Developing an Injury-Free 15 Hour Preservation Protocol of Donor Porcine Kidneys Using Normothermic Machine Perfusion.

Normothermic machine perfusion (NMP) offers a superior alternative to hypothermic preservation but is currently time limited. Extending this time could electivise transplantation and enable physiologic assessments of functionality. Porcine kidneys were retrieved, stored on ice for 3.5 hours before being placed onto a NMP circuit for 12 hours. Hemodynamics, biochemistry, and urine output were assessed. After 12 hours, kidneys were scored using the clinical assessment score. Biopsies were collected for histological assessment. Kidneys demonstrated continual improvements in hemodynamics. Perfusate sodium concentrations remained within physiologic parameters. Sodium bicarbonate increased over-time with corresponding decreases in lactate, demonstrating active renal gluconeogenesis and Cori cycle processes. Urine production began immediately and was sustained, indicating renal functionality. Under the clinical perfusion assessment score, all kidneys received a score of 1 and would be considered suitable for transplantation. Histological assessment revealed kidneys were injury free. Our NMP protocol safely preserves kidneys for over 15 hours. Successful perfusion was achieved with stable hemodynamics and biochemistry, with maintained urination. Importantly, kidneys remained in optimal health, with no evidence of injury. This may enable electivisation of transplantation, while reducing hypothermic injury.

The Use of Hemoglobin-Based Oxygen Carriers in Ex Vivo Machine Perfusion of Donor Organs for Transplantation.

There has been significant progress in the development of ex vivo machine perfusion for the nonischemic preservation of donor organs. However, several complications remain, including the logistics of using human blood for graft oxygenation and hemolysis occurring as a result of mechanical technology. Recently, hemoglobin-based oxygen carriers, originally developed for use as blood substitutes, have been studied as an alternative to red blood cell-based perfusates. Although research in this field is somewhat limited, the findings are promising. We offer a brief review of the use of hemoglobin-based oxygen carriers in ex vivo machine perfusion and discuss future directions that will likely have a major impact in progressing oxygen carrier use in clinical practice.

Renal hemofiltration prevents metabolic acidosis and reduces inflammation during normothermic machine perfusion of the vascularized composite allograft: A preclinical study.

INTRODUCTION: Recent experimental evidence suggests normothermic machine perfusion of the vascularized composite allograft results in improved preservation compared to static cold storage, with less reperfusion injury in the immediate post-operative period. However, metabolic acidosis is a common feature of vascularized composite allograft perfusion, primarily due to the inability to process metabolic by-products. We evaluated the impact of combined limb-kidney perfusion on markers of metabolic acidosis and inflammation in a porcine model. METHODS: Ten paired pig forelimbs were used for this study, grouped as either limb-only (LO, n = 5) perfusion, or limb-kidney (LK, n = 5) perfusion. Infrared thermal imaging was used to determine homogeneity of perfusion. Lactate, bicarbonate, base, pH, and electrolytes, along with an inflammatory profile generated via the quantification of cytokines and cell-free DNA in the perfusate were recorded. RESULTS: The addition of a kidney to a limb perfusion circuit resulted in the rapid stabilization of lactate, bicarbonate, base, and pH. Conversely, the LO circuit became progressively acidotic, correlating in a significant increase in pro-inflammatory cytokines. Global perfusion across the limb was more homogenous with LK compared to LO. CONCLUSION: The addition of a kidney during limb perfusion results in significant improvements in perfusate biochemistry, with no evidence of metabolic acidosis.

A post-preservation vascular flush removes significant populations of donor leukocytes prior to lung transplantation.

BACKGROUND: Donor leukocytes are intrinsically involved in acute lung allograft rejection, via self-presentation of donor antigens to recipient leukocytes. Therapeutic modalities to remove donor leukocytes are currently unavailable. We evaluated if a vascular flush immediately following preservation can be used for this purpose. METHODS: A post-preservation flush was performed with STEEN solution in n = 6 porcine lungs following static cold storage. The first 500 ml effluent from the left atrium was collected and an inflammatory profile performed. RESULTS: A total of 1.17 billion (±2.8 × 108) viable leukocytes were identified within the effluent. T cells were the dominant cell population, representing 82% of the total mobilised leukocytes, of which <0.01% were regulatory T cells. IL-18 was the most abundant cytokine, with a mean concentration of 84,216 pg (±153,552 pg). In addition, there was a mean concentration of 8819 ng (±4415) cell-free mitochondrial DNA. CONCLUSION: There is an immediate transfer of donor leukocytes, cytokines and damage-associated molecular patterns following reperfusion. Such a pro-inflammatory donor load may enhance alloantigen presentation and drive recipient alloimmune responses. A post-preservation flush may therefore be an effective method for reducing the immune burden of the donor lung prior to transplantation.

Hemodynamics and Metabolic Parameters in Normothermic Kidney Preservation Are Linked With Donor Factors, Perfusate Cells, and Cytokines.

Kidney transplantation is the best renal-replacement option for most patients with end-stage renal disease. Normothermic machine preservation (NMP) of the kidney has been studied extensively during the last two decades and implemented in clinical trials. Biomarker research led to success in identifying molecules with diagnostic, predictive and therapeutic properties in chronic kidney disease. However, perfusate biomarkers and potential predictive mechanisms in NMP have not been identified yet. Twelve discarded human kidneys (n = 7 DBD, n = 5 DCD) underwent NMP for up to 24 h. Eight were perfused applying urine recirculation (URC), four with replacement of urine (UR) using Ringer's lactate. The aim of our study was to investigate biomarkers (NGAL, KIM-1, and L-FABP), cells and cytokines in the perfusate in context with donor characteristics, perfusate hemodynamics and metabolic parameters. Cold ischemia time did not correlate with any of the markers. Perfusates of DBD kidneys had a significantly lower number of leukocytes after 6 h of NMP compared to DCD. Arterial flow, pH, NGAL and L-FABP correlated with donor creatinine and eGFR. Arterial flow was higher in kidneys with lower perfusate lactate. Perfusate TNF-α was higher in kidneys with lower arterial flow. The cytokines IL-1ß and GM-CSF decreased during 6 h of NMP. Kidneys with more urine output had lower perfusate KIM-1 levels. Median and 6-h values of lactate, arterial flow, pH, NGAL, KIM-1, and L-FABP correlated with each other indicating a 6-h period being applicable for kidney viability assessment. The study results demonstrate a comparable cytokine and cell profile in perfusates with URC and UR. In conclusion, clinically available perfusate and hemodynamic parameters correlate well with donor characteristics and measured biomarkers in a discarded human NMP model.

Pro-IL-1ß Is an Early Prognostic Indicator of Severe Donor Lung Injury During Ex Vivo Lung Perfusion.

BACKGROUND: Ex vivo lung perfusion (EVLP) is used to evaluate and recondition extended criteria donor lungs for transplantation. Interleukin-1ß (IL-1ß) has been identified as a prognostic indicator of nonrecovery during EVLP. This may be an effect of inflammasome activation or cellular necrosis following donation and graft preservation. Delineating the mechanism of IL-1ß release is required. METHODS: The inactive intracellular precursor molecule, pro-IL-1ß, was characterized along with the pro-IL-1ß processing enzyme, caspase-1, in the perfusate of n = 20 human lungs that had undergone EVLP (n = 10 lungs that failed to recover and were discarded versus n = 10 lungs that reconditioned and were transplanted). In an experimental porcine model, n = 8 lungs underwent EVLP and were randomized to receive either a specific NLRP3 inflammasome inhibitor or control. RESULTS: Significant increases in pro-IL-1ß and caspase-1 were observed in the perfusate from human lungs that did not recondition during EVLP compared with those that successfully reconditioned and were used for transplantation. Within the porcine EVLP, NLRP3 inflammasome inhibition reduced IL-1ß within the perfusate compared with controls, but this had no impact on lung function, hemodynamics, or inflammation. CONCLUSIONS: Our data suggest that pro-IL-1ß is passively released following cellular necrosis of the donor lung.

Non-ischemic Heart Preservation via Hypothermic Cardioplegic Perfusion Induces Immunodepletion of Donor Hearts Resulting in Diminished Graft Infiltration Following Transplantation.

Introduction: Many donor organs contain significant leukocyte reservoirs which upon transplantation activate recipient leukocytes to initiate acute rejection. We aimed to assess whether non-ischemic heart preservation via ex vivo perfusion promotes immunodepletion and alters the inflammatory status of the donor organ prior to transplantation. Methods: Isolated porcine hearts underwent ex vivo hypothermic, cardioplegic perfusion for 8 h. Leukocyte populations were quantified in left ventricle samples by flow cytometry. Cell-free DNA, cytokines, and chemokines were quantified in the perfusate. Tissue integrity was profiled by targeted proteomics and a histological assessment was performed. Heterotopic transplants comparing ex vivo hypothermic preservation and static cold storage were utilized to assess graft infiltration as a solid clinical endpoint. Results:Ex vivo perfusion significantly immunodepleted myocardial tissue. The perfusate displayed a selective, pro-inflammatory cytokine/chemokine pattern dominated by IFN-γ. The tissue molecular profile was improved following perfusion by diminished expression of nine pro-apoptotic and six ischemia-associated proteins. Histologically, no evidence of tissue damage was observed and cardiac troponin I was low throughout perfusion. Cell-free DNA was detected, the source of which may be necrotic/apoptotic leukocytes. Post-transplant graft infiltration was markedly reduced in terms of both leucocyte distribution and intensity of foci. Conclusions: These findings demonstrate that ex vivo perfusion significantly reduced donor heart immunogenicity via loss of resident leukocytes. Despite the pro-inflammatory cytokine pattern observed, a pro-survival and reduced ischemia-related profile was observed, indicating an improvement in graft viability by perfusion. Diminished graft infiltration was observed in perfused hearts compared with those preserved by static cold storage following 48 h of transplantation.

Ex Vivo Lung Perfusion Improves the Inflammatory Signaling Profile of the Porcine Donor Lung Following Transplantation.

BACKGROUND: Primary graft dysfunction and allograft rejection represent major caveats to successful lung transplantation. Reducing inflammation in donor lungs before transplantation may improve outcomes. Evidence exists that ex vivo lung perfusion (EVLP) can alter the donor lung environment, although the mechanisms remain unclear. This study aimed to characterize the inflammatory signaling profile of the lung following standard and EVLP transplant and delineate the immediate impact on the recipient circulation. METHODS: Female recipient pigs (n = 12) were randomized to undergo left lung transplantation from male donors either using the gold standard protocol (static cold storage) or following 3 hours of EVLP. The relative phosphorylation of 44 phosphokinases and the relative expression of 35 apoptosis-related molecules were profiled within the donor lung 24 hours posttransplantation. RESULTS: A global profile of mitochondrial salvage and cell survival was observed in the EVLP lung tissue compared with lungs undergoing standard transplantation. This included increased phosphorylation of downstream prosignaling kinases, including ERK1/2 and FAK. In addition, there was upregulated expression of the antiapoptotic proteins Bcl-2, HSP-70, LIVIN, and PON2 with downregulation of apoptosis inducing mitochondrial associated molecules, including clusterin, cytochrome C, and HTRA2/OMI. In the early postoperative period, there were significantly lower levels of circulating mitochondrial DNA in recipients receiving EVLP lungs compared with a standard transplant (P = 0.016). Genomic DNA did not differ between groups, with donor DNA undetectable at all time points. CONCLUSIONS: EVLP alters the inflammatory signaling profile of the donor lung before transplantation, with a global cell survival and antiapoptotic signature.

Ex-vivo flush of the limb allograft reduces inflammatory burden prior to transplantation.

BACKGROUND: Passenger leucocytes and inflammatory debris transferred from the donor limb to the recipient can induce allorecognition, which activates the host immune response. This is the first study to evaluate whether the transfer of this inflammatory burden can be reduced via post-preservation flush prior to revascularisation, and whether this is influenced by ischaemia. METHODS: Bilateral forelimbs from the same pig were procured and infused with preservation flush and stored on ice. Each limb from the same pig underwent a post-preservation intravascular flush with isotonic solution at either 2 or 6 h. Venous effluent underwent flow cytometry to phenotype leucocyte populations, with additional quantification of cytokines and cell-free DNA. RESULTS: We identified large populations of viable leucocytes in the flush effluent (8.65 × 108 ± 3.10 × 108 cells at 2 h and 1.02 × 109 ± 2.63 × 108 at 6 h). This comprised T cells, B cells, NK cells and monocytes. Post-preservation flush yielded significant concentrations of pro-inflammatory cytokines including IL-6, IL-18, GM-CSF, IL-1ß, IL1α and CXCL-8 and mitochondrial DNA. The regulatory cytokine, IL-10 was undetectable. CONCLUSIONS: This study supports the finding that a post-preservation flush removes leucocytes and inflammatory components that are responsible for direct presentation. This study also gives an indication of how ischaemia impacts on the inflammatory burden transferred to the recipient upon reperfusion.

The effect of 1.5 T cardiac magnetic resonance on human circulating leucocytes.

Aims: Investigators have proposed that cardiovascular magnetic resonance (CMR) should have restrictions similar to those of ionizing imaging techniques. We aimed to investigate the acute effect of 1.5 T CMR on leucocyte DNA integrity, cell counts, and function in vitro, and in a large cohort of patients in vivo. Methods and results: In vitro study: peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers, and histone H2AX phosphorylation (γ-H2AX) expression, leucocyte counts, and functional parameters were quantified using flow cytometry under the following conditions: (i) immediately following PBMC isolation, (ii) after standing on the benchside as a temperature and time control, (iii) after a standard CMR scan. In vivo study: blood samples were taken from 64 consecutive consenting patients immediately before and after a standard clinical scan. Samples were analysed for γ-H2AX expression and leucocyte counts. CMR was not associated with a significant change in γ-H2AX expression in vitro or in vivo, although there were significant inter-patient variations. In vitro cell integrity and function did not change with CMR. There was a significant reduction in circulating T cells in vivo following CMR. Conclusion: 1.5 T CMR was not associated with DNA damage in vitro or in vivo. Histone H2AX phosphorylation expression varied markedly between individuals; therefore, small studies using γ-H2AX as a marker of DNA damage should be interpreted with caution. Cardiovascular magnetic resonance was not associated with loss of leucocyte viability or function in vitro. Cardiovascular magnetic resonance was associated with a statistically significant reduction in viable leucocytes in vivo.

Characterizing the early inflammatory contribution of the donor kidney following reperfusion.

BACKGROUND: Donor kidneys contain a large reservoir of passenger leucocytes that contribute to acute rejection via direct alloantigen presentation and pro-inflammatory cytokine secretion. However, the early contribution of these cells following revascularization has not previously been described. We performed a secondary, high-volume preservation flush following cold storage to characterize the inflammatory contribution of the donor kidney upon reperfusion. METHODS: Porcine kidneys were retrieved using a protocol analogous to current UK clinical practice. Following 2 h of cold static preservation, kidneys underwent a secondary flush with Ringer's solution. The venous effluent was collected and leucocytes phenotyped via flow cytometry. Inflammatory mediators, including cytokines and cell-free DNA, were then assessed to determine the inflammatory contribution of the donor kidney. RESULTS: Upon reperfusion, a significant population of donor-derived CD45 + leucocytes mobilized from the renal vasculature via the renal vein [mean 4.738 × 10 8 (SD 1.348 × 10 8 )]. Within this population, T cells were dominant, representing >60% of the leucocyte repertoire. Granulocytes, monocytes and natural killer cells were also identified, but in comparatively lower numbers. Significant concentrations of cytokines and cell-free DNA were also eluted upon reperfusion. CONCLUSIONS: The donor kidney contains a significant immune load that rapidly mobilizes following reperfusion. Performing a secondary preservation flush prior to implantation may reduce this inflammatory burden via diversion of donor leucocytes and inflammatory mediators from entry into the recipient circulation. This may modulate direct presentation and reduce the inflammatory contribution of the donor kidney following transplantation.

Ex Vivo Normothermic Perfusion Induces Donor-Derived Leukocyte Mobilization and Removal Prior to Renal Transplantation.

INTRODUCTION: Ex vivo normothermic perfusion offers an alternative method of organ preservation, allowing donor kidneys to be reanimated and evaluated prior to transplantation. Beyond preservation, it can be used to characterize the immunological contribution of the donor kidney in isolation. Furthermore, it has the potential to be used as an immunomodulatory strategy to manipulate donor kidneys prior to transplantation. METHODS: Explanted porcine kidneys underwent 6 hours of perfusion. Sequential perfusate samples were collected and leukocytes characterized via flow cytometry. An inflammatory profile was generated via cytokine quantification. Cell-free DNA was also determined as markers of cell death. RESULTS: All kidneys functioned within normal parameters and met the criteria for transplantation at the end of perfusion. Throughout perfusion there were continuous increases in pro-inflammatory cytokines, including large concentrations of interferon-γ, suggesting that perfusion drives a significant inflammatory response. Increasing concentrations in cell-free DNA were also observed, suggesting cell death. During perfusion there was a marked cellular diapedesis of T cells, B cells, natural killer (NK) cells, and monocytes from the kidney into the circuit. Minor populations of granulocytes and macrophages were also detected. DISCUSSION: We demonstrate that ex vivo normothermic perfusion initiates an inflammatory cytokine storm and release of mitochondrial and genomic DNA. This is likely to be responsible for immune cell activation and mobilization into the circuit prior to transplantation. Interestingly this did not have an impact on renal function. These data therefore suggest that normothermic perfusion can be used to immunodeplete and to saturate the pro-inflammatory capacity of donor kidneys prior to transplantation.

Clinical Outcome of Patients Transplanted with Marginal Donor Lungs via Ex Vivo Lung Perfusion Compared to Standard Lung Transplantation.

BACKGROUND: Lung transplantation is limited by a scarcity of suitable donors resulting in high waiting list mortality. Ex vivo lung perfusion (EVLP) allows the evaluation and reconditioning of marginal donor lungs for use in transplantation. This study aimed to compare clinical outcome of patients transplanted with marginal organs by means of EVLP with a standard lung transplant cohort through a multicenter open trial. METHODS: Group 1 (n = 9) included patients transplanted using EVLP reconditioned marginal lungs. Group 2 (n = 46) consisted of date-matched patients transplanted using standard transplantation of acceptable lungs. The primary composite endpoint included acute rejection and infection at 12 months after transplantation. RESULTS: There was no significant difference in the overall incidence of acute rejection (P = 0.754) and the number of treated infection episodes (proven/probable pneumonia; P = 0.857/0.368 and proven/probable tracheobronchitis; P = 0.226/0.529) up to 12 months after transplantation, between group 1 and group 2. Additionally, there was no significant difference in early clinical outcome, including intensive care unit stay, hospital stay, and 1 year mortality between the two groups (P = 0.338, P = 0.112 and P = 0.372, respectively). DISCUSSION: This multicenter study demonstrates that EVLP is associated with no adverse effect on clinical outcome, including the incidence of acute rejection and infection after lung transplantation.

Mechanical removal of dendritic cell-generating non-classical monocytes via ex vivo lung perfusion.

BACKGROUND: Ex vivo lung perfusion (EVLP) is a novel procedure designed to rapidly assess and recondition unusable donor lungs for transplantation (LTx). EVLP may reduce graft immunogenicity and allorecognition via removal of passenger leukocytes. We aimed to explore this hypothesis using human EVLP and in vitro analysis. METHODS: Explanted human lungs (n = 7) underwent standard EVLP. Perfusate samples and the leukocyte filter were collected, and cells characterized via flow cytometry. Isolated alveolar monocytes (from post-LTx bronchoalveolar lavage) were differentiated to dendritic cells and characterized (n = 10). An in vitro (air epithelial-liquid endothelial) lung model was utilized to evaluate monocyte migration and differentiation within the lung. RESULTS: Non-classical monocytes (NCM, normally <1% of total white blood cell repertoire) mobilized within 30 minutes of EVLP and represented 80.04% of the passenger leukocyte population. This subset readily differentiated to dendritic cells and secreted pro-inflammatory cytokines (interferon-γ and interleukin-2) after stimulation. NCM rapidly diapedesed from the vascular bed to the alveolus and, when cultured on the alveolus, differentiated to dendritic cells with inflammatory phenotypes. CONCLUSIONS: The lung possesses a reservoir of NCM, which can readily diapedese to the alveolus or mobilize in the circulation. After activation, NCM differentiate to inflammatory dendritic cells with T-cell co-stimulatory capacity. EVLP may impart additional benefits after LTx via the removal of passenger monocytes, which may represent a previously unidentified beneficial mechanism of action.