Ex-vivo Lung Research Articles

Ex-Vivo Lung Perfusion Results In a 5% Increase in Lung Transplants

Introduction Ex-Vivo Lung Perfusion (EVLP) can be used to improve the performance of donor lungs determined unsuitable for transplant prior to recovery or after intraoperative evaluation. Methods This was a single OPO, single center study. Surgeons accepted lung offers for standard or EVLP recovery. Lungs were placed for EVLP recovery prior to the OR if there was no interest in standard recovery by local and national lung centers. Lungs were considered for EVLP on brain dead donors with a PaO2 of <300 mmHg and DCD donors. Additionally, EVLP was considered for lungs after intraoperative evaluation of lungs determined they were not acceptable for standard recovery. Results Over 4 years, 339 lung offers were accepted for transplant, 323 (95%) were accepted for standard recovery and 16 (5%) were accepted for EVLP recovery. There were 11 cases where lungs initially accepted for standard recovery were placed on EVLP. In 3 cases, lungs initially accepted for EVLP were recovered and transplanted without being placed on EVLP. Additionally, in 1 case, lungs initially accepted for EVLP were recovered and discarded without being placed on EVLP due to consolidation and pneumonia observed in the OR. Lungs placed on EVLP were transplanted 50% (12/24) of the time. Conclusion EVLP increased the total number of lungs in this study period by 5% (12/248). OPOs should consider utilizing EVLP to not only increase the lung donor pool but also to salvage lungs initially thought to be able to be recovered in the standard fashion. More research is needed to better understand EVLP effects on transplant outcomes and cost.

Ex-vivo lung perfusion: the model for the organ reconditioning hub.

Lung evaluation and reconditioning by ex-vivo lung perfusion (EVLP) is becoming increasingly established. We review strategies for broader implementation of this technology to transplant centers worldwide. The organ reconditioning hub model is a viable strategy for disseminating EVLP to small and large transplant centers given the well tolerated prolongation of preservation time afforded by EVLP. Regulatory and process issues remain hurdles to be overcome. EVLP demonstrates promise to increase lung utilization. Organ reconditioning hubs appear to be an efficient method of delivering this promise to all transplant centers, not necessarily only the largest ones. Organ allocation processes will need to adapt to this new paradigm of organ preservation and evaluation. Moreover, regulatory issues will need to be deliberated by the transplant community.

Ex Vivo Lung Perfusion Rehabilitates Sepsis-Induced Lung Injury

Sepsis is the number one cause of lung injury in adults. Exvivo lung perfusion (EVLP) is gaining clinical acceptance for donor lung evaluation and rehabilitation and may expand the use of marginal organs for transplantation. We hypothesized that 4 hours of normothermic EVLP would improve compliance and oxygenation in a porcine model of sepsis-induced lung injury. We used intravenous lipopolysaccharide (LPS) to induce a systemic inflammatory response in a porcine model of lung injury. Two groups of 4 animals each received a 2-hour infusion of LPS through the external jugular vein. Serial measurements of blood gases were performed every 30 minutes until the partial pressure of oxygen/fraction of inspired oxygen ratio dropped below 150 on two consecutive readings. Lungs were then randomized to treatment with 4 hours of normothermic EVLP with STEEN Solution (XVIVO Perfusion Inc, Englewood, CO) or 4 additional hours of invivo perfusion (control). Airway pressures and blood gases were recorded for calculation of dynamic lung compliance and partial pressure of oxygen/fraction of inspired oxygen ratios. EVLP was performed with hourly recruitment maneuvers and oxygen challenge. All animals reached a partial pressure of oxygen/fraction of inspired oxygen ratio of less than 150 mm Hg within 3 hours after start of the LPS infusion. Oxygenation and compliance in the control animals continued to decline during the 4-hour invivo perfusion period, and 3 of the 4 animals died of severe hypoxia within 4 hours. The EVLP group demonstrated significant improvements hour 1 to hour 4 in oxygenation (365.8 ± 53.0 vs 584.4 ± 21.0 mm Hg, p= 0.02) and dynamic compliance (9.0 ± 2.8 vs 15.0 ± 3.6, p= 0.02 mL/cm H2O). EVLP successfully rehabilitated LPS-induced lung injury in this preclinical porcine model and may thus provide a means to rehabilitate many types of acute lung injury.

(962) - Cytokine Levels in Steen Solution Perfusate Increase During Ex-Vivo Lung Perfusion (EVLP) of Lungs from Conventional Donors (Conv) and Uncontrolled Donation After Circulatory Determination of Death Donors (uDCDDs)

(962) - Cytokine Levels in Steen Solution Perfusate Increase During Ex-Vivo Lung Perfusion (EVLP) of Lungs from Conventional Donors (Conv) and Uncontrolled Donation After Circulatory Determination of Death Donors (uDCDDs)

(172) - High Levels of Cytokines in the Perfusate During Ex-Vivo Lung Perfusion Correlates with Persistence of Poor Graft Function After Lung Transplantation Using Reconditioned Grafts

(172) - High Levels of Cytokines in the Perfusate During Ex-Vivo Lung Perfusion Correlates with Persistence of Poor Graft Function After Lung Transplantation Using Reconditioned Grafts

(119) - Development of a Donor Lung Discard Score to Prompt Assessment for Ex-Vivo Lung Perfusion Reconditioning

(119) - Development of a Donor Lung Discard Score to Prompt Assessment for Ex-Vivo Lung Perfusion Reconditioning

(368) - Lessons Learned from the First Clinical Trial to Compare Outcomes of Lungs Transplanted from Uncontrolled Donation After Circulatory Determination of Death Donors (uDCDDs) Assessed by Ex-Vivo Lung Perfusion (EVLP) and CT Scan and Lungs Transplanted from Conventional Donors (CONV)

We obtained an Investigational Device Exemption (IDE) from the FDA and IRB approval to conduct a Phase II clinical trial to compare outcomes of lungs transplanted from uDCDDs to lungs transplanted from CONV donors.

(121) - Comparison of Two Strategies for Experimental Ex-Vivo Lung Perfusion - Cellular Solution and Open Atrium versus Acellular Solution and Closed Atrium

(121) - Comparison of Two Strategies for Experimental Ex-Vivo Lung Perfusion - Cellular Solution and Open Atrium versus Acellular Solution and Closed Atrium

(479) - Cell-Free DNA in Human Ex-Vivo Lung Perfusate as a Potential Biomarker to Predict the Risk of Primary Graft Dysfunction (PGD) in Lung Transplantation

(479) - Cell-Free DNA in Human Ex-Vivo Lung Perfusate as a Potential Biomarker to Predict the Risk of Primary Graft Dysfunction (PGD) in Lung Transplantation

Pyrrolidine dithiocarbamate administered during ex-vivo lung perfusion promotes rehabilitation of injured donor rat lungs obtained after prolonged warm ischemia.

Damaged lung grafts obtained after circulatory death (DCD lungs) and warm ischemia may be at high risk of reperfusion injury after transplantation. Such lungs could be pharmacologically reconditioned using ex-vivo lung perfusion (EVLP). Since acute inflammation related to the activation of nuclear factor kappaB (NF-κB) is instrumental in lung reperfusion injury, we hypothesized that DCD lungs might be treated during EVLP by pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. Rat lungs exposed to 1h warm ischemia and 2 h cold ischemia were subjected to EVLP during 4h, in absence (CTRL group, N = 6) or in presence of PDTC (2.5g/L, PDTC group, N = 6). Static pulmonary compliance (SPC), peak airway pressure (PAWP), pulmonary vascular resistance (PVR), and oxygenation capacity were determined during EVLP. After EVLP, we measured the weight gain of the heart-lung block (edema), and the concentration of LDH (cell damage), proteins (permeability edema) and of the cytokines IL-6, TNF-α and CINC-1 in bronchoalveolar lavage (BAL), and we evaluated NF-κB activation by the degree of phosphorylation and degradation of its inhibitor IκBα in lung tissue. In CTRL, we found significant NF-κB activation, lung edema, and a massive release of LDH, proteins and cytokines. SPC significantly decreased, PAWP and PVR increased, while oxygenation tended to decrease. Treatment with PDTC during EVLP inhibited NF-κB activation, did not influence LDH release, but markedly reduced lung edema and protein concentration in BAL, suppressed TNFα and IL-6 release, and abrogated the changes in SPC, PAWP and PVR, with unchanged oxygenation. In conclusion, suppression of innate immune activation during EVLP using the NF-κB inhibitor PDTC promotes significant improvement of damaged rat DCD lungs. Future studies will determine if such rehabilitated lungs are suitable for in vivo transplantation.

Ex Vivo Lung Perfusion: A Key Tool for Translational Science in the Lungs

Exvivo lung perfusion (EVLP) promises to be a comprehensive platform for assessment, reconditioning, and preservation of donor lungs and has been dramatically changing the face of clinical lung transplantation. Besides its increasing role in lung transplantation, EVLP has also been recognized as a useful tool for translational research involving the lungs. Based on recent remarkable evidence and experience using EVLP in lung transplantation, there is growing interest in, and expectations for, the use of EVLP beyond the field of lung transplantation. By combining EVLP with advances in regenerative medicine, stem cell biology, and oncology, the evolving technology of EVLP has tremendous potential to advance pulmonary medicine and science. In this review, we revisit recent advances in EVLP technology and research and discuss the future translation of EVLP applications into life-changing medicine.

Ex Vivo Lung Perfusion Model to Study Pulmonary Tissue Extracellular Microvesicle Profiles

Extracellular microvesicles (EVs) are being increasingly studied for their diagnostic potential. We investigated the feasibility of studying the kinetics and tissue-specific profiles of pulmonary EVs in the context of exvivo lung perfusion (EVLP) used for salvaging marginal lungs for transplantation. Perfusate from six marginal donor lungs placed on EVLP was collected at the following time points: 0, 10, and 60 minutes after and after perfusate exchange with Steen Solution; 120 and 180 minutes. Three lungs were successfully recovered for transplantation (transplant group), and three were not recoverable (nontransplant group). Perfusate EVs were isolated using methods of size exclusion chromatography, ultrafiltration, and ultracentrifugation. EVs were analyzed on NanoSight nanoparticle detector for quantity, size distribution, and surface expression of pulmonary tissue-specific markers. EV cargoes were profiled using mass spectrometry, reverse transcription polymerase chain reaction (RT-PCR), and Western blot analysis. Time course analysis showed EV presence by 10-minute time point. EV median size was smaller in the transplant group (165 nm versus 212 nm, p= 0.04). EV cargo analysis on Western blot analysis, RT-PCR, and NanoSight showed contribution from monocytes (CD14), endothelium (platelet endothelial cell adhesion molecule 1), and pulmonary parenchyma (epithelial cell adhesion molecule) into the perfusate total EV pool. Mass spectrometry showed differences in the EV protein cargoes of the transplant group versus the nontransplant group. EVLP system provides a platform to understand the kinetics of pulmonary EVs in an isolated fashion. Donor lung recovery may be associated with changes in EV size distribution and proteomic profiles. Pulmonary tissue-specific EV profiling using the EVLP system may provide insights into EV contribution to pulmonary pathologic processes.

Will ex-vivo lung perfusion allow a move towards daytime lung transplantation surgery?

Will ex-vivo lung perfusion allow a move towards daytime lung transplantation surgery?

First Evaluation of the New Thin Convex Probe Endobronchial Ultrasound Scope: A Human Ex Vivo Lung Study

Endobronchial ultrasonography (EBUS)-guided transbronchial needle aspiration allows for sampling of mediastinal lymph nodes. The external diameter, rigidity, and angulation of the convex probe EBUS renders limited accessibility. This study compares the accessibility and transbronchial needle aspiration capability of the prototype thin convex probe EBUS against the convex probe EBUS in human exvivo lungs rejected for transplant. The prototype thin convex probe EBUS (BF-Y0055; Olympus, Tokyo, Japan) with a thinner tip (5.9 mm), greater upward angle (170 degrees), and decreased forward oblique direction of view (20 degrees) was compared with the current convex probe EBUS (6.9-mm tip, 120 degrees, and 35 degrees, respectively). Accessibility and transbronchial needle aspiration capability was assessed in exvivo human lungs declined for lung transplant. The distance of maximum reach and sustainable endoscopic limit were measured. Transbronchial needle aspiration capability was assessed using the prototype 25G aspiration needle in segmental lymph nodes. In all evaluated lungs (n= 5), the thin convex probe EBUS demonstrated greater reach and a higher success rate, averaging 22.1 mm greater maximum reach and 10.3 mm further endoscopic visibility range than convex probe EBUS, and could assess selectively almost all segmental bronchi (98% right, 91% left), demonstrating nearly twice the accessibility as the convex probe EBUS (48% right, 47% left). The prototype successfully enabled cytologic assessment of subsegmental lymph nodes with adequate quality using the dedicated 25G aspiration needle. Thin convex probe EBUS has greater accessibility to peripheral airways in human lungs and is capable of sampling segmental lymph nodes using the aspiration needle. That will allow for more precise assessment of N1 nodes and, possibly, intrapulmonary lesions normally inaccessible to the conventional convex probe EBUS.

Outcomes after transplantation of lungs preserved for more than 12 h: a retrospective study

Ex-vivo lung perfusion (EVLP) can be used to extend overall lung preservation time by splitting one long cold ischaemic time into two shorter ones and interposing an additional EVLP time. We assessed the outcomes after clinical transplantation of lungs with more than 12 h of preservation time. For this retrospective study, we searched the Toronto Lung Transplant Program database for patients who had received at least one lung transplant between Jan 1, 2006, and April 30, 2015, at a single hospital in Toronto, Canada. We split the identified patients into those with a total preservation time of more than 12 h and those with a total preservation time of less than 12 h to act as the control group. Total preservation time was defined as the sum of first cold ischaemic time, EVLP time, and second cold ischaemic time. We excluded patients if they had received a heart-lung transplant or were younger than 18 years. In bilateral lung transplantations, we used the longer preservation time of the two lungs for analysis. Lung preservation was done according to present standards of care and EVLP was done according to the Toronto EVLP technique. The primary outcomes were survival and International Society for Heart and Lung Transplantation Primary Graft Dysfunction (PGD) grade at 72 h post-transplantation. We compared outcomes with our control group using univariable and multivariable models. We identified 906 patients who met eligibility criteria and had sufficient data for analysis (<12 h group [n=809]; mean lung preservation time 400·8 min [SD 121·8] vs >12 h group [n=97]; 875·7 min [109·0]). Median hospital and intensive-care unit length of stay were similar between the less than 12 h group and the more than 12 h group (hospital stay: 23 days [16-42] vs 25·5 days [17-50·25], p=0·60; intensive-care unit stay: 4 days [2-14] vs 4 days [2-16], p=0·53). PGD grade was also not different between the two groups at 72 h post-transplantation (p=0·85). There was also no difference in survival between the two groups as shown on Kaplan-Meier survival curves (p=0·61). Multivariable survival analysis using Cox's model showed increasing recipient age to be a significant variable affecting survival. Extension of graft preservation time beyond 12 h with EVLP does not negatively affect early lung transplantation outcomes. Extension of clinical lung preservation times might allow for more transplantations to be done as a result of improved facilitation and increased flexibility around timing of lung transplantation operations. None.

Emergence of a Wave of Wnt Signaling that Regulates Lung Alveologenesis by Controlling Epithelial Self-Renewal and Differentiation

Alveologenesis is the culmination of lung development and involves the correct temporal and spatial signals to generate the delicate gas exchange interface required for respiration. Using a Wnt-signaling reporter system, we demonstrate the emergence of a Wnt-responsive alveolar epithelial cell sublineage, which arises during alveologenesis, called the axin2+ alveolar type 2 cell, or AT2Axin2. The number of AT2Axin2 cells increases substantially during late lung development, correlating with a wave of Wnt signaling during alveologenesis. Transcriptome analysis, invivo clonal analysis, and exvivo lung organoid assays reveal that AT2sAxin2 promote enhanced AT2 cell growth during generation of the alveolus. Activating Wnt signaling results in the expansion of AT2s, whereas inhibition of Wnt signaling inhibits AT2 cell development and shunts alveolar epithelial development toward the alveolar type 1 cell lineage. These findings reveal a wave of Wnt-dependent AT2 expansion required for lung alveologenesis and maturation.

Evaluation of a New Ultrasound Thoracoscope for Localization of Lung Nodules in Ex Vivo Human Lungs

Localization of small, nonvisible and nonpalpable nodules is challenging during video-assisted thoracoscopic surgery. We evaluated the feasibility of using a new ultrasound thoracoscope to localize nodules in resected exvivo human lungs. The tumor was localized and measured in its greatest dimension with a prototype ultrasound thoracoscope (XLTF-UC180; Olympus Corporation, Tokyo, Japan) at different frequencies (5.0 to 12.0 MHz) and different lung specimen states (deflated, semiinflated). Measured tumor size and depth from lung surface were compared and correlated to the true diameter and depth from lung surface acquired from pathologic morphology. Exvivo evaluation was performed on 16 solid nodules and nine part solid ground-glass nodules. All tumors were successfully localized in the deflated lung specimens (average size, 13.7 ± 5.2 mm). The tumor boundaries were best evaluated with an ultrasound frequency of 10 MHz. Solid nodules were more easily visualized than ground-glass nodules. Part solid ground-glass nodules were not easily detected in the semiinflated specimen owing to peritumoral air surrounding the tumor. Tumor boundaries were also difficult to identify in deeply situated tumors and in lungs with underlying disease. A strong positive correlation existed between the ultrasound measurement and true measurement of tumor size (R2= 0.89, p < 0.001). The ultrasound thoracoscope can be used to localize nodules in resected human lungs. The clarity of the tumor boundaries is influenced by the tumor type and depth and the underlying pulmonary disease. Complete lung deflation and the use of 10 MHz ultrasound frequency optimize the visualization of target tumors.

Shear wave elastography reveals dispersion regimes in porous materials

Ultrasound shear wave elastography is a well established tool for characterization of biologic tissues. While it has found application in various medical disciplines such as oncology and urology its feasibility for pneumology still has to be shown. We provide experimental results of ultrasound shear wave elastography of porous materials in phantoms and ex-vivo lung tissue. Phantom foams immersed in water show a strong phase velocity dispersion with increasing frequency. Two regimes can be identified in the dispersion curves in the investigated frequency range from 50 to 700 Hz. These experimental results are in reasonable agreement with the theory of porous materials. The results from water-filled phantom foams are compared to gelatin-filled phantom foams. Finally, the phantom study is compared to surface waves dispersion curves of porcine lungs obtained with an ultra-fast optical camera and the first results of ex-vivo shear wave elastography in porcine lung. Ultrasound shear wave elastography is standard in non-porous organs such as muscle, liver, and breast tissue. Its application to porous materials could offer a noninvasive and nonionizing alternative for lung characterization.

Donation After Circulatory Death Lungs Transplantable Up to Six Hours After Ex Vivo Lung Perfusion

Despite the critical need for donor lungs, logistic and geographic barriers hinder lung utilization. We hypothesized that lungs donated after circulatory death subjected to 6 hours of cold preservation after exvivo lung perfusion (EVLP) would have similar outcomes after transplantation as lungs transplanted immediately after EVLP, and that both would perform superiorly compared with lungs transplanted immediately after procurement. Donor porcine lungs were procured after circulatory death and 15 minutes of warm ischemia. Three groups (n= 5 per group) were randomized: immediate left lung transplantation (Immediate group), EVLP for 4 hours followed by transplantation (EVLP group), or EVLP for 4 hours followed by 6 hours of cold preservation followed by transplantation (EVLP+Cold group). Lungs were reperfused for 2 hours before obtaining pulmonary vein samples for partial pressure of oxygen/fraction of inspired oxygen ratio calculations, airway pressures for compliance measurements, and wet/dry weight ratios. The partial pressure of oxygen/fraction of inspired oxygen ratios in the EVLP and EVLP+Cold groups were significantly improved compared with those in the Immediate group (429.7 ± 51.8 and 436.7 ± 48.2 versus 117.4 ± 22.9 mm Hg, respectively). In addition, dynamic compliance was significantly improved in the EVLP and EVLP+Cold groups compared with immediate group (26.2 ± 4.2 and 27.9 ± 3.5 versus 11.1 ± 2.4 mL/cmH2O, respectively). There were no differences in oxygenation capacity or dynamic compliance between the EVLP and EVLP+Cold groups. Inflammatory cytokine levels were significantly lower in the EVLP and EVLP+Cold groups. Lungs donated after circulatory death can be successfully transplanted as much as 6 hours after EVLP. Cold preservation of lungs after exvivo assessment and rehabilitation may improve organ allocation, even to distant recipients, without compromising allograft function.

Pharmacological Reconditioning of Marginal Donor Rat Lungs Using Inhibitors of Peroxynitrite and Poly (ADP-ribose) Polymerase During Ex Vivo Lung Perfusion.

Donor lungs obtained after prolonged warm ischemia (WI) may be unsuitable for transplantation due to the risk of reperfusion injury, but could be reconditioned using ex-vivo lung perfusion (EVLP). Key processes of reperfusion injury include the formation of reactive oxygen species (ROS)/nitrogen species (RNS) and the activation of poly(adenosine diphosphate-ribose) polymerase (PARP). We explored whether rat lungs obtained after WI could be reconditioned during EVLP using the ROS/RNS scavenger Mn(III)-tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) or the PARP inhibitor 3-aminobenzamide (3-AB). Rat lungs obtained after 3 hours cold ischemia (CI group, control), or 1 hour WI plus 2 hours CI (WI group) were placed in an EVLP circuit for normothermic perfusion for 3 hours. Lungs retrieved after WI were treated or not with 3-AB (1 mg/mL) or MnTBAP (0.3 mg/mL), added to the perfusate. Measurements included physiological variables (lung compliance, vascular resistance, oxygenation capacity), lung weight gain, levels of proteins, lactate dehydrogenase, protein carbonyl (marker of ROS), 3-nitrotyrosine (marker of RNS), poly(adenosine diphosphate-ribose) (PAR, marker of PARP activation) and IL-6, in the bronchoalveolar lavage or the lung tissue, and histology. In comparison to the CI group, the lungs from the WI group displayed higher protein carbonyls, 3-nitrotyrosine, PAR, lactate dehydrogenase and proteins in bronchoalveolar lavage, lung weight gain, perivascular edema, as well as reduced static compliance, but similar oxygenation. All these alterations were markedly attenuated by 3-AB and MnTBAP. After EVLP, lungs obtained after WI exhibit oxidative stress, PARP activation, and tissue injury, which are suppressed by pharmacological inhibitors of ROS/RNS and PARP.