications of NMP in kidney transplantation. Kidney NMP has been proven well tolerated and feasible in multiple studies. Two recent randomized controlled trials did not demonstrate a benefit of NMP compared to cold storage. The use of NMP may increase utilization through improved logistics. Graft assessment during perfusion may allow for well tolerated transplantation of marginal kidneys. Successful long-term perfusion up to 4 days of discarded kidneys has been performed. Gene therapies and treatments, including immune modification, have been carried out during kidney NMP. Normothermic perfusion has several applications to kidney transplantation including preservation, assessment, and treatment. Perfusion protocols viability criteria need to be defined. A portable, commercially available device is needed to increase clinical use. Further studies are needed to compare NMP to current preservation methods.

A shortage of available organs for liver transplantation has led transplant surgeons and researchers to seek for innovative approaches in hepatoprotection and improvement of marginal allografts. The most exciting development in the past decade has been continuous mechanical perfusion of livers with blood or preservation solution to mitigate ischemia-reperfusion injury in contrast to the current standard of static cold storage. Two variations of machine perfusion have emerged in clinical practice. During hypothermic oxygenated perfusion the liver is perfused using a red blood cell-free perfusate at 2–10°C. In contrast, normothermic machine perfusion mimics physiologic liver perfusion using a red blood cell-based solution at 35.5–037.5°C, offering a multitude of potential advantages. Putative effects of normothermic perfusion include abrogation of hyperfibrinolysis after reperfusion and inflammation, glycogen repletion, and regeneration of adenosine triphosphate. Research in normothermic machine perfusion focuses on development of biomarkers predicting allograft quality and susceptibility to ischemia-reperfusion injury. Moreover, normothermic perfusion of marginal allografts allows for application of a variety of therapeutic interventions potentially enhancing organ quality. Both methods need to be subjected to translational investigation and evaluation in clinical trials. A clear advantage is transformation of an emergency procedure at night into a planned daytime surgery. Current clinical trials suggest that normothermic perfusion not only increases the use of hepatic allografts but is also associated with milder ischemia-reperfusion injury, resulting in a reduced risk of early allograft dysfunction and less biliary complications, including ischemic cholangiopathy, compared to static cold storage. The aim of this review is to give a concise overview of normothermic machine perfusion and its current applications, benefits, and possible advances in the future.

Sequential hypothermic and normothermic perfusion preservation and transplantation of expanded criteria donor livers

Reply to: “The rescue of DCD rodent livers grafts: Is there HOPE?”

Novel cancer therapeutics have less than a 12% probability of translating from bench to bedside. Unwarranted toxicity and inadequate therapeutic delivery due to uptake by clearance organs, not predicted by current preclinical methods, have contributed towards this high rate of attrition. In the present work, we propose normothermic machine perfusion of human or human-sized organs as a more predictive, closer-to-human model to investigate drug pharmacokinetics and toxicity. Over the past decade, developments in the field of organ preservation for transplantation have enabled prolonged (>12 hours) normothermic machine perfusion (NMP) of isolated porcine or human organs ex vivo, maintaining quasi-physiological haemodynamic, synthetic and metabolic function using a packed red cell perfusate with physiological oxygenation and nutrient levels at normal body temperature. This preserves physiological processes such as metabolism and drug elimination, and enables easy access to tissue, blood and excreted biological fluids, with NMP livers producing bile and NMP kidneys producing urine. We hypothesise that this will provide a physiologically relevant platform to investigate drug pharmacokinetics and toxicity. We selected a widely used small-molecule chemotherapeutic (Irinotecan hydrochloride 2mg/ml, Medac, UK) which has seen decades of clinical use and benefits from extensive clinical pharmacokinetic data. The small-molecule drug was infused into isolated porcine and human livers and kidneys, with quantification of concentration time profiles of the prodrug and its main metabolites in plasma, bile and urine over 16-24 hours of NMP. In addition to irinotecan (CPT11), three of its metabolites (APC, SN38G, SN38) were successfully detected and quantified, demonstrating peak plasma concentrations (Cmax ~ 10,000 ng/mL, 1000 ng/mL, 100 ng/g, 30 ng/g), plasma decay rates and percentages of injected dose in bile (%ID ~20%, 8%, 25%, 1%) and urine (%ID ~20%, 0.06%, 0.5%,0.1%) that are comparable to clinical data. Drug-tissue toxicity could also be adequately replicated in the NMP model. In conclusion, we have demonstrated that human-sized isolated, normothermically perfused livers and kidneys accurately represent the clinically observed pharmacokinetic and toxicity profiles of an established small-molecule therapeutic. Further model validation is ongoing for biologics and other nanomedicines which are susceptible to clearance by the mononuclear phagocytic system or are hepato- or nephrotoxic. If this proves successful, normothermic machine perfusion of isolated porcine and human organs could greatly aid the early screening of candidate therapeutics and significantly enhance the pace and success rate with which they are translated into patients. Citation Format: Tamsyn Clark, Luca Bau, Fungai Dengu, Daniel Voyce, Robert Carlisle, Peter Friend, Constantin Coussios. Predicting clinical pharmacokinetics and toxicity of current and emerging oncology therapeutics by normothermic perfusion of isolated human-sized organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1369.