ABSTRACTIntroductionPreclinically, 24‐hour continuous Ex‐Situ Lung Perfusion (ESLP) is the longest duration achieved in large animal models and rejected human lungs. Here, we present our 36‐hour Negative Pressure Ventilation (NPV)‐ESLP protocol applied to porcine and rejected human lungs.MethodsFive sets of donor domestic pig lungs (45‐55 kg) underwent 36‐hour NPV‐ESLP. Two sets of clinically rejected human lungs were preserved on 36‐hour NPV‐ESLP. Graft function was assessed via physiologic parameters, edema formation, and cytokine profiles.ResultsPorcine and human lung function was stable with mean partial pressure of oxygen divided by the fraction of inspired oxygen (PaO2/FiO2; PF) ratios throughout preservation of 473±11.79 and 554.7±13.26, respectively (mean±standard error of the mean). In porcine lungs, mean compliance (Cdyn) during ESLP was 33.96±2.18, pulmonary artery pressure (PAP) 13.03±0.53, and pulmonary vascular resistance (PVR) 481.20 ±21.86. In human lungs, mean Cdyn was 82.68±3.54, PAP 6.00±0.33, and PVR 184.00±9.71. Average percentage weight‐gain was 34.47±13.22 in porcine lungs and 116.3±6.65 in rejected human lungs.ConclusionNPV‐ESLP can preserve porcine lungs and human lungs for 36‐hours with acceptable physiologic function. Greater weight‐gain in the human lungs is likely due to prolonged ischemic time prior to ESLP and use of an acellular perfusate. Continuous 36‐hour NPV‐ESLP could support therapies for endothelial protection and mitigate fluid accumulation.

Background Full-flow perfusion during prolonged ex situ lung perfusion (ESLP) results in unacceptable pulmonary edema formation. Clinical ESLP at 30% to 50% predicted cardiac output (CO) supports acceptable physiologic outcomes; however, progressive pulmonary edema still develops. Lower flow rates may provide equivalent physiologic preservation with less edema formation due to reduced hydrostatic pressures. We report our results of moderate-flow (MF; 30% CO) vs low-flow (LF; 10% CO) negative pressure ventilation (NPV)-ESLP with transplantation. Methods Twelve pig lungs underwent 12-hours of NPV-ESLP with 30% or 10% CO (n = 6/group). Three left lungs per group were transplanted post-ESLP and assessed in vivo over 4 hours. Lung function was assessed by physiologic parameters, weight-gain, and pro-inflammatory cytokine profiles. Results Results are MF vs LF (mean ± SEM). All lungs demonstrated acceptable oxygenation post-ESLP (454.2 ± 40.85 vs 422.7 ± 31.68, P = .28); however, after transplantation, the MF lungs demonstrated significantly better oxygenation (300.7 ± 52.26 vs 141.9 ± 36.75, P = .03). There was no significant difference in compliance after ESLP (21.38 ± 2.28 vs 16.48 ± 2.34, P = .08); however, pulmonary artery pressure (PAP; 10.89 ± 2.28 vs 21.11 ± 0.93, P = .06) and pulmonary vascular resistance (PVR; 438.60 ± 97.97 vs 782.20 ± 162.20, P = .05) were significantly higher in the LF group. Weight gain (%) post-ESLP and post-transplant was similar between groups (29.42 ± 5.72 vs 24.17 ± 4.42, P = .24; and 29.63 ± 7.23 vs 57.04 ± 15.78, P = .09). TNF-α and IL-6 were significantly greater throughout LF ESLP. Conclusions The MF NPV-ESLP results in superior lung function with less inflammation compared to LF NPV-ESLP.

Background Ex-Situ Lung Perfusion (ESLP) employs a membrane deoxygenator and mixed (N2/O2/CO2) or pure sweep gas (CO2) to target venous blood gas composition with physiologic pCO2 and pH. Clinically, mild permissive alkalosis counteracts elevated pulmonary vascular resistance (PVR) to improve perfusion. Increased PVR and pulmonary artery pressure (PAP) during ESLP mirrors rising pro-inflammatory cytokines. Increased hydrostatic pressure worsens edema and lung function. We report improved ESLP outcomes using mild permissive alkalosis. Methods Twelve juvenile pig lungs underwent 12-hour Negative Pressure Ventilation (NPV)-ESLP with a physiologic pH (Control: pH 7.35-7.45, n=6) or mild permissive alkalosis (pH+: pH 7.45-7.55, n=6) by varying sweep CO2 delivery. Three left lungs per group were transplanted and assessed over 4-hours. Results Five Control lungs failed on ESLP due to high PAPs, low compliance, and poor oxygenation. Repeat Controls (n=6) were performed to attain 12-hours of ESLP. There were no failures in the pH+ group. Results are pH+ vs Control. Oxygenation (PaO2/FiO2 454.2 vs 438.2; P = .37) and dynamic compliance (21.38 vs 22.22 mL/cmH2O; P = .41) were stable over 12-hour NPV-ESLP. Mean evaluation pH/pCO2/HCO3- was 7.50/15.6/14.5 vs 7.41/38.7/24.7. Control lungs required repeat THAM and milrinone boluses on ESLP to prevent acidosis and treat elevated PVR; this was not necessary in the pH+ group. Weight-gain/hour was similar (1.23% vs 1.38%; P = .37). Mean left lung PF ratios 4-hours post-transplantation were 301 mmHg vs 196 mmHg (P = .11). Control TNF-⍺ and IL-6 perfusate concentrations were significantly greater. Conclusions Mild permissive alkalosis porcine NPV-ESLP demonstrated more reliable preservation with reduced inflammation compared to a physiologic pH strategy.

BACKGROUND Ischemia/reperfusion injury (IRI) is an inherent problem in organ transplantation, owing to the obligate period of ischemia that organs must endure. Cyclosporine A (CsA), though better know as an immunosuppressant, has been shown to mitigate warm IRI in a variety of organ types, including the liver. However, there is little evidence for CsA in preventing hepatic IRI in the transplant setting. MATERIAL AND METHODS In the present study, we tested the effect of CsA on hepatic IRI in a large-animal ex vivo model of donation after circulatory death (DCD). Porcine donors were pre-treated with either normal saline control or 20 mg/kg of CsA. Animals were subject to either 45 or 60 minutes of warm ischemia before hepatectomy, followed by 2 or 4 hours of cold storage prior to reperfusion on an ex vivo circuit. Over the course of a 12-hour perfusion, perfusion parameters were recorded and perfusate samples and biopsies were taken at regular intervals. RESULTS Peak perfusate lactate dehydrogenase was significantly decreased in the lower-ischemia group treated with CsA compared to the untreated group (4220 U/L [3515-5815] vs 11 305 [10 100-11 674]; P=0.023). However, no difference was seen between controls and CsA-treated groups on other parameters in perfusate alanine or asparagine aminotransferase (P=0.912, 0.455, respectively). Correspondingly, we found no difference on midpoint histological injury score (P=0.271). CONCLUSIONS We found minimal evidence that CsA is protective against hepatic IRI in our DCD model.

Background Reliable 24-hour preservation is required to optimize the rehabilitation potential of Ex Situ Lung Perfusion (ESLP). Other ESLP protocols include fresh perfusate replacement to counteract an accumulation of deleterious by-products. We describe the results of our reliable 24-hour negative pressure ventilation (NPV)-ESLP protocol with satisfactory acute post-transplant outcomes and investigate perfusate exchange (PE) as a modification to enhance prolonged ESLP. Methods Twelve pig lungs underwent 24 hours of NPV-ESLP using 1.5L of cellular perfusate (500 mL packed red blood cells and 1 L buffered perfusate). The Control (n = 6) had no PE; the PE (n = 6) had 500 mL replaced after 12 hours of NPV-ESLP with 1000 mL fresh perfusate. Three left lungs per group were transplanted. Results Results are reported as Control vs PE (mean ± SEM). Both groups demonstrated stable and acceptable oxygenation during 24 hours of ESLP with final PF ratios of 527.5 ± 42.19 and 488.4 ± 35.38 (P = .25). Final compliance measurements were 20.52 ± 3.59 and 18.55 ± 2.91 (P = .34). There were no significant differences in pulmonary artery pressure after 24 hours of ESLP (10.02 ± 2.69 vs 14.34 ± 1.64, P = .10), and pulmonary vascular resistance only differed significantly at T12 (417.6 ± 53.06 vs 685.4 ± 81.19, P = .02). Percentage weight gain between groups was similar (24.32 ± 8.4 and 45.33 ± 7.76, P = .07). Post-transplant left lung oxygenation was excellent (327.3 ± 14.62 and 313.3 ± 15.38, P = .28). There was no significant difference in % weight gain of lungs post-transplant (22.20 ± 7.22 vs 14.36 ± 9.96, P = .28). Conclusion Acceptable lung function was maintained during 24-hour NPV-ESLP and post-transplant regardless of PE.

BACKGROUND:Cold ex situ lung perfusion (ESLP) has demonstrated improved preservation in small animal ESLP compared to normothermic ESLP and cold static preservation. We hypothesized that cold negative pressure ventilation (NPV)-ESLP would improve graft function in a porcine transplantation model. METHODS:Four perfusate temperatures were examined with 12 hours NPV-ESLP in a large animal transplantation model. Pig lungs were allotted to four groups: (1) Normothermia (38°C, n = 6); (2) profound hypothermia (10°C, n = 6); (3) moderate hypothermia (20°C, n = 3); (4) subnormothermia (32°C, n = 3). A fifth group subnormothermic low-flow (SNLF) perfusion was examined to assess the effect of reduced cardiac output with cold perfusion (32°C, 10% cardiac output, n = 6). RESULTS:Only Normothermic and SNLF groups demonstrated acceptable oxygenation after 12 hours NPV-ESLP and were transplanted. All other groups failed prematurely. After 12 hours of ESLP, Normothermic lungs demonstrated significantly greater dynamic compliance compared to SNLF lungs (P = .03). Edema formation post-ESLP was significantly worse in the SNLF group (P = .01). There was no significant difference in pulmonary artery pressures after ESLP (P = .10); however, pulmonary vascular resistance was significantly greater in the SNLF (P = .04). Isolated left lung oxygenation 4-hours post-transplant and left lung edema formation was not significantly different between Normothermic and SNLF post-transplant (P = .09). Proinflammatory cytokines were significantly greater during SNLF-ESLP (tumor necrosis factor alpha, P < .05). CONCLUSIONS:Prolonged normothermic (38°C) NPV-ESLP is superior to 10, 20, and 32°C perfusion. Normothermic ESLP of porcine lungs results in superior graft function and reduced inflammation versus SNLF-ESLP.

We sought to determine the role of donor blood circulating leukocytes in mediating oxidative stress and inflammation during normothermic ex situ heart perfusion (ESHP). Normothermic ESHP allows preservation of donated heart in a perfused, dynamic state, preventing ischemia. However, the cardiac function declines during ESHP, limiting the potential of this method for improvement of the outcomes of transplantation and expanding the donor pool. Extracorporeal circulation-related oxidative stress plays a critical role in the functional decline of the donor heart. Hearts from domestic pigs were perfused in working mode (WM, whole blood-based or leukocyte-depleted blood-based perfusate) or nonworking mode. Markers of oxidative stress and responsive glucose anabolic pathways were induced in the myocardium regardless of left ventricular load. Myocardial function during ESHP as well as cardioprotective mechanisms were preserved better in WM. Leukocyte-depleted perfusate did not attenuate tissue oxidative stress or perfusate proinflammatory cytokines and did not improve functional preservation. Although ESHP is associated with ongoing oxidative stress and metabolic alteration in the myocardium, preserved cardioprotective mechanisms in WM may exert beneficial effects. Leukocyte depletion of the perfusate may not attenuate inflammation and oxidative stress effectively or improve the functional preservation of the heart during ESHP.

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short-and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the in vivo validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the in vivo validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

Introduction: Primary graft dysfunction (PGD) can occur early following lung transplantation (LT) and correlates with acute and chronic lung allograft rejection. Abrupt inflammatory and oxidative stress responses occur within minutes of LT. This ischemia-reperfusion injury (IRI) is believed to be responsible for initiating PGD. Interestingly, adiponectin (APN) a bioactive peptide, has significant anti-inflammatory and cytoprotective properties to pulmonary endothelium and airway epithelium. Thus, gene delivery of APN to donated lungs may decrease the incidence of PGD from IRI. We aim to assess the feasibility and early post-transplant effect of adenovirus-mediated adiponectin gene therapy to donor lungs. Methods: Male Lewis rats (350-450 g) were randomly allocated into 3 groups (N=5/ group), where they underwent in vivo transbronchial instillation of 1) Control: 400 µL of PBS+10% glycerol, 2) Ad-mCherry: adenovirus-mCherry 400 µL of 1.5*1010 PFU, and 3) Ad-APN: adenovirus-adiponectin 400 µL of 1.5*1010 PFU. After 24 h, transduced lungs were procured, flushed with ice-cold low-potassium dextran, and preserved in cold ischemia for ~3 h. Then, left donor lungs were orthotopically transplanted into recipient male Sprague Dawley rats (350-450 g). Following 2 h of reperfusion, blood gas samples were analyzed from the aorta and left pulmonary vein. Finally, recipient rats were euthanized, plasma and lung tissue were collected for wet/dry ratio and further analysis. Results: Target gene expression in Ad-APN and Ad-mCherry lungs was confirmed using tissue immunofluorescent staining. During the 2 h reperfusion, all recipient rats had stable oxygen saturation (SPO2% >90). The mean arterial partial pressure of oxygen (pO2) for groups 1-3 was 191.0, 193.4, and 231.5 mmHg; the estimated % contribution of donor lung to recipient pO2 was 73.2, 46.0, and 46.5, respectively. On average, wet/dry ratio of left donor lung was 8.7 for Control, 9.5 for Ad-mCherry, and 6.7 for the Ad-APN group. Plasma multiplex cytokine assay showed elevated levels of pro-inflammatory cytokines: IL-6, IL-1β, and IL-18 in Ad-mCherry compared to Control and Ad-APN. Monocyte chemoattractant protein (MCP-1) secretion was significantly higher in the Ad-mCherry group than APN (p = 0.02). Conclusion: Adenovirus treated lungs with Ad-mCherry, and Ad-APN exhibited comparable lung function post-transplant. Of interest, APN expression modulated the vector-induced inflammation in the Ad-APN group. Furthermore, APN induced the downregulation of MCP-1 secretion by alveolar macrophages. This will impair the chemoattraction of recipient monocytes and neutrophils, therefore potentially attenuating lung allograft inflammation and IRI post transplantation.

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

BackgroundHeart transplantation (HTx) is a recognized therapy for terminal heart failure patients; however, it is limited by availability of suitable donors and quality of cardiac grafts. To expand heart donor pool, normothermic ex situ heart perfusion (ESHP) has emerged as a platform for reconditioning marginal donor hearts and consequently increasing the number and quality of organs available for transplant. It also allows continuous functional and metabolic assessment of donor hearts before transplantation. Myocardial function is highly dependent on myocardial perfusion. The coronary autoregulation is essential to maintain normal cardiac function. Our objective is to investigate coronary autoregulation during normothermic ESHP and find ways to protect myocardial function during heart preservation.MethodsThe hearts of anesthetized and intubated domestic breed pigs (N=12) will be harvested after cold cardioplegia arrest. After being mounted on a custom ESHP apparatus, the procured hearts will be perfused ex situ in a beating state in Langendorff mode for 1 hour and then will be switched to working heart system (heart rate=100 beats/minute) to be perfused at 37°C for 12 hours (whole blood based perfusate). Cardiac functional parameters was monitored in the entire interval. Coronary vascular resistance (CVR) was calculated as an indicator of coronary artery function. Myocardial oxygen consumption (MVO2) was determined by coronary blood flow and oxygen extraction as a metabolic parameter. At 1 and 5 hours of perfusion, loading of the heart was varied by increasing or decreasing left atrial pressure to observe the corresponding response in coronary artery flow.ResultsDuring 12 hours normothermic perfusion, cardiac function declined over time as indicated by the cardiac index change at 5 and 11 hours compared with its baseline (p<0.05); myocardial oxygen consumption was decreased significantly (p<0.01, Fig. A). The coronary blood flow increased over time and coronary vascular resistance decreased, with significantly difference at T5 and T11 compared with T1 respectively (p<0.01, Fig. B). At T1 of perfusion, coronary artery flow shows strong correlation with left ventricle stroke work (p<0.01, R2=0.79, Fig. C); however, at T5, the regression line is significantly different (p<0.01), with relative lower slope (R2=0.36, Fig. D), which means as myocardial function demand increases, coronary artery flow increases less proportion than that in T1.ConclusionOur data suggests that regulation of coronary artery function is disturbed during ESHP leading to apparent excessive coronary blood flow over time. This is in contrast to a gradual reduction in myocardial function over time, suggesting that there is loss of coronary autoregulation. The theory of coronary autoregulation states that as myocardial functional demand increases, coronary blood flow increases as well. However, our data reveals a poor correlation between hemodynamics stress and coronary flow suggesting that this autoregulation phenomenon is disturbed. Whether the loss of coronary artery regulation cause the decline of cardiac function or the mechanism of disturbed autoregulation need to be further investigated.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BACKGROUND: Ex situ heart perfusion (ESHP) preserves the donated heart in a perfused, beating condition preventing cold storage-related ischemia and provides a platform to evaluate myocardial viability during preservation. However, myocardial function declines gradually during ESHP. Extracorporeal circulation systems are associated with the induction of systemic inflammatory and stress responses. Our aim was to evaluate the incidence of inflammation and induction of endoplasmic reticulum stress responses during an extended period of ESHP. METHODS: Cardiac function, myocardial tissue injury, markers of inflammation, oxidative stress, and endoplasmic reticulum stress were assessed in healthy pig hearts, perfused for 12 hours either in non working mode (non-WM=7) or working mode (WM, n=6). RESULTS: Cardiac function declined during ESHP but was significantly better preserved in the hearts perfused in WM (median 11-hour cardiac index/1-hour cardiac index: WM=27% versus non-WM=9.5%, P=0.022). Myocardial markers of endoplasmic reticulum stress were expressed higher in ESHP hearts compared with in vivo samples. The proinflammatory cytokines and oxidized low-density lipoprotein significantly increased in the perfusate throughout the perfusion in both perfusion groups. The left ventricular expression of the cytokines and malondialdehyde was induced in non-WM, whereas it was not different between WM and in vivo. CONCLUSIONS: Myocardial function declines during ESHP regardless of perfusion mode. However, ESHP in WM may lead to superior preservation of myocardial function and viability. Both inflammation and endoplasmic reticulum stress responses are significantly induced during ESHP and may contribute to the myocardial functional decline, representing a potential therapeutic target to improve the clinical donor heart preservation.

BACKGROUND:Normothermic ex vivo lung perfusion (EVLP) has been used successfully to evaluate and recondition marginal donor lungs; however, multiple barriers continue to prevent its widespread adoption. We sought to develop a common hospital ingredient-derived perfusate (CHIP) with equivalent functional and inflammatory characteristics to a standard Krebs-Henseleit buffer with 8% serum albumin-derived perfusate (KHB-Alb) to improve access and reduce costs of ex vivo organ perfusion. METHODS:Sixteen porcine lungs were perfused using negative pressure ventilation (NPV) EVLP for 12 hours in a normothermic state and were allocated equally to 2 groups: KHB-Alb vs CHIP. Physiological parameters, cytokine profiles, and edema formation were compared between treatment groups. RESULTS:Perfused lungs in both groups demonstrated equivalent oxygenation (partial pressure of arterial oxygen/fraction of inspired oxygen ratio >350 mm Hg) and physiological parameters. There was equivalent generation of tumor necrosis factor-α and IL-6, irrespective of perfusate solution used, when comparing CHIP vs KHB-Alb. Pig lungs developed equivalent edema formation between groups (CHIP: 15.8 ± 4.8%, KHB-Alb 19.5 ± 4.4%, P > .05). CONCLUSION:A perfusate derived of common hospital ingredients provides equivalent results to a standard Krebs-Henseleit buffer with 8% serum albumin-based perfusate in NPV-EVLP.

Although lung transplant remains the only option for patients with end-stage lung failure, short preservation times result in an inability to meet patient demand. Successful cryopreservation may ameliorate this problem; however, very little research has been performed on lung cryopreservation due to the inability to prevent ice nucleation or growth. Therefore, this research sought to characterize the efficacy of a small-molecule ice recrystallization inhibitor (IRI) for lung cryopreservation given its well-documented ability to control ice growth. Sprague-Dawley heart-lung blocks were perfused at room temperature using a syringe-pump. Cytotoxicity of the IRI was assessed through the subsequent perfusion with 0.4% (w/v) trypan blue followed by formalinfixation. Ice control was assessed by freezing at a chamber rate of 5 degrees C/min to 20 degrees C and cryofixation using a low-temperature fixative. Post-thaw cell survival was determined by freezing at a chamber rate of 5 degrees C/min to 20 degrees C and thawing in a 37 degrees C water bath before formalin-fixation. In all cases, samples were paraffinembedded, sliced, and stained with eosin. The IRI studied was found to be non-toxic, as cell membrane integrity following perfusion was not significantly different than controls (p = 0.9292). Alveolar ice grain size was significantly reduced by the addition of this IRI (p = 0.0096), and the addition of the IRI to DMSO significantly improved post-thaw cell membrane integrity when compared to controls treated with DMSO alone (p = 0.0034). The techniques described here provide a low-cost solution for rat ex vivo lung perfusion which demonstrated that the ice control and improved post-thaw cell survival afforded by IRI-use warrants further study.

BACKGROUND:Myocardial function declines in a time-dependent fashion during ex situ heart perfusion. Cell death and metabolic alterations may contribute to this phenomenon, limiting the safe perfusion period and the potential of ex situ heart perfusion to expand the donor pool. Our aim was to investigate the etiology of myocardial functional decline in ex situ perfused hearts. METHODS:Cardiac function, apoptosis, effectors and markers of cell death, and metabolic function were assessed in healthy pig hearts perfused for 12 hours. These hearts were perfused in nonworking mode or working mode. RESULTS:Cardiac function declined during ex situ heart perfusion regardless of perfusion mode but was significantly better preserved in the hearts perfused in working mode (11-hour cardiac index/1-hour cardiac index: working mode, 33%; nonworking mode, 10%; p = 0.025). The rate of apoptosis was higher in the ex situ perfused hearts compared with in vivo samples (apoptotic cells: in vivo, 0.13%; working mode, 0.54%; nonworking mode, 0.88%; p < 0.001), but the absolute values were low and out of proportion to the decline in function in either group. Myocardial dysfunction at the end of the perfusion interval was partially rescued by delivery of a pyruvate bolus. CONCLUSIONS:A significant decline in myocardial function occurs over time in hearts preserved ex situ that is out of proportion to the magnitude of myocyte cell death present in dysfunctional hearts. Alterations in myocardial substrate utilization during prolonged ex situ heart perfusion may contribute to this phenomenon and represent an avenue to improve donor heart preservation.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Background. Extended periods of ex vivo lung perfusion (EVLP) lead to several inadvertent consequences including accumulation of lactate and increasing electrolyte concentrations in the perfusate. We sought to determine whether continuous hemodialysis (CHD) of the perfusate would be a suitable modality for improving ionic homeostasis in extended EVLP without compromising functional outcomes. Methods. Twelve porcine lungs were perfused using EVLP for 24 hours. All lungs were ventilated with negative pressure ventilation. Lungs in the treatment group (n = 6) underwent continuous hemodialysis of the perfusate. Functional parameters, edema formation, and histopathologic analysis were used to assess graft function. Electrolyte and lactate profiles were also followed to assess the efficiency of hemodialysis. Results. Lungs in both treatment and control groups demonstrated stable and acceptable oxygenation to 24 hours. Lungs demonstrated a decrease in compliance over time. There was no difference in oxygenation and compliance between groups. CHD-EVLP lungs had higher pulmonary vascular resistance and pulmonary artery pressures. Despite increased perfusion pressures, weight gain at both 11 and 23 hours was not different between groups. Perfusate sodium and lactate concentrations were significantly lower in the CHD-EVLP group. Conclusion. The addition of continuous hemodialysis to EVLP did not improve graft function up to 24 hours despite improved maintenance of perfusate composition.

IntroductionHeart transplantation remains the standard treatment for advanced heart failure. However, waiting lists for donor heart continue to rise all over the world due to the discrepancy between the demand and supply of suitable organs. Ex‐vivo heart perfusion (EVHP) has been proposed as a means improving heart preservation and expanding the donor pool. The clinically available EVHP performs in non‐working mode (NWM). Alternatively, EVHP in working mode (WM) may be better in terms of function assessment and preservation.ObjectivesThe generation of circulating mediators of inflammation during EVHP has not been previously investigated. We hypothesized that inflammatory mediators will be activated during EVHP and that the inflammatory response would play a role in declining of donor heart function.MethodsThe procured porcine hearts were perfused ex vivo in a beating state for 12 hours (normothermic, whole blood‐based perfusate, no steroids) on a custom EVHP apparatus. Group 1 hearts (n=9) were perfused in a working mode (left atrial pressure=6 mmHg, heart rate=100 beats/minutes) for the entire EVHP interval. Group 2 hearts (n=6) were briefly transitioned into a working mode at hours T1, T5, and T11 for inflammatory and functional assessment, but were otherwise perfused in a non‐working mode (left atrial pressure=0 mmHg). In vivo hearts (n=4) without perfusion were treated as baseline control. Cardiac functional parameters were compared between two perfusion modes groups. The pro‐inflammatory cytokine levels in the perfusate and myocardial tissue were measured by ELISA and western blot and compared between two groups.ResultsMyocardial function declined over time but the function parameters were better preserved in WM (T11 cardiac index (mL/minute/gram): WM=6.9±1.0 vs NWM=2.0±1.2, p=0.02; LV stroke work (mm Hg·mL): WM=1012.5±245.7 vs NWM=303.4±121.6, p=0.03). The perfusate concentration of pro‐inflammatory cytokines TNF‐α, IL‐6, IL‐8, IL‐1α, IL‐1β, IL‐18 increased significantly during the 12‐hour perfusion interval (p<0.05), but were not significantly different between WM and NWM groups. The IL‐6 and TNF‐α increased in the left ventricular tissue after perfusion compared to baseline myocardial samples (p<0.05), with no significant difference between WM and NWM.ConclusionsA significant pro‐inflammatory cytokine response is generated during prolonged EVHP in the perfusate and left ventricular tissue, independent of perfusion mode. The inflammatory responses may play role in declining of cardiac function. Further studies are warranted to elucidate the stimulants, consequences of inflammatory responses (I.E. on energy metabolism), and methods to mitigate these effects.Support or Funding InformationCanadian National Transplant Research Program, Canadian Institute of Health Research Grant