This research was funded on behalf of The Hospital Research Foundation.

<ol>
	<li>Chambers, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+international+thoracic+organ+transplant+registry+of+the+international+society+for+heart+and+lung+transplantation:+Thirty-fifth+adult+lung+and+heart-lung+transplant+report-2018;+focus+theme:+Multiorgan+transplantation.">The international thoracic organ transplant registry of the international society for heart and lung transplantation: Thirty-fifth adult lung and heart-lung transplant report-2018; focus theme: Multiorgan transplantation.</a> The Journal of Heart and Lung Transplantation. 37 (10), 1169-1183 (2018).</li><li>Valapour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OPTN/SRTR+2017+annual+data+report:+Lung.">OPTN/SRTR 2017 annual data report: Lung.</a> American Journal of Transplantation. 19, 404-484 (2019).</li><li>Chambers, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+registry+of+the+international+society+for+heart+and+lung+transplantation:+Thirty-fourth+adult+lung+and+heart-lung+transplantation+report-2017;+focus+theme:+Allograft+ischemic+time.">The registry of the international society for heart and lung transplantation: Thirty-fourth adult lung and heart-lung transplantation report-2017; focus theme: Allograft ischemic time.</a> The Journal of Heart and Lung Transplantation. 36 (10), 1047-1059 (2017).</li><li>Klein, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+donation+and+utilization+in+the+united+states,+1999-2008.">Organ donation and utilization in the united states, 1999-2008.</a> American Journal of Transplantation. 10 (4), 973-986 (2010).</li><li>Singh, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+of+refusals+for+donor+quality,+organ+utilization,+and+survival+after+lung+transplantation.">Sequence of refusals for donor quality, organ utilization, and survival after lung transplantation.</a> The Journal of Heart and Lung Transplantation. 38 (1), 35-42 (2019).</li><li>Bhorade, S. M., Vigneswaran, W., McCabe, M. A., Garrity, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liberalization+of+donor+criteria+may+expand+the+donor+pool+without+adverse+consequence+in+lung+transplantation.">Liberalization of donor criteria may expand the donor pool without adverse consequence in lung transplantation.</a> The Journal of Heart and Lung Transplantation. 19 (12), 1199-1204 (2000).</li><li>Snell, G. I., Griffiths, A., Levvey, B. J., Oto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Availability+of+lungs+for+transplantation:+Exploring+the+real+potential+of+the+donor+pool.">Availability of lungs for transplantation: Exploring the real potential of the donor pool.</a> The Journal of Heart and Lung Transplantation. 27 (6), 662-667 (2008).</li><li>Cypel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normothermic+ex+vivo+lung+perfusion+in+clinical+lung+transplantation.">Normothermic ex vivo lung perfusion in clinical lung transplantation.</a> The New England Journal of Medicine. 364 (15), 1431-1440 (2011).</li><li>Wallinder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+results+in+transplantation+of+initially+rejected+donor+lungs+after+ex+vivo+lung+perfusion:+A+case-control+study.">Early results in transplantation of initially rejected donor lungs after ex vivo lung perfusion: A case-control study.</a> European Journal of Cardio-Thoracic Surgery. 45 (1), 40-45 (2014).</li><li>Cypel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience+with+the+first+50+ex+vivo+lung+perfusions+in+clinical+transplantation.">Experience with the first 50 ex vivo lung perfusions in clinical transplantation.</a> The Journal of Thoracic and Cardiovascular Surgery. 144 (1), 1200-1206 (2012).</li><li>Buchko, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+parenteral+nutrition+in+ex+vivo+lung+perfusion:+Addressing+metabolism+improves+both+inflammation+and+oxygenation.">Total parenteral nutrition in ex vivo lung perfusion: Addressing metabolism improves both inflammation and oxygenation.</a> American Journal of Transplantation. 19 (12), 3390-3397 (2019).</li><li>Andreasson, A. S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+inflammation+and+tissue+injury+markers+in+perfusate+and+bronchoalveolar+lavage+fluid+during+human+ex+vivo+lung+perfusion.">Profiling inflammation and tissue injury markers in perfusate and bronchoalveolar lavage fluid during human ex vivo lung perfusion.</a> European Journal of Cardio-Thoracic Surgery. 51 (3), 577-586 (2017).</li><li>Sadaria, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+expression+profile+in+human+lungs+undergoing+normothermic+ex-vivo+lung+perfusion.">Cytokine expression profile in human lungs undergoing normothermic ex-vivo lung perfusion.</a> The Annals of Thoracic Surgery. 92 (2), 478-484 (2011).</li><li>Ricard, J. D., Dreyfuss, D., Saumon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilator-induced+lung+injury.">Ventilator-induced lung injury.</a> European Respiratory Journal. 42, 2-9 (2003).</li><li>Aboelnazar, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+pressure+ventilation+decreases+inflammation+and+lung+edema+during+normothermic+ex-vivo+lung+perfusion.">Negative pressure ventilation decreases inflammation and lung edema during normothermic ex-vivo lung perfusion.</a> The Journal of Heart and Lung Transplantation. 37 (4), 520-530 (2018).</li><li>Lai-Fook, S. J., Rodarte, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleural+pressure+distribution+and+its+relationship+to+lung+volume+and+interstitial+pressure.">Pleural pressure distribution and its relationship to lung volume and interstitial pressure.</a> Journal of Applied Physiology. 70 (3), 967-978 (1991).</li><li>Buchko, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+transplantation+using+negative+pressure+ventilation+ex+situ+lung+perfusion+with+extended+criteria+donor+lungs.">Clinical transplantation using negative pressure ventilation ex situ lung perfusion with extended criteria donor lungs.</a> Nature Communications. 11 (1), 5765 (2020).</li><li>Buchko, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost+perfusate+alternative+for+ex+vivo+perfusion.">A low-cost perfusate alternative for ex vivo perfusion.</a> Transplantation Proceedings. 52 (10), 2941-2946 (2020).</li><li>Forgie, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+lung+orthotopic+transplantation+in+a+juvenile+porcine+model+for+ESLP.">Left lung orthotopic transplantation in a juvenile porcine model for ESLP.</a> The Journal of Visualized Experiments. , (2021).</li></ol>

DHF holds patents on ex situ organ perfusion technology and methods. DHF and JN are founders and major shareholders of Tevosol, Inc.

There are several critical surgical steps along with troubleshooting needed to ensure a successful ESLP run. Juvenile porcine lungs are extremely delicate compared to adult human lungs, so the procuring surgeon must be cautious when handling porcine lungs. It is critical to attempt a &#34;no-touch&#34; technique to avoid causing trauma and atelectasis when dissecting out the lungs. &#34;No-touch&#34; means using the bare minimum amount of manual manipulation of the lungs during procurement. Recruitment maneuvers while on...

Lung transplantation (LTx) remains the standard of care for end-stage lung disease1; however, LTx has significant limitations including inadequate donor organ utilization2 and a waitlist mortality of 40%3, which is higher than any other solid organ transplant4,5. Donor organ utilization rates are low (20-30%) due to organ quality concerns. Excessive geographic transportation distance compounded by stringent donor organ acceptance criteria exacerbates these quality concerns. LTx also trails other solid organ transplants in terms of long-term graft and patient outcomes2. Primary graft dysfunction (PGD), most often caused by ischemic reperfusion injury (IRI), represents the leading cause of 30-day mortality and morbidity post-LTx and increases the risk for chronic graft dysfunction6,7. Efforts to decrease IRI and extend safe transport times are paramount to improve patient outcomes.
Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. ESLP facilitates the preservation, assessment, and reconditioning of donor lungs before transplantation. It has exhibited satisfactory short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs, contributing to an increase in the number of suitable donor lungs for LTx, with organ utilization rates increasing by 20% in some centres8,9,10. Compared to the current clinical standard for LTx, cold static preservation (CSP), ESLP offers several advantages: organ preservation time is not limited to 6 h, evaluation of organ function is possible before implantation, and due to continuous organ perfusion, modifications can be made to the perfusate that optimizes organ function11.
The vast majority of current ESLP devices designed for human use utilize positive pressure ventilation (PPV); however, recent literature has indicated that this ventilation strategy is inferior to negative pressure ventilation (NPV) ESLP, with PPV inducing more significant ventilator-induced lung injury12,13,14,15. In both human and porcine lungs, NPV-ESLP exhibits superior organ function when compared to positive pressure ex situ lung perfusion (PPV-ESLP) across various physiological domains, including pro-inflammatory cytokine production, pulmonary edema, and bullae formation15. The homogenous distribution of intrathoracic pressure across the entire lung surface in NPV-ESLP has been suggested as a significant factor underlying this advantage15,16. In addition to its pre-clinical benefits, the clinical safety and feasibility of NPV-ESLP have been demonstrated in a recent clinical trial17. Utilizing a novel NPV-ESLP device, twelve extended criteria donor human lungs were successfully preserved, evaluated, and subsequently transplanted with 100% 30-day and 1-year survival.
The objective of the present manuscript is to demonstrate a working protocol of our lab&#39;s NPV-ESLP device using juvenile porcine lungs under normothermic conditions for 12 h of duration. The surgical retrieval is covered in detail, and our custom software platform&#39;s initiation, management, and termination are also described. The strategy for tissue collection and the management of the samples is also explained.

<table><tbody><tr><td>0 ETHIBOND Green 1 x 36&quot; Endo Loop 0</td><td>ETHICON</td><td>D8573</td><td></td></tr><tr><td>2-0 SILK Black 12&quot; x 18&quot; Strands</td><td>ETHICON</td><td>SA77G</td><td></td></tr><tr><td>ABL 800 FLEX Blood Gas Analyzer</td><td>Radiometer</td><td>989-963</td><td></td></tr><tr><td>Adult-Pediatric Electrostatic Filter HME - Small</td><td>Covidien</td><td>352/5877</td><td></td></tr><tr><td>Arterial Filter</td><td>SORIN GROUP</td><td>01706/03</td><td></td></tr><tr><td>Backhaus Towel Clamp</td><td>Pilling</td><td>454300</td><td></td></tr><tr><td>Biomedicus Pump</td><td>Maquet</td><td>BPX-80</td><td></td></tr><tr><td>Cable Ties &amp;ndash; White 12&amp;rdquo;</td><td>HUASU International</td><td>HS4830001</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher Scientific</td><td>C69-500G</td><td></td></tr><tr><td>Cooley Sternal Retractor</td><td>Pilling</td><td>341162</td><td></td></tr><tr><td>CUSHING Gutschdressing Forceps</td><td>Pilling</td><td>466200</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G5767-500G</td><td></td></tr><tr><td>Deep Deaver Retractor</td><td>Pilling</td><td>481826</td><td></td></tr><tr><td>Debakey Straight Vascular Tissue Forceps</td><td>Pilling</td><td>351808</td><td></td></tr><tr><td>Debakey-Metzenbaum Dissecting</td><td>Pilling</td><td>342202</td><td></td></tr><tr><td>Scissors</td><td>Pilling</td><td>342202</td><td></td></tr><tr><td>Endotracheal Tube 9.0mm CUFD</td><td>Mallinckrodt</td><td>9590E</td><td>Cuff removed for ESLP apparatus</td></tr><tr><td>Flow Transducer</td><td>BIO-PROBE</td><td>TX 40</td><td></td></tr><tr><td>Human Albumin Serum</td><td>Grifols Therapeutics</td><td>2223708</td><td></td></tr><tr><td>Infusion Pump</td><td>Baxter</td><td>AS50</td><td></td></tr><tr><td>Inspire 7 M Hollow Fiber Membrane Oxygenator</td><td>SORIN GROUP</td><td>K190690</td><td></td></tr><tr><td>Intercept Tubing 1/4&quot; x 1/16&quot; x 8&#039;</td><td>Medtronic</td><td>3108</td><td></td></tr><tr><td>Intercept Tubing 3/8&quot; x 3/32&quot; x 6&#039;</td><td>Medtronic</td><td>3506</td><td></td></tr><tr><td>Intercept Tubing Connector 3/8&quot; x 1/2&quot;</td><td>Medtronic</td><td>6013</td><td></td></tr><tr><td>MAYO Dissecting Scissors</td><td>Pilling</td><td>460420</td><td></td></tr><tr><td>Medical Carbon Dioxide Tank</td><td>Praxair</td><td>5823115</td><td></td></tr><tr><td>Medical Nitrogen Tank</td><td>Praxair</td><td>NI M-K</td><td></td></tr><tr><td>Medical Oxygen Tank</td><td>Praxair</td><td>2014408</td><td></td></tr><tr><td>Organ Chamber</td><td>Tevosol</td><td></td><td></td></tr><tr><td>PlasmaLyte A</td><td>Baxter</td><td>TB2544</td><td></td></tr><tr><td>Poole Suction Tube</td><td>Pilling</td><td>162212</td><td></td></tr><tr><td>Potassium Phosphate</td><td>Fischer Scientific</td><td>P285-500G</td><td></td></tr><tr><td>Scale</td><td>TANITA</td><td>KD4063611</td><td></td></tr><tr><td>Silicon Support Membrane</td><td>Tevosol</td><td></td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>792519-1KG</td><td></td></tr><tr><td>Sodium Chloride 0.9%</td><td>Baxter</td><td>JB1324</td><td></td></tr><tr><td>Sorin XTRA Cell Saver</td><td>SORIN GROUP</td><td>75221</td><td></td></tr><tr><td>Sternal Saw</td><td>Stryker</td><td>6207</td><td></td></tr><tr><td>Surgical Electrocautery Device</td><td>Kls Martin</td><td>ME411</td><td></td></tr><tr><td>Temperature Sensor probe</td><td>Omniacell Tertia Srl</td><td>1777288F</td><td></td></tr><tr><td>THAM Buffer</td><td>Thermo Fisher Scientific</td><td>15504020</td><td>made from UltraPureTM Tris</td></tr><tr><td>TruWave Pressure Transducer</td><td>Edwards</td><td>VSYPX272</td><td></td></tr><tr><td>Two-Lumen Central Venous Catheter 7fr</td><td>Arrowg+ard</td><td>CS-12702-E</td><td></td></tr><tr><td>Vorse Tubing Clamp</td><td>Pilling</td><td>351377</td><td></td></tr><tr><td>Willauer-Deaver Retractor</td><td>Pilling</td><td>341720</td><td></td></tr><tr><td>Yankauer Suction Tube</td><td>Pilling</td><td>162300</td><td></td></tr></tbody></table>

normothermic negative pressure ventilation ex situ lung perfusion: evaluation of lung function and metabolism

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.

At the beginning of lung perfusion and ventilation (preservation mode), the lungs will generally have a low pulmonary artery pressure (&#60; 10 mmHg) and low dynamic compliance (&#60; 10 mL/mmHg) as the perfusate warms to normothermia. Yorkshire pigs weighing 35-50 kg typically results in lungs weighing 350-500 g. During the first hour of NPV-ESLP, the measured expiratory tidal volumes (TVe) are 0-2 mL/kg, and the inspiratory tidal volumes (TVi) are 100-200 mL. TVe generally reaches 4-6 mL/kg within 3-6 h, and after that...

Watch this Scientific Journal Video about Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism at JoVE.com

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

normothermic-negative-pressure-ventilation-ex-situ-lung-perfusion

Research

JoVE Journal

Medicine

2.1K Views.  University of Alberta. Dr. Darren Freed creëerde dit ESLP-apparaat en het maakt gebruik van gepatenteerde negatieve drukventilatietechnologie en -software, die nergens anders op het gebied van ESLP-onderzoek te zien is. Ons lab heeft eerder aangetoond dat negatieve druk ventilatie minder longletsel veroorzaakt tijdens ESLP in vergelijking met positieve druk beademing. Dit is waarschijnlijk te wijten aan het meer fysiologische mechanisme van negatieve drukventilatie.Dit apparaat en protocol zijn ontworpen om...

Video: Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

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.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Isolated Lung Perfusion System in the Rabbit Model

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.

The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

Since the establishment of lung transplantation as a therapeutic strategy for advanced lung diseases, the scientific community is faced with the problem of a low number of lungs considered viable for the donation process. In recent decades, however, this scenario has been positively changed, given the development of ex vivo lung perfusion (EVLP) as a strategy for evaluating and reconditioning marginal lungs. The establishment of EVLP in large transplant centers has favored an increase in the number of lung transplants, both by increasing the diagnostic accuracy of lung function and by constituting an effective platform for the reconditioning of lung grafts. In this context, faced with ethical and logistical issues, as well as in the study of immunological factors associated with lung transplantation, the development of rodent EVLP models has become important, given their reliability, the possibility of genetic manipulation, and lower costs. This paper describes a protocol for establishing a rat EVLP model and shows the inflammatory profile associated with the perfused lungs. This will help propagate knowledge about the rat EVLP model, promoting our understanding of the biological responses associated with that revolutionary technique.

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

Here, we describe the necessary steps for establishing a rat EVLP model and show the inflammatory profile associated with the perfused lungs. The aim is to propagate knowledge and experiences about the rat EVLP model, enabling the integral understanding of the biological responses associated with that revolutionary technique.

Since the establishment of lung transplantation as a therapeutic strategy for advanced lung diseases, the scientific community is faced with the problem ...

Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics

Mechanical ventilation is widely used and requires specific knowledge for understanding and management. Health professionals in this field may feel insecure and lack knowledge because of inadequate training and teaching methods. Therefore, the objective of this article is to outline the steps involved in generating an ex vivo porcine lung model to be used in the future, to study and teach lung mechanics. To generate the model, five porcine lungs were carefully removed from the thorax following the guidelines of the Animal Research Ethics Committee with adequate care and were connected to the mechanical ventilator through a tracheal cannula. These lungs were then subjected to the alveolar recruitment maneuver. Respiratory mechanics parameters were recorded, and video cameras were used to obtain videos of the lungs during this process. This process was repeated for five consecutive days. When not used, the lungs were kept refrigerated. The model showed different lung mechanics after the alveolar recruitment maneuver every day; not being influenced by the days, only by the maneuver. Therefore, we conclude that the ex vivo lung model can provide a better understanding of lung mechanics and its effects, and even of the alveolar recruitment maneuver through visual feedback during all stages of the process.

Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics

We present an ex vivo pig lung model for the demonstration of pulmonary mechanics and alveolar recruitment maneuvers for teaching purposes. The lungs can be used for more than one day (up to five days) with minimal changes in pulmonary mechanics variables.

Mechanical ventilation is widely used and requires specific knowledge for understanding and management. Health professionals in this field may feel ...

Advancing Lung Transplantation with PolyhHb-Based Perfusion in Rat EVLP Models

Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. However, new and exciting technology, such as ex vivo lung perfusion (EVLP), offers lung transplant providers extended assessment for marginal donor allografts. This dynamic assessment platform has led to an increase in lung transplantation and has allowed providers to use donors that were previously discarded, thus expanding the donor pool. Current perfusion techniques use cellular or acellular perfusates, and both have distinct advantages and disadvantages. Perfusion composition is critical to maintaining a homeostatic environment, providing adequate metabolic support, decreasing inflammation and cellular death, and ultimately improving organ function. Perfusion solutions must contain sufficient protein concentration to maintain appropriate oncotic pressure. However, current perfusion solutions often lead to fluid extravasation through the pulmonary endothelium, resulting in inadvertent pulmonary edema and damage. Thus, it is necessary to develop novel perfusion solutions that prevent excessive damage while maintaining proper cellular homeostasis. Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat EVLP. The goal of this study is to provide the lung transplant community with key information in designing and developing novel perfusion solutions, as well as the proper protocols to test them in clinically relevant translational transplant models.

Author Spotlight: Utilizing Next-Generation Polymerized Human Hemoglobin for Improved Donor Lung Evaluation and Preservation in Rats

Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat ex vivo lung perfusion.

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. ...

Evaluation of Apoptosis and Lung Injury in DCD Donor Lungs Preserved by EVLP

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after cardiac death (DCD). However, a critical challenge remains in maximizing the number of viable lungs that meet stringent criteria for clinical use. Ex vivo lung perfusion (EVLP) emerges as a promising technology to evaluate and potentially improve the function of donated lungs. This paper describes the development of a cost-effective and reproducible small animal experimental platform specifically designed for EVLP research. This platform offers a high degree of customization, enabling researchers to tailor experimental parameters to address specific research questions within the field of EVLP. The team established a rat model to simulate DCD and implemented a two-stage preservation approach. Lungs were subjected to static cold storage using a perfusate solution at 4 &#176;C for 4 h, followed by normothermic EVLP for another 4 h. Researchers evaluated various lung function parameters, including pulmonary vascular resistance, pulmonary dynamic compliance, blood gas measurements of the perfusate, and apoptotic cell death in lung tissues. The study demonstrates that EVLP could significantly improve the functional performance of DCD lungs compared to simple cold storage at 4 &#176;C, suggesting that EVLP has the potential to mitigate ischemia-reperfusion injury and enhance graft function following circulatory death, thereby increasing the pool of transplantable lungs.

The objective is to develop a new ex vivo lung perfusion (EVLP) model in rats that can sustain consistent lung function for 4 h throughout EVLP and furnish the industry with implementation specifics that will offer a sturdy method for applying EVLP to rodent models.

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after ...

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro models. Laboratories wishing to use isolated lungs need to be aware of important steps and inherent challenges to establish a setup that is affordable, reliable, and that can be easily adapted to fit the topic of interest. This paper describes a DIY (do it yourself) model for ex vivo rat lung ventilation and perfusion to study drug and gas effects on pulmonary vascular tone, independent of changes in cardiac output. Creating this model includes a) the design and construction of the apparatus, and b) the lung isolation procedure. This model results in a setup that is more cost-effective than commercial alternatives and yet modular enough to adapt to changes in specific research questions. Various obstacles had to be resolved to ensure a consistent model that is capable of being used for a variety of different research topics. Once established, this model has proven to be highly adaptable to different questions and can easily be altered for different fields of study.

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs.

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro ...

Biology

Evaluation of Lung Function in an Ex vivo Murine Model

Respiratory mechanics are a key area of study in defining and treating lung pathologies by assessing functional lung capacity. Lung mechanics can be evaluated through various lung maneuvers that involve different oscillatory waveforms. When applied to the lungs, these maneuvers measure multiple variables, such as pressure, volume, and flow, based on the response to the waveforms. These signals are then computed and analyzed to determine parameters such as hysterisivity, resistance, compliance, tissue damping, and tissue elastance, providing a detailed assessment of overall lung function. The analysis of respiratory mechanics is particularly important in evaluating donor lungs for lung transplantation. The present protocol is the first of its kind, offering a comprehensive and reproducible stepwise method for assessing respiratory mechanics using an ex vivo murine model. It includes details on the selected animal model, lung recovery, storage and preservation, and experimentation using a forced oscillation technique-based system. Additionally, it outlines data analysis, clinical significance, and the applications of the forced oscillation technique in studying an ex vivo model.

The present protocol describes a stepwise method for analyzing the respiratory mechanics of an ex vivo murine model using the forced oscillation technique (FOT).

Respiratory mechanics are a key area of study in defining and treating lung pathologies by assessing functional lung capacity. Lung mechanics can be ...

Ex vivo Lung Perfusion: A Technique for Pulmonary Research in a Rabbit Model

Source: Pacheco-Baltazar, A., et al., <a href="https://www.jove.com/v/62734">Isolated Lung Perfusion System in the Rabbit Model.</a> J. Vis. Exp. (2021).
This video shows an ex vivo technique for isolated lung perfusion. It is a tool for pulmonary research that will aid studies of various physiological and pathological mechanisms involved in lung transplantation procedures.

Source: Pacheco-Baltazar, A., et al., Isolated Lung Perfusion System in the Rabbit Model. J. Vis. Exp. (2021).
This video shows an ex vivo technique for ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Samenvatting

Meer video's verkennen