Normothermic Ex Vivo Lung Perfusion Research Articles

Subnormothermic ex vivo lung perfusion possibly protects against ischemia-reperfusion injury via the mTORC-HIF-1α pathway.

Ex vivo lung perfusion (EVLP) is a useful technique for evaluating and repairing donor lungs for transplantation. However, studies examining the effects of perfusate temperature on graft function are limited. Thus, this study aimed to examine these effects during EVLP on ischemic-reperfusion injury in the donor lung. Twenty-four male Sprague-Dawley rats were randomly divided into three groups, as follows: no treatment (sham group, n=5), normothermic EVLP (37 °C, n=5), and subnormothermic EVLP (30 °C, n=5). Lung function analyses, including oxygen capacity (OC), compliance, and pulmonary vascular resistance (PVR), were performed hourly during EVLP. Further, after 4 h of EVLP, histological evaluation of the right lobe was performed using the lung injury severity (LIS) scale. The expression levels of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-18 were evaluated. Metabolomic analysis of left lung tissues was conducted using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) after 4 h of EVLP in the EVLP groups and after 1 h of cold preservation in the sham group. Compared with those in the normothermic group, in the subnormothermic group, functional parameters during EVLP and subsequent histologic results were significantly superior, expression levels of inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-18 were significantly lower, and glycolytic activity was significantly decreased. Furthermore, expression levels of mammalian target of rapamycin complex (mTORC), hypoxia-inducible factor (HIF) 1α, and nucleotide-binding domain, leucine-rich-containing family pyrin domain containing 3 (NLRP3) and its effector caspase-1 were significantly lower in the subnormothermic group than in the normothermic group. EVLP with subnormothermic perfusion improves lung graft function by reducing the expression of pro-inflammatory cytokines and glycolytic activity during EVLP. Additionally, EVLP can be a useful target for the improvement of graft function after transplantation.

Novel Polymerized Human Serum Albumin For Ex Vivo Lung Perfusion.

Ex vivo lung perfusion (EVLP) is a method of organ preservation to expand the donor pool by allowing organ assessment and repair. Perfusion solution composition is crucial to maintaining and improving organ function during EVLP. EVLP compared perfusates supplemented with either polymeric human serum albumin (PolyHSA) or standard human serum albumin (HSA). Rat heart-lung blocks underwent normothermic EVLP (37°C) for 120 minutes using perfusate with 4% HSA or 4% PolyHSA synthesized at a 50:1 or 60:1 molar ratio of glutaraldehyde to PolyHSA. Oxygen delivery, lung compliance, pulmonary vascular resistance (PVR), wet-to-dry ratio, and lung weight were measured. Perfusion solution type (HSA or PolyHSA) significantly impacted end-organ metrics. Oxygen delivery, lung compliance, and PVR were comparable among groups (P > 0.05). Wet-to-dry ratio increased in the HSA group compared to the PolyHSA groups (both P < 0.05) suggesting edema formation. Wet-to-dry ratio was most favorable in the 60:1 PolyHSA-treated lungs compared to HSA (P < 0.05). Compared to using HSA, PolyHSA significantly lessened lung edema. Our data confirm that the physical properties of perfusate plasma substitutes significantly impact oncotic pressure and the development of tissue injury and edema. Our findings demonstrate the importance of perfusion solutions and PolyHSA is an excellent candidate macromolecule to limit pulmonary edema.

Endothelial protection in lung grafts through heparanase inhibition during ex vivo lung perfusion in rats

We hypothesized that enhancing glycocalyx preservation would reduce endothelial damage in lung grafts during ex-vivo lung perfusion (EVLP) leading to better transplant outcomes. In this study, we characterized the effects of inhibiting heparanase (HPSE), an enzyme responsible for glycocalyx shedding, on lung quality during EVLP. Human clinical EVLP perfusate from lung graft patients was utilized to identify a potential association between glycocalyx integrity in grafted lung tissue and clinical data. In addition, we performed pre-clinical studies in which rat lungs underwent normothermic EVLP for 4 hours with/without HPSE inhibitors, heparin (1,000-U/h) or heparastatin (SF4; 1-μM), added to the perfusate. After 4-hours EVLP, left lungs were transplanted into syngeneic rats then evaluated for graft quality 2-hours after reperfusion. Clinically, increased degradation of syndecan-1 was identified in dysfunctional grafts during EVLP. Levels of heparan sulfate in perfusate after EVLP were associated with incidence of graft dysfunction after transplantation. In the pre-clinical rat study, SF4 effectively inhibited HPSE activity, and significantly attenuated dissociated glycocalyx levels, endothelial dysfunction, edema, and inflammation in lungs during EVLP compared to both controls and heparin groups. High-doses of heparin demonstrated markedly increased perfusate syndecan-1 concentrations and deteriorated lung quality during EVLP compared with controls. Post-transplant graft function and inflammation were significantly improved in SF4-treated group compared to those in both control and heparin-treated groups. This study demonstrated that HPSE activity inhibition by SF4 can improve graft preservation during EVLP by protecting the glycocalyx and endothelial function, leading to better lung function following transplantation.

Transient hyperthermia during ex vivo lung perfusion has no protective effect in rat model

BackgroundAlthough ex vivo lung perfusion (EVLP) is a useful technique for evaluating and repairing donor lungs for transplantation, EVLP itself can lead to inflammation in the lung. Heat shock proteins (HSPs) have anti-inflammatory effects and can reduce ischemic reperfusion injury in the donor's lungs after transplantation. In this study, the effects of transient hyperthermia during EVLP on the expression of HSPs and inflammatory pathways were examined. MethodsFifteen male Sprague–Dawley rats were randomly divided into three groups: sham (n = 5), normothermic EVLP (37 °C, n = 5), and transient hyperthermia during EVLP (42 °C, n = 5). Lung function analyses regarding PaO2/FiO2 ratio, compliance, and pulmonary vascular resistance were conducted. The expression levels of HSPs and inflammatory cytokines were also evaluated. The degree of lung injury was histopathologically evaluated. Transcriptome analysis was performed on lung tissues from the sham (n = 2), normothermic EVLP (n = 2), and heat stress-EVLP (n = 2) groups. ResultsThere were no significant differences in functional or histological parameters between the three groups. The expression of HSPs had significantly increased, especially that of HSPs 40 and 60 in the heat stress EVLP group; this was consistent with the inflammatory response. Inflammatory cytokine levels were significantly higher during EVLP and intensified with transient hyperthermia. ConclusionTransient hyperthermia during EVLP has no protective effect on the donor lung graft or activation of the inflammatory pathway at the gene level.

Metabolomic fingerprinting of porcine lung tissue during pre-clinical prolonged ex vivo lung perfusion using in vivo SPME coupled with LC-HRMS

Normothermic ex vivo lung perfusion (NEVLP) has emerged as a modernized organ preservation technique that allows for detailed assessment of donor lung function prior to transplantation. The main goal of this study was to identify potential biomarkers of lung function and/or injury during a prolonged (19 h) NEVLP procedure using in vivo solid-phase microextraction (SPME) technology followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The use of minimally invasive in vivo SPME fibers for repeated sampling of biological tissue permits the monitoring and evaluation of biochemical changes and alterations in the metabolomic profile of the lung. These in vivo SPME fibers were directly introduced into the lung and were also used to extract metabolites (on-site SPME) from fresh perfusate samples collected alongside lung samplings. A subsequent goal of the study was to assess the feasibility of SPME as an in vivo method in metabolomics studies, in comparison to the traditional in-lab metabolomics workflow. Several upregulated biochemical pathways involved in pro- and anti-inflammatory responses, as well as lipid metabolism, were observed during extended lung perfusion, especially between the 11th and 12th hours of the procedure, in both lung and perfusate samples. However, several unstable and/or short-lived metabolites, such as neuroprostanes, have been extracted from lung tissue in vivo using SPME fibers. On-site monitoring of the metabolomic profiles of both lung tissues through in vivo SPME and perfusate samples on site throughout the prolonged NEVLP procedure can be effectively performed using in vivo SPME technology.

Continuous Endogenous Exhaled CO Monitoring by Laser Spectrometer in Human EVLP Before Lung Transplantation.

Endogenous production of carbon monoxide (CO) is affected by inflammatory phenomena and ischemia-reperfusion injury. Precise measurement of exhaled endogenous CO (eCO) is possible thanks to a laser spectrometer (ProCeas® from AP2E company). We assessed eCO levels of human lung grafts during the normothermic Ex-Vivo Lung Perfusion (EVLP). ProCeas® was connected in bypass to the ventilation circuit. The surgical team took the decision to transplant the lungs without knowing eCO values. We compared eCO between accepted and rejected grafts. EVLP parameters and recipient outcomes were also compared with eCO values. Over 7 months, eCO was analyzed in 21 consecutive EVLP grafts. Two pairs of lungs were rejected by the surgical team. In these two cases, there was a tendency for higher eCO values (0.358 ± 0.52 ppm) compared to transplanted lungs (0.240 ± 0.76 ppm). During the EVLP procedure, eCO was correlated with glucose consumption and lactate production. However, there was no association of eCO neither with edema formation nor with the PO2/FiO2 ratio per EVLP. Regarding post-operative data, every patient transplanted with grafts exhaling high eCO levels (>0.235 ppm) during EVLP presented a Primary Graft Dysfunction score of 3 within the 72 h post-transplantation. There was also a tendency for a longer stay in ICU for recipients with grafts exhaling high eCO levels during EVLP. eCO can be continuously monitored during EVLP. It could serve as an additional and early marker in the evaluation of the lung grafts providing relevant information for post-operative resuscitation care.

Normothermic &lt;i&gt;ex vivo&lt;/i&gt; perfusion of isolated lungs in an experiment using a russian-made perfusion system

According to global health statistics, respiratory diseases, together with infectious complications and hereditary lung diseases, rank as the third leading cause of death. Today, lung transplantation (LTx) is a well-recognized modality of treatment for end-stage chronic lung disease. However, the number of LTx surgeries performed is much lower than other solid organs. This is due to the high requirements for the potential donor and characteristics of the lung graft, reflecting the efficiency of gas exchange function. Non-compliance with the selection criteria leads to deselection of donors, which, according to various estimates, occurs in 80–85% of cases. One of the ways to increase the number of lung transplant surgeries is to restore them to the level of optimal gas exchange parameters, which can be achieved and objectively assessed during normothermic ex vivo lung perfusion (EVLP). EVLP is becoming increasingly common at leading transplantation centers in Europe and North America. This has significantly increased the number of transplant surgeries as a result of using lungs procured from suboptimal donors and rehabilitated via EVLP. In our pilot study, the developed Russian-made mechanical circulatory support system showed that performing normothermic EVLP for isolated lungs under experimental conditions is feasible. Basic and optimized perfusion protocols have fully shown that they are reliable and efficient.

Electrical Impedance as a Noninvasive Metric of Quality in Allografts Undergoing Normothermic Ex Vivo Lung Perfusion.

Ex vivo lung perfusion (EVLP) increases the pool of suitable organs for transplant by facilitating assessment and repair at normothermia, thereby improving identification of quality of marginal organs. However, there exists no current objective approach for assessing total organ edema. We sought to evaluate the use of electrical impedance as a metric to assess total organ edema in lungs undergoing EVLP. Adult porcine lungs (40 kg) underwent normothermic EVLP for 4 hours. To induce varying degrees of lung injury, the allografts were perfused with either Steen, a modified cell culture media, or 0.9% normal saline. Physiologic parameters (peak airway pressure and compliance), pulmonary artery and left atrial blood gases, and extravascular lung water measurements were evaluated over time. Wet-to-dry ratios were evaluated postperfusion. Modified Murray scoring was used to calculate lung injury. Impedance values were associated with lung injury scores ( p = 0.007). Peak airway pressure ( p = 0.01) and PaO 2 /FiO 2 ratios ( p = 0.005) were both significantly associated with reduced impedance. Compliance was not associated with impedance ( p = 0.07). Wet/dry ratios were significantly associated with impedance and Murray Scoring within perfusion groups of Steen, Saline, and Modified Cell Culture ( p = 0.0186, 0.0142, 0.0002, respectively). Electrical impedance offers a noninvasive modality for measuring lung quality as assessed by tissue edema in a porcine model of normothermic EVLP. Further studies evaluating the use of impedance to assess organ edema as a quality marker in human clinical models and abdominal organs undergoing ex vivo perfusion warrant investigation.

Normothermic ex vivo lung perfusion outperforms conventional cold preservation in a deceased rat lung

BackgroundUtilization rate of donor lungs in China is constrained by the inefficiency of conventional cold preservation (COLD) in lung transplantation. We assessed the performance of normothermic ex vivo lung perfusion (EVLP) in lung donors following cardiac death (DCD) with fresh EVLP perfusate.MethodsA total of 24 adult Sprague-Dawley (SD) rats were randomized into three groups (EVLP, COLD, and control groups) with 8 rats each. Fresh EVLP perfusate was prepared for lung perfusion. The pH, electrolyte concentration, perfusate penetration pressure during irrigation, physiological function, integrity of barrier function, pathological changes, and expressions of inflammation-related cytokines of donor lungs were examined.ResultsThe pH, electrolyte concentration, osmotic pressure of the perfusate were kept stable for 4 hours after EVLP was administered. Pulmonary arterial pressure (PAP), pulmonary venous pressure (PVP), pulmonary venous oxygenation index (PaO2/FiO2), and end carbon dioxide partial pressure (PCO2) in the EVLP group were all within acceptable limits. After 4 hours, the oxygenation index was significantly higher in the EVLP group compared with that of the COLD group (P=0.0032). The EVLP group had a decreased wet/dry weight ratio (P=0.0155) and Evans blue dye content (P=0.0075) compared to that of the COLD group. Pathological examination showed more obvious lung damage in the COLD group than in the EVLP group. However, there was no difference in protein expression of high mobility group box 1 (HMGB1) and toll-like receptor 4 (TLR4) between the EVLP group and the COLD group.ConclusionsFresh lung perfusate could be used for EVLP to protect and repair donor lungs. Normothermic EVLP performed better than conventional cold preservation. EVLP might be powerful for lung quality maintenance and may have potential for anti-inflammation.

Gene Expression Change Related Inflammation Pathway during Rat EVLP and Heat Stress

<h3>Purpose</h3> Although Ex Vivo lung perfusion (EVLP) is a useful technique for evaluating and repairing the donor lung in lung transplantation, it can cause various lung injuries. In this study, we investigated the inflammation pathways that induces lung injury during EVLP through RNA sequencing. And, we examined how perfusate temperature during EVLP changes the inflammatory pathways. <h3>Methods</h3> Fifteen male Sprague Dawley rats were randomly divided into a sham group(n=5), normothermic EVLP (37°C,n=5) and hyperthermic group (42°C, n=5). In hyperthermic group, 5 minutes of heat stress (42°C) was given during 4 hours EVLP. Transcriptome analysis was performed with lung tissues of sham (n=2), normothermic EVLP (n=2), and hyperthermic EVLP (n=2). <h3>Results</h3> A total of 7172 genes were significantly (P<0.01) changed in the control group, and most of these changed genes were related to pathways of immune activation. In hyperthermic EVLP group, the expression of genes related to inflammation was higher than normothermic group and the genes related to defense mechanism against hypoxia or apoptosis was lower. And these genetic change was confirmed that the opposite direction was shown according to the temperature of the perfusate. <h3>Conclusion</h3> In this study, we identified inflammation pathways that causes lung injuries during EVLP at gene level using RNA sequencing. This technique could be useful to select proper treatments for lung injury during EVLP by present the injury pathway corresponding to a specific temperature of perfusate.

Conditioned Medium from Human Amnion-Derived Mesenchymal Stromal/Stem Cells Attenuating the Effects of Cold Ischemia-Reperfusion Injury in an In Vitro Model Using Human Alveolar Epithelial Cells.

The clinical results of lung transplantation (LTx) are still less favorable than other solid organ transplants in both the early and long term. The fragility of the lungs limits the procurement rate and can favor the occurrence of ischemia-reperfusion injury (IRI). Ex vivo lung perfusion (EVLP) with Steen SolutionTM (SS) aims to address problems, and the implementation of EVLP to alleviate the activation of IRI-mediated processes has been achieved using mesenchymal stromal/stem cell (MSC)-based treatments. In this study, we investigated the paracrine effects of human amnion-derived MSCs (hAMSCs) in an in vitro model of lung IRI that includes cold ischemia and normothermic EVLP. We found that SS enriched by a hAMSC-conditioned medium (hAMSC-CM) preserved the viability and delayed the apoptosis of alveolar epithelial cells (A549) through the downregulation of inflammatory factors and the upregulation of antiapoptotic factors. These effects were more evident using the CM of 3D hAMSC cultures, which contained an increased amount of immunosuppressive and growth factors compared to both 2D cultures and encapsulated-hAMSCs. To conclude, we demonstrated an in vitro model of lung IRI and provided evidence that a hAMSC-CM attenuated IRI effects by improving the efficacy of EVLP, leading to strategies for a potential implementation of this technique.

Normothermic Ex-Vivo Lung Perfusion (Evlp) Requiring Pulmonary Artery (Pa) Reconstruction after its Main Trunk Accidental Division during Harvesting

Normothermic ex-vivo lung perfusion (EVLP) is an established strategy to increase donor pool, improving graft quality assessment, facilitating self-repair mechanisms, providing a platform for active drug treatments and safely prolonging the preservation time.

Pegylated-Catalase Is Protective in Lung Ischemic Injury and Oxidative Stress

Lung transplant ischemia-reperfusion injury is typified by toxic metabolites and oxygen free radicals leading to worse graft function. Catalase is an enzyme involved in oxidative-stress detoxification. We hypothesize that direct delivery of highly concentrated polyethylene glycol-catalase (PEG-CAT) during normothermic exvivo lung perfusion (EVLP) significantly reduces ischemia-reperfusion injury. To demonstrate protection, primary culture porcine endothelial cells were treated with PEG-CAT (0 to 1250 U/mL) in a model of oxidative stress (400 μM H2o2). Invivo, rat lungs were subjected to 0 hours or 1 hour of warm ischemic injury and 2 hours of EVLP with or without PEG-CAT. Perfusate was collected throughout the perfusion duration and tissue was collected at the end. Tissue and perfusate underwent analysis for markers of apoptosis and a biometric signature of lung health. Uptake of PEG-CAT into primary endothelial cells was demonstrated with Alexa Fluor 488-labeled PEG-CAT. Oxidatively stressed cells pretreated with PEG-CAT had significantly decreased cytotoxicity and caspase 3/7 activity and increased cell viability and cell membrane integrity. In a rat model of warm ischemia with EVLP, PEG-CAT improved allograft viability as measured by indications of cell membrane integrity (lactate dehydrogenase and hyaluronic acid), presence of vasoconstrictive peptides (endothelin-1 and big endothelin-1) released from endothelial cells, and reduced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling). Invitro and exvivo, PEG-CAT protects against oxidative stress-induced cytotoxicity, maintains cellular metabolism, and mitigates lung ischemia-reperfusion in an experimental model. Together, these data suggest that PEG-CAT is a potential therapeutic target for donor organs at risk for ischemia-reperfusion injury.

A Novel Negative Pressure-Flow Waveform to Ventilate Lungs for Normothermic Ex Vivo Lung Perfusion.

Ex vivo lung perfusion (EVLP) is increasingly used to treat and assess lungs before transplant. Minimizing ventilator induced lung injury (VILI) during EVLP is an important clinical need, and negative pressure ventilation (NPV) may reduce VILI compared with conventional positive pressure ventilation (PPV). However, it is not clear if NPV is intrinsically lung protective or if differences in respiratory pressure-flow waveforms are responsible for reduced VILI during NPV. In this study, we quantified lung injury using novel pressure-flow waveforms during normothermic EVLP. Rat lungs were ventilated-perfused ex vivo for 2 hours using tidal volume, positive end-expiratory pressure (PEEP), and respiratory rate matched PPV or NPV protocols. Airway pressures and flow rates were measured in real time and lungs were assessed for changes in compliance, pulmonary vascular resistance, oxygenation, edema, and cytokine secretion. Negative pressure ventilation lungs demonstrated reduced proinflammatory cytokine secretion, reduced weight gain, and reduced pulmonary vascular resistance (p < 0.05). Compliance was higher in NPV lungs (p < 0.05), and there was no difference in oxygenation between the two groups. Respiratory pressure-flow waveforms during NPV and PPV were significantly different (p < 0.05), especially during the inspiratory phase, where the NPV group exhibited rapid time-dependent changes in pressure and airflow whereas the PPV group exhibited slower changes in airflow/pressures. Lungs ventilated with PPV also had a greater transpulmonary pressure (p < 0.05). Greater improvement in lung function during NPV EVLP may be caused by favorable airflow patterns and/or pressure dynamics, which may better mimic human respiratory patterns.

Anti-inflammatory exosomes mediate the beneficial effect of mesenchymal stem cell therapy on lung preservation

Background & Aim Mesenchymal stem cell (MSCs) therapy protects donor lungs against ischemic-reperfusion injury. We recently reported that local infusion of human umbilical cord-derived MSCs during donor lung ablation significantly conserved lung function. The anti-inflammatory effect of MSCs is mainly exerted through a paracrine mechanism. Thus, in the present study, we investigated whether the preservation capacity of MSCs is related to the secretion of anti-inflammatory exosomes. Methods, Results & Conclusion The rat lung preservation model settled before consisted on: cardiac arrest, warm ischemia (2h) in which cells were administered at 1h via the pulmonary artery (1 × 10^6 cells or vehicle), cold ischemia, and normothermic ex-vivo lung perfusion with ventilation. Here, we assessed 3 experimental groups right after the warm ischemia period (2h): vehicle, MSCs, and exosomes (released by 1 × 10^6 cells). Exosomes were chromatographically purified from MSCs conditioned medium and their anti-inflammatory activity was evaluated, in vitro, based on their ability to suppress the acquisition of M1 phenotype in LPS-stimulated RAW264.7 cells. Physiologic data (pressure-volume curves) were acquired right after the cardiac arrest and at the end of the warm ischemia time and lung biopsies were taken to study edema formation, neutrophil infiltration, redox profile, and inflammatory molecular markers. As expected, exosomes were positive for human-specific CD63 and CD81, and showed the tipical size and morphology by electron microscopy. Exosomes also exposed their anti-inflammatory activity by significantly reducing IL-6 secretion in LPS-stimulated macrophages by 60%. Previously, in the complete lung preservation model (4.5h), we observed that MSCs administration facilitated organ preservation by significantly reducing the drop on lung compliance (∼50% vs vehicle treated group, p Taking in consideration our previous data and these new results we conclude that MSCs-derived exosomes have a key role in lung preservation mediated by MSCs. Consequently, we postulate that exosomes may represent an innovative cell free therapy for organ preservation.

An extracellular oxygen carrier during prolonged pulmonary preservation improves post-transplant lung function

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. 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. 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 mmHg vs 343 ± 27 mmHg, 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. The use of an EOC during an extended pulmonary preservation period led to significantly superior early post-transplant lung function.

Successful Transplantation of Porcine Lungs Following 3 Days of Preservation Using a Modified Cold Static Method Paired with Intermittent Normothermic Ex Vivo Lung Perfusion (EVLP)

We have recently showed the superiority of 10°C cold static preservation (CSP) compared to standard 4°C. Despite that, CSP cannot be prolonged unlimited. Here, we hypothesized that combining CSP at 10°C with 2 short cycles of normothermic EVLP (a "recharge" period) would further extend donor lung preservation. Donor lungs (n=3) from Yorkshire pigs (28-35kg) were flushed with 4°C LPD and subsequently preserved using 10°C CSP and intermittent normothermic EVLP (Fig 1A). After a total of 72h of preservation, a left lung transplant was performed followed by 4h of reperfusion. At 4h of reperfusion, isolated graft assessment was performed by clamping the contralateral pulmonary artery and bronchus. To evaluate the contribution of the EVLP recharge periods, 2 control lungs were preserved solely with 10°C CSP for 72h and assessed using normothermic EVLP. After 3-days of preservation, post-transplant graft function was excellent. Systemic P/F ratio after excluding the contra-lateral lung was 430 ± 57 mmHg. Lung function was also stable during the intermittent EVLP periods (Fig 1B) and after transplantation (Fig 1C). No pulmonary edema was observed in the bronchoscopic assessment after transplant. Lungs preserved purely in CSP for 72h were assessed on EVLP and failed immediately with the development of massive pulmonary edema. We demonstrate for the first time the feasibility of 3-day lung preservation leading to excellent early post-transplant outcomes. Further experiments are being performed to confirm these positive findings and underlying mechanisms. The combination of a modified CSP method (10°C) and intermittent EVLP can open new opportunities to further prolong organ preservation and provide time for advanced lung treatments and repair.

Inflammatory Response after Immediate versus Delayed Ex-Vivo Lung Perfusion in a Porcine Cardiac Arrest Donation Model

Ex Vivo Lung Perfusion (EVLP) is a promising tool for evaluation and reconditioning in marginal donor lungs. However, the optimal protocol is discussed controversially. It is our hypothesis that different EVLP protocols and different perfusion solutions result in different cytokine patterns during EVLP. Following cardiac arrest and 30 min of no-touch warm ischemia, pig lungs were harvested, flushed and stored in cold preservation solutions. Different groups were processed as immediate long EVLP (I-EVLP, 12 h), delayed short EVLP (4 h) after 9 hours of cold storage (D-EVLP). Lungs were perfused with different acellular solutions: Steen solution or modified HTK solution containing either dextran(CD) or dextran and albumin (CDA). Pulmonary gas exchange, biochemical and structural parameters were recorded hourly. Cytokine / chemokine levels were analyzed in perfusates at baseline and after 1 and 4 h of EVLP. ∆ pO2 (pulmonary venous - pulmonary arterialpO2) was higher in D-EVLP group, especially at h 1: I-EVLP: 259 ± 132, D-EVLP: 291 ± 96 and h 2: I-EVLP: 228 ± 125, D-EVLP: 252 ± 116 (n.s.). Compared to D-EVLP, lungs that underwent EVLP with CD and CDA showed significantly higher oxygen capacity (∆ pO2 CD: 402.79 +30.33 mmHg, CDA: 414.86 + 9.77 mmHg). Within 4 h ELVP, concentrations of IFN-γ, TNF-a, IL-1α/β, IL-10, CXCL8 (IL-8) and IL-18 increased significantly (all p<0.05) in the four groups. Of note, no significant differences could be detected between I- vs. D-EVLP lungs and CD vs. CDA solutions. In a non-heart beating porcine lung donor model with relevant ischemic tissue damage, lungs were perfused with normothermic EVLP. Even after 9 hours cold static preservation (CSP) and 4 hours EVLP lung, function is comparable to direct EVLP. The modified HTK solution with dextran and dextran/ albumin had significantly better oxygenation. Cytokines and chemokines including the cell death marker IL-18 increase during 4 h of EVLP but a delay before EVLP may not dramatically worsen the inflammatory response.

Developing Universal Blood Type Donor Lungs Using Ex Vivo ABO Enzymatic Treatment

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). 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. 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. 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.

Subnormothermic Ex Vivo Lung Perfusion Improves Graft Preservation in Rat Ex Vivo Lung Perfusion Model

Current standard for organ assessment with machine perfusion before lung transplantation are performed with normothermic temperature. However, subnormothermic condition is already in clinical use with several solid organs. We investigated the impact of subnormothermic perfusion in a rat ex vivo lung perfusion (EVLP) model. Lungs retrieved from 12 outbred Sprague Dawley male rats followed by one hour of cold ischemic storage. All lungs were then perfused in a rat EVLP system for 4 hours. Lung grafts underwent EVLP at two different perfusion temperature (normothermic at 37°C, N=6 or subnormothermic at 28°C, N=6). EVLP physiology and biochemistry were monitored. Oxygenation, pulmonary vascular resistance and dynamic lung compliance were significantly better in the 28°C group compared to the 37°C group (p<0.01) (Figure 1). Potassium ion and lactate concentrations in the subnormothermic perfusion were almost similar with the normothermic perfusion. The tissue ATP at 28°C group showed better ATP preservation than 37°C group. Findings from this preliminary study demonstrated that subnormothermic EVLP might be an alternative to normothermic EVLP in terms of better graft protection. This observation should be validated in large animal model.