Gradual Rewarming Preservation of Liver and Kidney Grafts

The aim of this thesis was to study the role of gradual rewarming on enhancing organ quality in organs with low quality such as ECD and especially DCD during the organ preservation phase and before implantation. This thesis also addresses an improvement in gradual rewarming protocol by adding a hemoglobin-based oxygen carrier (HBOC) to the perfusion solution in the preclinical kidney and liver rodent models

Hemostatic system activation and reperfusion injury in liver machine preservation and transplantation of extended criteria donor livers

For patients with end-stage liver disease and certain types of hepatic malignancies, liver transplantation is universally accepted as the most effective and only curable treatment available. The success of liver transplantation has ironically however, become one of the greatest challenges faced by transplant health professionals worldwide as a great discrepancy continues to exist between the supply of suitable donor livers for transplantation and the demand. In an effort to tackle the donor organ scarcity, a significant proportion of additional donor organs has resulted from the increased reliance on the use of extended criteria donor (ECD) livers. Such livers include; livers from older donors, livers that exceed the traditionally accepted degree of steatosis (or in lay terms; “fatty livers”) and livers donated after circulatory death (DCD). In fact, in 2018 in the Netherlands, more than 50% of deceased donor liver transplants were derived from DCD donors. Several studies have shown that selective use of ECD livers results in successful transplantation procedures and acceptable survival rates following transplantation. Nevertheless, a higher incidence of post-transplant morbidity such as early allograft dysfunction, (severe) intraoperative bleeding and life threatening biliary complications, have been reported after transplantations with ECD livers. Studies have shown that these are largely attributed to by ischemia-reperfusion injury (IR injury) incurred by these organs during the procurement, preservation and implantation processes. Machine perfusion (MP) is a promising alternative preservation modality that allows for the storage of donor organs under conditions simulating in vivo physiology. Therefore, ischemia, and subsequently, ischemia-reperfusion injury is minimized. In this thesis more insight is provided into (1) the incidence of bleeding and coagulation problems in DCD transplantation, (2) machine perfusion as a method to (better) preserve ECD-DCD donor livers to reduce intra- and postoperative complications and (3) the effect of machine perfusion on the coagulation system and on ischemia-reperfusion damage in ECD-DCD donor livers

Gradual Rewarming with Gradual Increase in Pressure during Machine Perfusion after Cold Static Preservation Reduces Kidney Ischemia Reperfusion Injury

In this study we evaluated whether gradual rewarming after the period of cold ischemia would improve organ quality in an Isolated Perfused Kidney Model. Left rat kidneys were statically cold stored in University of Wisconsin solution for 24 hours at 4 degrees C. After cold storage kidneys were rewarmed in one of three ways: perfusion at body temperature (38 degrees C), or rewarmed gradually from 10 degrees C to 38 degrees C with stabilization at 10 degrees C for 30 min and rewarmed gradually from 10 degrees C to 38 degrees C with stabilization at 25 degrees C for 30 min. In the gradual rewarming groups the pressure was increased stepwise to 40 mmHg at 10 degrees C and 70 mmHg at 25 degrees C to counteract for vasodilatation leading to low perfusate flows. Renal function parameters and injury biomarkers were measured in perfusate and urine samples. Increases in injury biomarkers such as aspartate transaminase and lactate dehydrogenase in the perfusate were lower in the gradual rewarming groups versus the control group. Sodium re-absorption was improved in the gradual rewarming groups and reached significance in the 25 degrees C group after ninety minutes of perfusion. HSP-70, ICAM-1, VCAM-1 mRNA expressions were decreased in the 10 degrees C and 25 degrees C groups. Based on the data kidneys that underwent gradual rewarming suffered less renal parenchymal, tubular injury and showed better endothelial preservation. Renal function improved in the gradual rewarming groups versus the control group

Heterogeneous Indications and the Need for Viability Assessment: An International Survey on the Use of Machine Perfusion in Liver Transplantation

Although machine perfusion (MP) is being increasingly adopted in liver transplantation, indications, timing, and modality are debated. To investigate current indications for MP a web-based Google Forms survey was launched in January 2021 and addressed to 127 experts in the field, identified among first and corresponding Authors of MP literature in the last 10 years. The survey presented 10 real-life cases of donor-recipient matching, asking whether the liver would be accepted (Q1), whether MP would be used in that particular setting (Q2) and, if so, by which MP modality (Q3) and at what timing during preservation (Q4). Respondents could also comment on each case. The agreement was evaluated using Krippendorff's alpha coefficient. Answers from 39 (30.1%) participants disclosed significant heterogeneity in graft acceptance, MP indications, technique, and timing. Agreement between respondents was generally poor (Q1, α = 0.11; Q2, α = 0.14; Q3, α = 0.12, Q4, α = 0.11). Overall, respondents preferred hypothermic MP and an end-ischemic approach in 56.3% and 81.1% of cases, respectively. A total of 18 (46.2%) participants considered only one MP approach, whereas 17 (43.6%) and 3 (7.7%) considered using alternatively 2 or 3 different techniques. Of 38 comments, 17 (44.7%) were about the use of MP for graft viability assessment before implantation. This survey shows considerable variability in MP indications, emphasizing the need to identify scenarios of optimal utilization for each technique. Viability assessment emerges as a fundamental need of transplant professionals when considering the use of MP. Keywords: ex situ organ perfusion; machine perfusion; normothermic regional perfusion; organ preservation; viability assessment

Modifying Donor Organ Retrieval and Preservation to Enhance Transplant Outcomes

The last 1-2 decades have seen remarkable advances in organ procurement and preservation practices, especially with renewed enthusiasm for machine perfusion (MP) technology. However, cold static storage (CS) remains the most popular world-wide approach for the preservation of organs such as the kidneys, liver, and pancreas, largely due to its simplicity. It is clear that CS techniques have limited potential for further improvement, and will likely be supplanted and/or supplemented with MP technologies over the coming years due to the reparative, resuscitative, and assessment capabilities afforded by MP. This is especially important as we increase our utilisation of marginal and/or donation after circulatory death (DCD) organs to meet the ever-increasing demand requirements for transplantation. This dissertation explores selected aspects of abdominal organ procurement and preservation as targets for improvement and/or modification with the aim to enhance recipient transplantation outcomes. The kidney is used as a model organ for the development and exploration of MP as a means to ameliorate transplant organ ischaemia-reperfusion injury (IRI), including through the targeted delivery of anti-IRI drugs. In contrast, the optimization of CS protocols, including identification of ideal perfusion fluids and in situ perfusion routes, forms the basis for liver and pancreas transplantation work in this thesis. Such investigations are necessary to promote uniformity of practice between centres, and allow appropriate comparisons between MP and CS. The kidney MP work was guided by a systematic review and meta-analysis comparing MP and CS in the clinical and pre-clinical setting. Although hypothermic MP (HMP) was shown to enhance short-term graft outcomes, results were equivocal with respect to graft survival, especially in the DCD setting. Preliminary evidence indicated the potential superiority of normothermic MP (NMP) above HMP or CS, which may be further enhanced by using NMP as a conduit for directed drug delivery to the kidney to ameliorate IRI. We therefore developed and optimized a local NMP set-up using a series of porcine kidneys, which was then utilized to deliver the anti-IRI agent CD47-blocking antibody (αCD47Ab) in a porcine DCD model. The significant potential of this agent was initially confirmed by testing in a murine model of severe warm IRI, including its comparative efficacy to two other promising IRI agents, soluble complement receptor 1 (sCR1), and recombinant thrombomodulin, and also sCR1 in combination with αCD47Ab. αCD47Ab was successfully delivered to porcine DCD kidneys using NMP, with subsequent downstream positive impacts upon renal perfusion, and some functional and IRI-related parameters. The clinical utilisation of renal NMP has so far been limited to the UK, and this modality has not been tested in human kidneys in Australasia. Furthermore, the mechanistic basis of brief renal NMP is not entirely clear. Therefore, and as a prelude to a phase I clinical trial, NMP was tested in discarded deceased donor human kidneys. Fifteen kidneys were obtained from 10 donors, and successfully underwent NMP. NMP was especially effective for assessing and improving DCD kidneys discarded for poor macroscopic perfusion at retrieval. Flow cytometry analyses showed evidence of a massive passenger leukocyte efflux during NMP. In paired kidney analyses, one hour of NMP was shown to be superior to CS alone after simulated transplantation using ex vivo whole allogeneic blood reperfusion, in terms of renal perfusion and functional parameters. Whole transcriptome RNA sequencing revealed NMP-mediated induction of protective stress and inflammatory-related pathways, in addition to a reduction in cell death pathways. Accordingly, immunofluorescence techniques confirmed a reduction in cell death and IRI in NMP kidneys compared to their CS counterparts. CS and procurement techniques formed the basis of liver and pancreas transplantation-related studies conducted for this thesis. Firstly, we showed that blood transfusion requirements can be significantly reduced in recipients if the pancreas is retrieved using ultrasonic shears (Harmonic Scalpel), implying a reduction in procedural risk and recipient sensitization. Two systematic reviews and meta-analyses were then conducted to ascertain optimal in situ perfusion/preservation fluids, and perfusion routes, during procurement of pancreatic and hepatic allografts. There was a lack of overwhelming evidence favouring any specific preservation fluid, although University of Wisconsin solution will likely remain the solution of choice, especially for the pancreas. Furthermore, in standard criteria donors, aortic-only perfusion was found to produce equivalent liver transplant outcomes in comparison to dual (aorto-portal perfusion). However, existing studies included small patient numbers and short periods of follow-up. We therefore compared aortic and dual perfusion during liver retrieval using the Australia and New Zealand Liver Transplant Registry, which provided a much larger patient cohort with prolonged follow-up. This study confirmed the equivalence of aortic-only and dual perfusion in standard criteria liver donors, however there was also evidence indicating the superiority of dual perfusion in a subset of suboptimal/higher risk donors. Overall, this thesis expounds upon the putative benefits of NMP in kidney transplantation, including by directed drug delivery targeting the IRI cascade, and also enhances our understanding of optimal perfusion routes and preservation fluids for the liver and pancreas. The ultimate aim is to facilitate expansion of the donor pool whilst simultaneously enhancing recipient transplantation outcomes through the evidence-based implementation of technologies and techniques in a unified and coordinated manner

Use of mesenchymal stem cells for preconditioning of kidney grafts in an ex-vivo kidney perfusion model

Introduction: One of the biggest issues in transplantation today is the lack of suitable transplantable organs for an increasing number of patients on the transplant waiting lists. Efforts to address this problem are made by the including organs from extended-criteria and donation-after-cardiac death donors, however these organs show inferior outcomes. Advances in organ preservation are needed to focus on techniques to assess and optimise organ viability prior to transplantation. Methods: This research focuses on organ preconditioning using hypothermic- and normothermic ex-vivo machine perfusion technology in combination with stem cell treatment. A transplational approach ranging from small animal models via porcine to human models was used with an intent to facilitate translation of findings into clinical practice. A portable rapid sampling micro-dialysis (rsMD) device was tested which might enable live monitoring of kidney grafts during preconditioning periods and help estimate the condition of organs. Results: In the first head to head comparison between hypothermic and normothermic machine perfusion for organ preconditioning ex-vivo, an advantage for hypothermic perfusion was detected in the porcine model. Contrary to this, in the transplational model using human organs, the outcome was dependent on demographic backgrounds of the organs; especially the cold ischemia time organs were exposed to had an impact on which perfusion modality was better for an organ. RsMD can successfully provide detailed real-time information on tissue and organ viability at all timepoints of organ preservation. Delivery of Mesenchymal Stem Cells (MSC) into grafts ex-vivo by machine perfusion was possible for the hypothermic- as well as the normothermic setting. MSCs could be traced within the grafts, but did not immediately alter their function. Conditioned Mesenchymal Stem Cell media can influence the phenotype of immune cells, e.g. macrophages. Dependent on the genetic background of MSCs, the obtained conditioned media showed to influence macrophages in different ways. Conclusion: This research has been successful in its overall objective to introduce Mesenchymal Stem Cells into ex-vivo machine perfusion settings. By doing so the goal would be to alleviate ischemia reperfusion injury of organs for transplantation and to thereby expand the pool of acceptable donor organs.Open Acces

Heterogeneous indications and the need for viability assessment:An international survey on the use of machine perfusion in liver transplantation

Although machine perfusion (MP) is being increasingly adopted in liver transplantation, indications, timing, and modality are debated. To investigate current indications for MP a web-based Google Forms survey was launched in January 2021 and addressed to 127 experts in the field, identified among first and corresponding Authors of MP literature in the last 10 years. The survey presented 10 real-life cases of donor–recipient matching, asking whether the liver would be accepted (Q1), whether MP would be used in that particular setting (Q2) and, if so, by which MP modality (Q3) and at what timing during preservation (Q4). Respondents could also comment on each case. The agreement was evaluated using Krippendorff's alpha coefficient. Answers from 39 (30.1%) participants disclosed significant heterogeneity in graft acceptance, MP indications, technique, and timing. Agreement between respondents was generally poor (Q1, α = 0.11; Q2, α = 0.14; Q3, α = 0.12, Q4, α = 0.11). Overall, respondents preferred hypothermic MP and an end-ischemic approach in 56.3% and 81.1% of cases, respectively. A total of 18 (46.2%) participants considered only one MP approach, whereas 17 (43.6%) and 3 (7.7%) considered using alternatively 2 or 3 different techniques. Of 38 comments, 17 (44.7%) were about the use of MP for graft viability assessment before implantation. This survey shows considerable variability in MP indications, emphasizing the need to identify scenarios of optimal utilization for each technique. Viability assessment emerges as a fundamental need of transplant professionals when considering the use of MP.</p

Improved Normothermic Machine Perfusion After Short Oxygenated Hypothermic Machine Perfusion of Ischemically Injured Porcine Kidneys

Background: In an era where global kidney shortage has pushed the field of transplantation towards using more marginal donors, modified kidney preservation techniques are currently being reviewed. Some techniques require further optimization before implementation in full scale transplantation studies. Using a porcine donation after circulatory death kidney model, we investigated whether initial kidney hemodynamics improved during normothermic machine perfusion if this was preceded by a short period of oxygenated hypothermic machine perfusion (oxHMP) rather than static cold storage (SCS). Methods: Kidneys subjected to 75 minutes of warm ischemia were randomly assigned to either SCS (n = 4) or SCS + oxHMP (n = 4), with a total cold storage time of 240 minutes. Cold preservation was followed by 120 minutes of normothermic machine perfusion with continuous measurement of hemodynamic parameters and renal function. Results: oxHMP preserved kidneys maintained significantly lower renal resistance throughout the normothermic machine perfusion period compared to SCS kidneys (P < 0.001), reaching lowest levels at 60 minutes with means of 0.71 ± 0.35 mm Hg/mL/min/100 g (SCS) and 0.45 ± 0.15 mm Hg/mL/min/100 g (oxHMP). Accordingly, the oxHMP group had a higher mean renal blood flow versus SCS kidneys (P < 0.001). oxHMP kidneys had higher oxygen consumption during normothermic machine perfusion compared to SCS preserved kidneys (P < 0.001). Creatinine clearance remained similar between groups (P = 0.665). Conclusions: Preceding oxHMP significantly improved initial normothermic machine perfusion hemodynamics and increased total oxygen consumption. With the long period of warm ischemia, immediate kidney function was not observed, reflected by the findings of low creatinine clearance in both groups

Heterogeneous indications and the need for viability assessment:An international survey on the use of machine perfusion in liver transplantation

Although machine perfusion (MP) is being increasingly adopted in liver transplantation, indications, timing, and modality are debated. To investigate current indications for MP a web-based Google Forms survey was launched in January 2021 and addressed to 127 experts in the field, identified among first and corresponding Authors of MP literature in the last 10 years. The survey presented 10 real-life cases of donor–recipient matching, asking whether the liver would be accepted (Q1), whether MP would be used in that particular setting (Q2) and, if so, by which MP modality (Q3) and at what timing during preservation (Q4). Respondents could also comment on each case. The agreement was evaluated using Krippendorff's alpha coefficient. Answers from 39 (30.1%) participants disclosed significant heterogeneity in graft acceptance, MP indications, technique, and timing. Agreement between respondents was generally poor (Q1, α = 0.11; Q2, α = 0.14; Q3, α = 0.12, Q4, α = 0.11). Overall, respondents preferred hypothermic MP and an end-ischemic approach in 56.3% and 81.1% of cases, respectively. A total of 18 (46.2%) participants considered only one MP approach, whereas 17 (43.6%) and 3 (7.7%) considered using alternatively 2 or 3 different techniques. Of 38 comments, 17 (44.7%) were about the use of MP for graft viability assessment before implantation. This survey shows considerable variability in MP indications, emphasizing the need to identify scenarios of optimal utilization for each technique. Viability assessment emerges as a fundamental need of transplant professionals when considering the use of MP.</p

Preserving organ function of marginal donor kidneys

Niertransplantatie is de beste medische behandeling voor patiënten die lijden aan eindstadium nierfalen. De afgelopen decennia is de samenstelling van de overleden donorpool radicaal veranderd, zodanig dat er steeds meer organen beschikbaar komen van oudere donoren, die vaak al meerdere aandoeningen in de voorgeschiedenis hebben, of van zogenaamde non-heart beating donoren bij wie de orgaanuitname pas kan beginnen wanneer het hart al enkele minuten stil heeft gestaan. Zulke orgaandonoren worden ook wel marginale donoren genoemd. Dit proefschrift beschrijft de resultaten van klinische en pre-klinische studies op het gebied van niertransplantatie. In deze studies wordt de invloed die verscheidene karakteristieken van overleden orgaandonoren op het transplantatieresultaat hebben gekwantificeerd. Tevens wordt het effect onderzocht van interventies vóór of gedurende orgaanpreservatie, die zijn gericht op het beter conserveren van de orgaankwaliteit voorafgaand aan de transplantatie. Daarnaast beschrijft het proefschrift een studie waarin biomarkers worden gemeten in de orgaanpreservatievloeistof en een andere studie waarin de vasculaire weerstand wordt bepaald tijdens machinale preservatie van donornieren. Deze beide studies hebben als doel het voorspellen van de vitaliteit en de functie van het orgaan na transplantatie. Hoewel de resultaten van de studies in dit proefschift betrekking hebben op nieren afkomstig van alle typen overleden donoren, zijn ze het meest relevant voor marginale donornieren. Aangezien de functie en levensduur van zulke nieren na transplantatie vaak suboptimaal zijn, is extra informatie over hun kwaliteit nog vóór transplantatie belangrijk. Tevens zijn nieuwe interventies die de orgaanfunctie ná transplantatie verbeteren noodzakelijk