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Although lung transplant is a life-saving therapy for some patients, primary graft dysfunction (PGD) is a leading cause of mortality and morbidity soon after a transplant. Ischemia reperfusion injury is known to be one of the most critical factors in PGD development. PGD is by definition an acute lung injury syndrome that occurs during the first 3 d following lung transplantation. To successfully translate laboratory discoveries to clinical practice, a reliable and practical large animal model is critical. This protocol describes a surgical technique for swine lung transplantation and postoperative management for a further 3 d post transplant. The protocol includes the background and rationale, required supplies, and a detailed description of the donor operation, transplant surgery, postoperative care, and sacrifice surgery. A pig lung transplant model is reliably produced in which the recipients survive for 3 d post transplant. This 3-d survival model can be used by lung transplant researchers to assess the development of PGD and to test therapeutic strategies targeting PGD. In total, the protocol requires 5 h for the surgeries, plus ~2 h in total for the postoperative care.

Cold static preservation on ice (~4°C) remains the clinical standard of donor organ preservation. However, mitochondrial injury develops during prolonged storage, which limits the extent of time that organs can maintain viability. We explored the feasibility of prolonged donor lung storage at 10°C using a large animal model and investigated mechanisms related to mitochondrial protection. Functional assessments performed during ex vivo lung perfusion demonstrated that porcine lungs stored for 36 hours at 10°C had lower airway pressures, higher lung compliances, and better oxygenation capabilities, indicative of better pulmonary physiology, as compared to lungs stored conventionally at 4°C. Mitochondrial protective metabolites including itaconate, glutamine, and N-acetylglutamine were present in greater intensities in lungs stored at 10°C than at 4°C. Analysis of mitochondrial injury markers further confirmed that 10°C storage resulted in greater protection of mitochondrial health. We applied this strategy clinically to prolong preservation of human donor lungs beyond the currently accepted clinical preservation limit of about 6 to 8 hours. Five patients received donor lung transplants after a median preservation time of 10.4 hours (9.92 to 14.8 hours) for the first implanted lung and 12.1 hours (10.9 to 16.5 hours) for the second. All have survived the first 30 days after transplantation. There was no grade 3 primary graft dysfunction at 72 hours after transplantation, and median post-transplant mechanical ventilation time was 1.73 days (0.24 to 6.71 days). Preservation at 10°C could become the standard of care for prolonged pulmonary preservation, providing benefits to both patients and health care teams.

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Neutrophil extracellular traps in ex vivo lung perfusion perfusate predict the clinical outcome of lung transplant recipients To the Editor: Lung transplantation is lifesaving for patients with end-stage lung diseases. However, a scarcity of donor lungs suitable for transplantation limits the number of procedures that can be performed. Furthermore, major obstacles to successful lung transplantation still exist; primary graft dysfunction (PGD), the leading cause of early morbidity and mortality post-lung transplant, develops in ∼15-30% of recipients [1]. Normothermic ex vivo lung perfusion (EVLP) maintains donor lungs in a physiological state through perfusion and ventilation, so that the lungs can be assessed, treated and accepted for transplantation, thereby increasing the donor pool and alleviating the severe shortage of viable donor lungs [2]. In addition to donor lung assessment, EVLP provides a unique platform to study cellular processes occurring in donor lungs. EVLP perfusate is a unique, noninvasive source of biological sampling, which offers valuable information about the state of the donor lung. Rapid, molecular diagnostic testing could be an important addition to assessment of donor lungs, and may help improve outcome [3-7]. Detection of injurious markers in EVLP perfusate may also help inform future targeted treatments for the lungs, which could be administered directly to the organ through the EVLP circuit. In order to develop a reliable molecular assessment of donor lungs, a stable, easily quantifiable biomarker that reflects donor lung injury is required. Neutrophil extracellular traps (NETs) are structures comprised of extracellular neutrophil DNA in a complex with antimicrobial granular proteins, such as neutrophil elastase (NE) and myeloperoxidase. Neutrophils form NETs in response to innate immune challenges. Although NETs are generally considered antibacterial, several recent studies suggest that the cytotoxicity of NETs can be highly detrimental to lung and other tissue through complement activation, exposed histone cytotoxicity, promotion of thrombosis, and propagation of neutrophil recruitment and activation [8-11]. NETs have been specifically implicated in a variety of lung disease and injury states, including ventilator-induced lung injury and PGD [12, 13]. NETs play an important role in lung injury, but their role in lung transplantation is largely unknown. As NETs are a quantifiable measure of the innate immune response to a variety of inflammatory stimuli, this study aimed to determine whether NETs are detectable in EVLP perfusate, indicating their production by neutrophils in donor lungs. Furthermore, this study assessed the ability of NETs to serve as a relevant biomarker of donor lung injury. We report that elevated NET levels in donor lung EVLP perfusate are associated with worse recipient outcomes. Written, informed consent was obtained, and this study was approved by the University Health Network Research Ethics Board. Clinical perfusate samples from consecutive EVLP cases that w...

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Ex vivo lung perfusion (EVLP) is an excellent platform to apply novel therapeutics, such as gene and cell therapies, before lung transplantation. We investigated the concept of human donor lung engineering during EVLP by combining gene and cell therapies. Premodified cryopreserved mesenchymal stromal cells with augmented anti-inflammatory interleukin-10 production (MSC ) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSC IL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSC IL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSC to poor metabolic, and especially acidic, conditions decreased their IL-10 production.In conclusion, we found that "off-the-shelf" MSC IL-10 therapy of human lungs during EVLP is safe and feasible, and results in rapid IL-10 elevation, and that the acidic target-tissue microenvironment may compromise the efficacy of cell-based therapies.