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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 (MSCIL-10) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSCIL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSCIL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSCIL-10 to poor metabolic, and especially acidic, conditions decreased their IL-10 production. In conclusion, we found that “off-the-shelf” MSCIL-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., Graphical abstract, Mesenchymal stromal cells with augmented anti-inflammatory IL-10 production were administered to human lungs during ex vivo lung perfusion, a platform that enables application of novel therapeutics to donor lungs before transplantation. IL-10 production was rapid but compromised by lung injury and acidosis, indicating that the local target-tissue microenvironment affects cell-based therapies.

Background Ex vivo lung perfusion (EVLP) is an isolated organ assessment technique that has revolutionized the field of lung transplantation and enabled a safe increase in the number of organs transplanted. The objective of this study was to develop a protein-based assay that would provide a precision medicine approach to lung injury assessment during EVLP. Methods Perfusate samples collected from clinical EVLP cases performed from 2009 to 2019 were separated into development (n=281) and validation (n=57) sets to derive and validate an inflammation score based on IL-6 and IL-8 protein levels in perfusate. The ability of an inflammation score to predict lungs suitable for transplantation and likely to produce excellent recipient outcomes (time on ventilator ≤ three days) was assessed. Inflammation scores were compared to conventional clinical EVLP assessment parameters and associated with outcomes, including primary graft dysfunction and patient care in the ICU. Results An inflammation score accurately predicted the decision to transplant (AUROC 68% [95% CI 62-74]) at the end of EVLP and those transplants associated with short ventilator times (AUROC 73% [95% CI 66-80]). The score identified lungs more likely to develop primary graft dysfunction at 72-hours post-transplant (OR 4.0, p=0.03). A model comprised of the inflammation score and ∆PO2 was able to determine EVLP transplants that were likely to have excellent recipient outcomes, with an accuracy of 87% [95% CI 83-92]. Conclusions The adoption of an inflammation score will improve accuracy of EVLP decision-making and increase confidence of surgical teams to determine lungs that are suitable for transplantation, thereby improving organ utilization rates and patient outcomes.

The shortage of organ donors remains the major limiting factor in lung transplant, with the number of patients on the waiting list largely exceeding the number of available organ donors. Another issue is the low utilization rate seen in some types of donors. Therefore, novel strategies are continuously being explored to increase the donor pool. Advanced age, smoking history, positive serologies, and size mismatch are common criteria that decrease the rate of use when it comes to organ utilization. Questioning these limitations is one of the purposes of this review. Challenging these limitations by adapting novel donor management strategies could help to increase the rate of suitable lungs for transplantation while still maintaining good outcomes. A second goal is to present the latest advances in organ donation after controlled and uncontrolled cardiac death, and also on how to improve these lungs on ex vivo platforms for assessment and future specific therapies. Finally, pushing the limit of the donor envelope also means reviewing some of the recent improvements made in lung preservation itself, as well as upcoming experimental research fields. In summary, donor lung optimization refers to a global care strategy to increase the total numbers of available allografts, and preserve or improve organ quality without paying the price of early-, mid-, or long-term negative outcomes after transplantation.

For patients with end-stage lung disease, lung transplantation is a lifesaving therapy. Currently however, the number of patients who require a transplant exceeds the number of donor lungs available. One of the contributing factors to this is the conservative mindset of physicians who are concerned about transplanting marginal lungs due to the potential risk of primary graft dysfunction. Ex vivo lung perfusion (EVLP) technology has allowed for the expansion of donor pool of organs by enabling assessment and reconditioning of these marginal grafts before transplant. Ongoing efforts to optimize the therapeutic potential of EVLP are underway. Researchers have adopted the use of different large and small animal models to generate translational preclinical data. This includes the use of rejected human lungs, pig lungs, and rat lungs. In this review, we summarize some of the key current literature studies relevant to each of the major EVLP model platforms and identify the advantages and disadvantages of each platform. The review aims to guide investigators in choosing an appropriate species model to suit their specific goals of study, and ultimately aid in translation of therapy to meet the growing needs of the patient population.

Background The use of a novel extracellular oxygen carrier (EOC) preservation additive known as HEMO2Life has recently been shown to lead to a superior preservation of different types of solid organs. Our study aimed to investigate the effect of this EOC on extending lung preservation time and its mechanism of action. Methods Donor pigs were randomly allocated to either of the following 2 groups (n = 6 per group): (1) 36 hours cold preservation or (2) 36 hours cold preservation with 1 g/liter of EOC. The lungs were evaluated through 12 hours of normothermic ex vivo lung perfusion (EVLP) followed by a left-single lung transplant into a recipient pig. Grafts were reperfused for 4 hours, followed by right pulmonary artery clamping to assess graft oxygenation function. Results During EVLP assessment, EOC-treated lungs showed improvements in physiologic parameters, whereas the control lungs deteriorated. After a total of 48 hours of preservation (36 hours cold + 12 hours normothermic EVLP), transplanted grafts in the treatment group displayed significantly better oxygenation than in the controls (PaO2/FiO2: 437 ± 36 mm Hg vs 343 ± 27 mm Hg, p = 0.041). In addition, the use of EOC led to significantly less edema formation (wet-to-dry ratio: 4.95 ± 0.29 vs 6.05 ± 0.33, p = 0.026), less apoptotic cell death (p = 0.041), improved tight junction preservation (p = 0.002), and lower levels of circulating IL-6 within recipient plasma (p = 0.004) compared with non-use of EOC in the control group after transplantation. Conclusion The use of an EOC during an extended pulmonary preservation period led to significantly superior early post-transplant lung function.

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

Background Transmission of latent human cytomegalovirus (HCMV) via organ transplantation with post-transplant viral reactivation is extremely prevalent and results in substantial adverse impact on outcomes. Therapies targeting the latent reservoir within the allograft to mitigate viral transmission would represent a major advance. Here, we delivered an immunotoxin (F49A-FTP) that targets and kills latent HCMV aiming at reducing the HCMV reservoir from donor lungs using ex-vivo lung perfusion (EVLP). Methods HCMV seropositive human lungs were placed on EVLP alone or EVLP+1mg/L of F49A-FTP for 6 hours (n=6 , each). CD14+ monocytes isolated from biopsies pre and post EVLP underwent HCMV reactivation assay designed to evaluate viral reactivation capacity. Off-target effects of F49A-FTP were studied evaluating cell death markers of CD34+ and CD14+ cells using flow cytometry. Lung function on EVLP and inflammatory cytokine production were evaluated as safety endpoints. Results We demonstrate that lungs treated ex-vivo with F49A-FTP had a significant reduction in HCMV reactivation compared to controls, suggesting successful targeting of latent virus (76% median reduction in F49A-FTP vs. 15% increase in controls, p=0.0087). Furthermore, there was comparable cell death rates of the targeted cells between both groups, suggesting no off-target effects. Ex-vivo lung function was stable over 6 hours and no differences in key inflammatory cytokines were observed demonstrating safety of this novel treatment. Conclusions Ex-vivo F49A-FTP treatment of human lungs targets and kills latent HCMV, markedly attenuating HCMV reactivation. This approach demonstrates the first experiments targeting latent HCMV in a donor organ with promising results towards clinical translation.

A large proportion of controlled donation after circulatory death (cDCD) donor lungs are declined because cardiac arrest does not occur within a suitable time after the withdrawal of life-sustaining therapy. Improved strategies to preserve lungs after asystole may allow the recovery team to arrive after death actually occurs and enable the recovery of lungs from more cDCD donors. The aim of this study was to determine the effect of donor positioning on the quality of lung preservation after cardiac arrest in a cDCD model. Cardiac arrest was induced by withdrawal of ventilation under anesthesia in pigs. After asystole, animals were divided into 2 groups based on body positioning (supine or prone). All animals were subjected to 3 hours of warm ischemia. After the observation period, donor lungs were explanted and preserved at 4°C for 6 hours, followed by 6 hours of physiologic and biological lung assessment under normothermic ex vivo lung perfusion. Donor lungs from the prone group displayed significantly greater quality as reflected by better function during ex vivo lung perfusion, less edema formation, less cell death, and decreased inflammation compared with the supine group. A simple maneuver of donor prone positioning after cardiac arrest significantly improves lung graft preservation and function.

Purpose of review Within the last decade, ex-vivo lung perfusion (EVLP) has become a widespread technology used for organ assessment and reconditioning within clinical transplantation. This review aims to offer insights toward future applications and developments in regards to its utility. Recent findings The intervention of EVLP is a well-tolerated method to effectively allow for extended preservation periods. The thoughtful usage of EVLP can therefore be used to optimize operating room logistics and progress lung transplantation toward becoming a more elective procedure. EVLP has also demonstrated itself as an excellent platform for targeted therapies. Prolonged perfusion achieved through further platform stability will allow for time-dependent molecular therapies. Lastly, EVLP allows for the opportunity to perform advanced diagnostics within an isolated setting. Sophistication of point-of-care technologies will allow for accurate predictive measures of transplant outcomes within the platform. Summary The future of EVLP involves usage of the system as a preservation modality, utilizing advanced diagnostics to predict transplant outcome, and performing therapeutic interventions to optimize organ quality. The generation of clinical data to facilitate and validate these approaches should be performed by transplant centers, which have acquired significant experience using EVLP within their clinical activity.

Availability of organs is a limiting factor for lung transplantation, leading to substantial mortality rates on the wait list. Use of organs from donors with transmissible viral infections, such as hepatitis C virus (HCV), would increase organ donation, but these organs are generally not offered for transplantation due to a high risk of transmission. Here, we develop a method for treatment of HCV-infected human donor lungs that prevents HCV transmission. Physical viral clearance in combination with germicidal light-based therapies during normothermic ex-vivo Lung Perfusion (EVLP), a method for assessment and treatment of injured donor lungs, inactivates HCV virus in a short period of time. Such treatment is shown to be safe using a large animal EVLP-to-lung transplantation model. This strategy of treating viral infection in a donor organ during preservation could significantly increase the availability of organs for transplantation and encourages further clinical development., Organs from donors with transmissible viral infections, such as hepatitis C virus (HCV), are not offered for transplantation due to a high risk of transmission. Here, Galasso et al. develop a method for treatment of HCV-infected human donor lungs that is safe and prevents HCV transmission in the pig model.

Background Despite numerous reports demonstrating the efficacy of exogenous surfactant therapy during lung transplantation, this strategy remains absent in routine clinical use. Here, we systematically review and meta-analyze the effect of exogenous surfactant on respiratory pathophysiological variables during lung transplantation. Methods To identify relevant clinical and pre-clinical studies, we performed an electronic search of MEDLINE, EMBASE, and Cochrane CENTRAL from inception to June 11, 2021. In addition, research-in-progress databases were searched. Randomized and non-randomized adult and pediatric clinical studies and animal experiments that compared the use of surfactant for lung transplantation with a control group were included. The primary outcome was the ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2 ratio). Results From 1,041 citations, we identified 35 studies, of which 6 were clinical studies and 29 were pre-clinical. Thirty-two studies were included in the quantitative analysis. The administration of surfactant therapy during clinical lung transplantation significantly improved PaO2/FiO2 ratio in recipients (mean difference [MD] 93 mmHg, 95% confidence interval [CI] 25–160 mmHg, p Conclusions Exogenous surfactant therapy improves post-transplant lung function; however, its effects on clinical outcomes remain uncertain. High-quality randomized controlled trials are needed to determine whether the physiologic benefits of surfactant therapy affect patient-important outcomes in lung transplant recipients.

Background: Effective treatment of respiratory infections continues to be a major challenge. In high doses (³160 ppm), inhaled Nitric Oxide (iNO) has been shown to act as a broad-spectrum antimicrobial agent in preliminary studies. However, the safety of prolonged in vivo implementation of high-dose iNO therapy has not been studied. Herein we aim to explore the feasibility and safety of delivering continuous high-dose iNO over an extended period of time using an in vivo animal model. Methods: Yorkshire pigs were randomized to one of the following two groups: group 1, standard ventilation; and group 2, standard ventilation + continuous iNO 160 ppm + methylene blue (MB) as intravenous bolus, whenever required, to maintain metHb

Introduction High-dose nitric oxide (NO) has been shown effective against a variety of micro-organisms in vitro, including common bacteria found in donor organs. However, clinical obstacles related to its implementation in vivo are the formation of methemoglobin and the accumulation of toxic nitrogen compounds. Ex vivo lung perfusion (EVLP) is a platform that allows for organ maintenance with an acellular perfusion solution, thus overcoming these limitations. The present study explores the safety of continuous high-dose inhaled (iNO) during EVLP for an extended period of 12 hours. Methods Lungs procured from Yorkshire pigs were randomized into control (standard ventilation) and treatment (standard ventilation + 200 ppm iNO) groups, then perfused with an acellular solution for 12 hours (n = 4/group). Lung physiology and biological markers were evaluated. Results After 12 hours of either standard EVLP or EVLP + 200 ppm iNO, we did not notice any significant physiologic difference between the groups: pulmonary oxygenation (P = .586), peak airway pressures (P = .998), and dynamic (P = .997) and static (P = .908) lung compliances. In addition, no significant differences were seen among proinflammatory cytokines measured in perfusate and lung tissue. Importantly, most common toxic compounds were kept at safe levels throughout the treatment course. Conclusions High-dose inhaled NO delivered continuously over 12 hours appears to be safe without inducing any significant pulmonary inflammation or deterioration in lung function. These findings support further efficacy studies to explore the use of iNO for the treatment of infections in donor lungs during EVLP.

KEY POINTS Demand for organs continues to exceed their availability, both in Canada and around the world.[1][1] Optimizing the medical management of potential organ donors is an important strategy for enhancing organ supply. The explicit goals for management of deceased organ donors in the intensive

The application of ex vivo lung perfusion (EVLP) has significantly increased the successful clinical use of marginal donor lungs. While large animal EVLP models exist to test new strategies to improve organ repair, there is currently no rat EVLP model capable of maintaining long-term lung viability. Here, we describe a new rat EVLP model that addresses this need, while enabling the study of lung injury due to cold ischemic time (CIT). The technique involves perfusing and ventilating male Lewis rat donor lungs for 4 h before transplanting the left lung into a recipient rat and then evaluating lung function 2 h after reperfusion. To test injury within this model, lungs were divided into groups and exposed to different CITs (i.e., 20 min, 6 h, 12 h, 18 h and 24 h). Experiments involving the 24-h-CIT group were prematurely terminated due to the development of severe edema. For the other groups, no differences in the ratio of arterial oxygen partial pressure to fractional inspired oxygen ([Formula: see text]/[Formula: see text]) were observed during EVLP; however, lung compliance decreased over time in the 18-h group ( P = 0.012) and the [Formula: see text]/[Formula: see text] of the blood from the left pulmonary vein 2 h after transplantation was lower compared with 20-min-CIT group ( P = 0.0062). This new model maintained stable lung function during 4-h EVLP and after transplantation when exposed to up to 12 h of CIT.

There is a shortage of lungs available to meet the needs of patient who require lung transplantation. Ex vivo lung perfusion (EVLP) has allowed for the assessment and reconditioning of extended criteria and high-risk donor lungs, allowing for an increased volume of lung transplants. Recently, an invested interest in the utilization donation after cardiac death (DCD) donors has surfaced, in which EVLP has played an important role. Clinical studies have shown that EVLP can recondition donor lungs that initially would be unacceptable for transplantation. In a recent study, the intervention of EVLP was also shown to extend the window of total organ preservation time. Preclinical studies have shown promising results for EVLP to serve as a therapeutic platform to modulate inflammation, reduce edema, and treat infections and aspiration injuries. In addition, studies have shown the potential to recondition lungs of donation after cardiac death donors which suffer from additional injury in comparison to donation after brain death donors. EVLP is dynamic platform that can serve to treat and array of issues prior to transplantation. Indeed, the investigation of these therapeutic arsenals will soon allow physicians to expand the donor organ pool and improve transplantation outcomes.

Purpose This study explores feasibility and safety of using enzymes (FpGalNAc deacetylase & FpGalNase) to remove ABO-A antigens from donor lungs during ex vivo lung perfusion (EVLP). The enzymes have been reported to efficiently convert ABO-A blood to O (Withers, Nat Microbiol, 2019). Methods Human lungs (ABO-A1, n=5), declined for transplant, were treated with the enzymes during EVLP. Tissues were sampled before and after. The distribution and expression level of ABO antigens were analyzed by immunohistochemistry and flow cytometry. Lung function was monitored for side-effects. Immediate post-transplant immune response was simulated in an ex vivo model of hyperacute lung rejection, with introduction of ABO-O plasma (carrying ABO antibodies) as the surrogate for the recipient circulation (Fig.1H, n=1). The physiology and histology of lungs were analyzed for anti-A-induced immune responses. Results The histo-blood type A (BTA) antigens in the A1 lungs are observed primarily on endothelial and epithelial cells. BTA were cleared remarkably well from treated lungs (Fig.1A). The enzymes convert vascular BTA to the blood group H (BTH) antigens found in ABO-O tissue (Fig.1B). Over 97% of endothelial BTA antigens were removed within the typical 4h clinical EVLP timeframe using very low dose (1 µg/mL) enzymes (Fig.1C). No acute side-effects were seen (Fig.1D-G). The rejection model showed that the 3-hour treatment of human lung with low dose enzymes prevented the damage triggered by type O plasma (presumably due to anti-A antibodies) as observed in the physiology and histology of the control lung (Fig.1I-J). Conclusion Ex vivo enzymatic treatment can efficiently remove ABO-A antigen in donor lungs without acute side-effects. Preliminary results show that the treatment can prevent ABO mismatch-induced early damages. The treatment can potentially allow expansion of ABO-incompatible lung transplantation, leading to significant improvements in logistics and fairness of organ allocation.

Effective treatment of respiratory infections continues to be a major challenge. In high doses (≥160 ppm), inhaled Nitric Oxide (iNO) has been shown to act as a broad-spectrum antimicrobial agent, including its efficacy in vitro for coronavirus family. However, the safety of prolonged in vivo implementation of high-dose iNO therapy has not been studied. Herein we aim to explore the feasibility and safety of delivering continuous high-dose iNO over an extended period of time using an in vivo animal model. Yorkshire pigs were randomized to one of the following two groups: group 1, standard ventilation; and group 2, standard ventilation + continuous iNO 160 ppm + methylene blue (MB) as intravenous bolus, whenever required, to maintain metHb 2 remained

Purpose We explored the feasibility and safety of using recently developed enzymes (FpGalNAc deacetylase and FpGalNase) to remove histo-blood type antigens from donor lungs during ex vivo lung perfusion (EVLP). The enzymes have been reported to efficiently convert Type A whole blood to O (Withers, Nature Microbiology, 2019). Methods Human type A red blood cells (RBCs, n=5/group) and arteries (aorta and pulmonary arteries, n=3/group) were treated with enzyme-containing EVLP solution and lung preservation solution. Human donor lungs (type A) declined for transplantation were treated with the enzymes (1µg/mL) during normothermic EVLP for 1 and 3 h. Lung physiological parameters were monitored throughout treatment. Cell and tissue samples were taken before and after treatment. The distribution and expression levels of blood type antigens were analyzed by flow cytometry and immunohistochemistry. Results The enzymes achieved over 99% antigen removal from RBC within 1h at a dose of 4 µg/mL (Fig1A) in EVLP solution and lung preservation solution. The histo-blood type antigens in human arteries were observed primarily on endothelial cells - blood type A (BTA) antigens co-localized with endothelial marker CD31 (Fig1B, Pre-treatment). In vitro static treatment of arteries demonstrated 92±1% of BTA antigen removal with 1 µg/mL enzyme (Fig1B). Blood type antigen in human lungs were observed on both endothelial and epithelial cells. Within 1h of perfusion, human lungs treated with enzyme had remarkable clearance of intravascular BTA antigens (Fig1C). Similar results were observed after 3h treatment. No acute physiological side effects were observed from the enzymatic treatment. Conclusion Ex vivo enzymatic treatment can efficiently remove blood type antigen in donor lungs without acute side effects. This treatment has potential to be developed as an adjuvant therapy in ABO-incompatible organ transplantation, minimizing the need for recipient antibody removal procedures or augmented immunosuppression.

Purpose The majority of potential donor lungs are not being used. The primary reason for underutilization is the concern for primary graft dysfunction (PGD). There is currently no clinically available therapy for PGD. Alpha 1 antitrypsin (A1AT) is a serine protease inhibitor with anti-inflammatory and cytoprotective properties. We and others have shown benefits of A1AT in small and large animal lung transplant studies. Before performing clinical trials, evidence of therapeutic efficacy in human lungs would be valuable. We tested the effect of A1AT given during ex vivo lung perfusion (EVLP) to human lungs rejected for transplantation. Methods Double lung blocks rejected for transplantation (n=8) were divided and placed on separate EVLP circuits for 12h. Lungs were randomly assigned to receive A1AT or placebo, with the contralateral lung serving as control for the treated lung. Outcome measures included: hourly physiologic lung function, perfusate loss, wet-dry weight ratio, inflammatory mediators, endothelin-1 (ET-1), and zonula occludens tight junction protein‐1 (ZO‐1, immunofluorescence staining). Results The A1AT-treated group demonstrated significantly better lung function: higher pO2 and compliance (Fig A), lower pulmonary artery pressure (-0.4 mmHg p= 0.04) and vascular resistance (-30.4 dynes·s cm−5 p= 0.01). Perfusate loss, a surrogate for lung permeability, was lower in the A1AT group, as was the wet-dry ratio (Fig B). A1AT also decreased ET-1 levels in perfusate (Fig C) and increased ZO-1 expression on endothelial cells (Fig D). Conclusion In this study we demonstrated that human A1AT was able to improve the quality of severly injuried human lungs, likely through endothelial cell protection. The encouraging results justify a clinical trial, in order to improve donor lung quality and clinical outcomes in lung transplantation. Our study also illustrates that testing selected drugs on injured human lungs on EVLP is a viable strategy prior to clinical application.

Purpose Ex-vivo lung perfusion (EVLP) maintains marginal donor lungs at body temperature with ventilation and circulating perfusate, allowing for functional assessment prior to transplantation. Prolonged EVLP would allow for advanced time-dependent therapies for donor lung repair and reconditioning. We hypothesized that the addition of a dialysis machine to the EVLP circuit would maintain homeostasis of the donor lung and prolong EVLP duration. Methods Porcine donor lungs (n=3) were extracted and placed on the EVLP platform for 36 hours or until termination criteria (dynamic compliance Results Dialysis successfully prevented an increase in electrolyte levels (Fig 1C) and maintained glucose and lactate levels at baseline (1C). EVLP was prolonged in the dialysis group with a mean duration of EVLP reaching 32±6.93 h in the E+D group compared to 18.67±3.27 h in the historical control group (Fig 1D). Percent lung survival at 24h of perfusion was 100% in the E+D group, while only 20% was seen in historical controls (Fig 1B). Both lungs which survived to 36h of EVLP presented excellent lung function based on assessment by EVLP. Conclusion Dialysis may preserve lung function and length of EVLP by maintaining homeostasis of the lung. Following the results of this encouraging pilot study, a formal blinded experiment is being performed and is underway.

Purpose Post-transplant CMV reactivation from latently-infected lung allografts is extremely prevalent and results in inferior long-term outcomes. CMV latently infected cells ubiquitously express a viral surface protein, US28. We aimed to evaluate the therapeutic effects of a novel immunotoxin (F49A-FTP) that targets US28 in human lungs during EVLP, with the ultimate goal of minimizing or preventing CMV transmission and reactivation post-transplantation. Methods Human lungs rejected for transplantation were randomly placed on EVLP alone or EVLP+1mg/L of F49A-FTP (n=6 each) for 6 hours. Biopsies were collected pre and post EVLP and subjected to an in vitro assay designed to evaluate viral reactivation capacity (Fig 1A). Based on US28’s homology to the human CX3CR1 chemokine receptor, potential off-target effects of the toxin were studied evaluating cell death markers of CD34+ and CD14+ using flow cytometry, and the ratio of cells post:pre perfusion were calculated. Lung function on EVLP and inflammatory cytokine production were evaluated as safety endpoints. Results Delivery of F49A-FTP during EVLP showed no acute toxic events demonstrated by no significant differences in lung physiology and levels of IL-8, IL1-b and IFN-γ between the groups. Furthermore, while control lung tissue demonstrated a median increase from baseline of 32% (Range -16% to +112.5%) in viral reactivation, treated lungs demonstrated a significant median reduction in CMV reactivation of 76% (Range -15% to -99.9%) (Fig 1B, p=0.0087). No off-target effects were noted upon F49A-FTP delivery, demonstrated by similar amounts of cell death between the groups (Fig 1C). Conclusion We demonstrate that ex vivo F49A-FTP treatment of human lungs on EVLP markedly attenuates CMV reactivation and could potentially be used to treat donor organs prior to transplant. Ongoing in vivo reactivation experiments using the EVLP treated samples in a humanized mouse will further validate these findings.

Purpose Ex-vivo lung perfusion (EVLP) maintains marginal donor lungs at body temperature with ventilation and circulating perfusate, allowing for functional assessment prior to transplantation. Prolonged EVLP would allow for advanced time-dependent therapies for donor lung repair and reconditioning. We hypothesized that the addition of a dialysis machine to the EVLP circuit (dEVLP) would maintain homeostasis of the donor lung and prolong EVLP duration. Methods Porcine donor lungs (n=6) were extracted and placed on the EVLP platform for 36 hours or until termination criteria (dynamic compliance Results EVLP was prolonged in the dialysis group with a mean duration of EVLP reaching 30.83±0.57 h in the dEVLP group compared to 19.6±2.61 h in the historical control group (Fig 1D). Percent lung survival at 24h of perfusion was 100% in the dEVLP group, while only 20% was seen in historical controls (Fig 1B). Dialysis successfully prevented an increase in electrolyte levels (Fig 1C), maintained glucose and lactate levels at baseline (1C), and maintained physiological parameters (Fig 1D). Both lungs which survived to 36h of EVLP presented excellent lung function based on assessment by EVLP. Conclusion Dialysis may preserve lung function and length of EVLP by maintaining homeostasis of the lung. Following the results of this encouraging proof of concept study, a formal blinded experiment is underway.

Purpose Improved real-time assessment of donor lung quality during Ex Vivo Lung Perfusion (EVLP) could enhance safe utilization of lungs for transplantation. Near-infrared (NIR) fluorescent imaging with indocyanine green (ICG) has been used in various clinical intraoperative applications to evaluate tissue perfusion. We investigated the feasibility of NIR imaging during EVLP and aimed to detect and quantify lung perfusion and permeability. Methods Pig lungs with extended 24-hour cold ischemia (n=4) and human lungs rejected from clinical transplantation (n=2) were assessed with NIR imaging during EVLP. Serial NIR imaging was performed with SPY Elite imaging system after ICG (0.3, 0.6 or 1.0 mg) administration through pulmonary artery (Fig A). Tissue perfusion was quantified using SPYQ analysis software, and tissue ICG accumulation was correlated with lung damage. Results Strong NIR signal was detected immediately after ICG administration with 0.6 mg (pig lungs) or 1.0 mg (human lungs) doses resulting in optimal fluorescence for tissue perfusion evaluation. In contrast to the transient kinetics and fast liver metabolism during clinical in vivo ICG administration, a single ICG dose during EVLP resulted in stable NIR signal for over 10 hours. Perfusion quantification of pig lungs with extended cold preservation revealed peripheral areas with compromised tissue perfusion that gradually recovered over time with recruitment of additional pulmonary vascular beds (Fig B). ICG accumulation was detected in sites with damage and increased vascular permeability in pig and human lungs (Fig C and D). Conclusion NIR imaging with a clinically-approved intravascularly-delivered ICG fluorescent probe can dynamically quantify tissue perfusion and detect damage-induced vascular permeability. This novel and promising imaging modality could be used during EVLP to assess lung quality, or to determine effects of therapeutics that target tissue perfusion and vascular permeability.

Purpose Donor to recipient CMV mismatch leads to high incidence of CMV infection post lung transplant leading to inferior long-term patient outcomes. CMV latently infected cells ubiquitously express a surface marker, US28. We aimed to evaluate the therapeutic effects of a novel fusion toxin protein (F49A-FTP) that targets US28 in human lungs during EVLP, with the ultimate goal of minimizing or preventing CMV reactivation post transplantation. Methods Human lungs rejected for transplantation were randomly placed on EVLP alone or EVLP+1mg/L of F49A-FTP (n=6 each) for 6 hours. Biopsies were collected to evaluate viral reactivation using an in vitro assay that induces latent CMV to reactivate to an infectious phase. Since US28 has 38% homology to the human CX3CR1 chemokine receptor, potential off-target effects of the toxin were studied using flow cytometry for cell death assessment of CD34+ and CD14+ cells. Lung function on EVLP was evaluated as a safety endpoint. Results F49A-FTP was delivered through the PA on EVLP with no acute toxic events based on physiological parameters (Figure 1A). We performed an in vitro assay using tissue samples obtained after EVLP to stimulate CMV to reactivate in order to assess efficacy of the drug. After primary culture from lung tissue samples, relative reactivation events (assessed with IFF staining for IE protein of lytic CMV, Figure 1B) occurred in 100% of controls vs. only 0.04±0.02% of treated lungs. For off-target analysis, we performed flow cytometry and calculated the ratio of post:pre perfusion CD34+ and CD14+ cells (Figure 1C). There were no changes in number of cells for both groups: CD34+ ratio 0.8±0.5 vs. 0.6±0.5 (control and F49A-FTP, respectively, p=0.4974); CD14+ ratio 1.54±0.74 vs. 1.66±1.13 (control and F49A-FTP, respectively, p=0.8660). Conclusion Our study demonstrates that ex vivo F49A-FTP treatment of human lungs on EVLP markedly attenuates CMV reactivation. Ongoing ex vivo and in vivo reactivation experiments will confirm these findings.

Purpose Ex vivo lung perfusion (EVLP) enables novel therapeutic approaches to repair damaged lungs for transplantation. We investigated the concept and feasibility of elective cell therapy for donor lungs. Dosing, efficacy and transgene expression kinetics of engineered mesenchymal stromal cell therapy were examined in pig EVLP and transplantation. Methods Human umbilical cord perivascular mesenchymal stromal/stem cells were transduced with an adenoviral vector encoding human IL-10 (MSC-hIL-10 cells), cryopreserved and recovered 1h before use. Pig lungs with 4 to 24-hour cold ischemia (n=5) were connected to EVLP for 6 (n=3) or 12 hours (n=2) and MSC-hIL-10 cells (40-150 × 106) were administered through the pulmonary artery after 1h of EVLP. Left single lung transplantation was performed after EVLP and the grafts were monitored up to 4h (n=4) and 3d (n=1) after reperfusion. Human IL-10 levels were determined by ELISA and fluorescence in situ hybridization with human-specific ALU probes was used to track MSC-hIL-10 cells. Results Supra-therapeutic levels of hIL-10 were present in EVLP perfusate within minutes after MSC-hIL10 cell administration and the levels consistently increased during EVLP (Figure 1A). Recipient plasma hIL-10 levels were elevated 1h after lung transplantation, remained high for 4h and gradually decreased to low levels by 3d after transplantation (Figure 1C). Elevated tissue and bronchoalveolar lavage hIL-10 levels were observed in the transplanted lung and remained low in the recipient native lung. MSC-hIL-10 cells were detected in the donor lung during EVLP and after transplantation (Figure 1B). Conclusion Engineered mesenchymal stromal cell therapy during EVLP is a novel and practical “off-the-shelf” strategy to achieve rapid transient therapeutic levels of soluble cytokines such as IL-10 for pre-transplant lung repair and protection. Ongoing studies will determine the therapeutic effect and longer-term fate of MSC-hIL-10 cells delivered during EVLP.

Purpose Extracorporeal membrane oxygenation, veno-venous configuration (VV-ECMO) is an effective intervention to improve gas exchange in patients with severe respiratory failure, refractory to conventional treatments, or as a bridge to lung transplantation. However, optimal mechanical ventilation (MV) strategies during VV-ECMO to minimize ventilator-induced lung injury are undetermined. While there is general consensus in minimizing tidal ventilation, the potential benefit of high positive end-expiratory pressure (PEEP) strategies in optimizing cardiopulmonary interaction and reducing the severity of lung injury remains unclear.We hypothesize that PEEP-induced alveolar recruitment will reduce lung injury during VV-ECMO for ARDS, in comparison to atelectatic lungs. Methods Yorkshire pigs weresedated, paralyzed, mechanically ventilated,and cannulated for VV-ECMO support. Lung injury was done by two serial bronchoscopic instillations of gastric juice to achieve a P/F ratio Results During VV-ECMO, gas exchange and hemodynamic parameters remained stable and comparable in the two groups, despite the very low tidal volume delivered (HP 54 ml ±; LP 41 ml ±11). Lung volume was higher in the HP compared with the LP group, and remained unchanged during the 5 hours of VV-ECMO support. At the end of the 5 hours of ECMO support, in the dependent lung areas the W/D was significantly higher in the HP compared to LP group (HP 11.1, 95% CI: 10.5-11.7; LP 9.2, 95% CI: 8.2-10.3); in the non-dependent areas the W/D was significantly higher in the LP compared to HP group (LP 6.3, 95% CI: 5.6-6.9; HP 5.3, 95% CI: 4.9-5.8). Conclusion VV-ECMO for severe acute respiratory failure MV with very low tidal volume and LP reduces regional lung edema in the dependent areas, while HP reduces edema.More studies are required to characterize the mechanisms of these findings.