Name: Thermal Preconditioning During Ex-vivo Lung Perfusion for the Rehabilitation of Damaged Lung Grafts before Transplantation (Video) | JoVE Journal
Uploaded: 2025-10-31T13:30:00.000+00:00
Duration: 9 min 34 s
Description: Learn thermal preconditioning for lung graft rehabilitation using an ex-vivo rat perfusion model. This method activates protective mechanisms to improve transplant success without drugs.

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5 minutes

Supported by a grant from the Swiss National Science Foundation (Nr 310030_212252) to TK and LL).

The authors have no conflicts of interest to declare.

The development of a reproducible and physiologically relevant rat EVLP model represents a significant step forward in lung transplantation research. Despite significant species differences between the rat and human lung (recently reviewed in1), notably the greater vulnerability of the rat lung, the rat EVLP model offers pharmacological and non-pharmacological interventions with high reproducibility and low cost. EVLP offers a unique platform to evaluate donor lung function, simulate clinical injury scenarios such as ischemia-reperfusion injury, and implement therapeutic interventions prior to transplantation2,39,40. In this context, the rat EVLP model enables high-throughput mechanistic investigations under tightly controlled conditions33. In recent years, several groups have contributed to the refinement of rat EVLP techniques. Nelson et al. provided foundational procedural guidance39. Wang et al. proposed a modular EVLP setup tailored to mimic donation after circulatory death (DCD) simulation41. Gouchoe et al. explored novel perfusate formulations using artificial oxygen carriers42 and Oliveira et al. focused on inflammation after long cold ischemia preservation38. Building on this existing knowledge, our current protocol introduces a novel, non-pharmacological strategy, namely thermal preconditioning, designed to activate endogenous protective mechanisms for the reconditioning of rat lung grafts damaged by warm ischemia. Our damaged lung model, which simulates DCD conditions, was performed in male Sprague-Dawley rats33.
The establishment of this protocol required careful attention to several technically demanding steps that critically influence lung viability and reproducibility. Lung retrieval must be performed with minimal manipulation to avoid parenchymal injury. The depth of anesthesia is paramount to prevent gasping-induced pulmonary congestion and poor exsanguination. Stringent avoidance of air bubbles during cannulation and circuit priming is essential, as even small emboli can precipitate edema43. Specific troubleshooting advice for common protocol challenges is indicated in Table 3. While ventilation and perfusion settings can be adjusted within safe limits, lung-protective strategies must be maintained to prevent barotrauma or volutrauma44. The perfusate flow can be increased up to 20% of the estimated cardiac output in rats, compared to 40% in larger models like pigs and humans, but must be carefully titrated45. In our protocol, we use lower perfusate flow (7.5% of theoretical cardiac output), to prevent surges of pulmonary artery pressure and the development of early pulmonary edema in warm ischemic damaged lungs, thereby allowing extended preparation. Although some laboratories administer corticosteroids during perfusion to reduce inflammation46,47, their known anti-inflammatory effects could confound some experimental outcomes, and we therefore do not use them in our model to preserve the ability to study lung injury and inflammatory processes.
Thermal preconditioning requires precise temperature control. Because heat is lost along the tubing from reservoir to lung, the perfusate temperature must be monitored at the inflow cannula, and the water-bath setting kept slightly above the desired inflow temperature to achieve the target within the preparation. Sensors are calibrated annually using a Multi-Channel PCE-T 1200 Digital Thermometer to ensure accuracy.
Our findings also address key limitations of conventional heat therapy. Systemic heat application is constrained by imprecise temperature regulation, systemic side effects, and lack of organ specificity. EVLP circumvents these barriers by providing a controlled, localized environment, allowing precise delivery of thermal stress directly to the lung tissue29. This method enables rigorous modulation of both the magnitude and duration of the thermal stress, two critical parameters for optimizing the protective cellular response without triggering thermal injury. Moreover, our EVLP platform allows multiple downstream evaluations, including molecular, histological, and functional analyses. Importantly, lungs subjected to EVLP can also be transplanted, allowing for the direct evaluation of post-EVLP graft function in vivo29. Thus, the model supports assessment at both the isolated-organ and post-transplantation levels. In this context, the integration of thermal preconditioning, a transient exposure to 41.5 &#176;C during EVLP, emerges as a promising intervention. By stimulating a heat shock response and other stress-adaptive pathways, this method enhances graft resilience without pharmacological confounders. Our results show that thermal preconditioning improves lung graft physiology, reduces edema formation and the release of inflammatory mediators and biomarkers of cellular injury (as exemplified by the reduced levels of von Willebrand factor in the EVLP perfusate of heat-treated lungs). Morphological analyses further support the protective effect of this intervention, with reduced structural damage and preserved tissue integrity. Most experimental rat EVLP data have been generated in males, limiting sex-specific insight; two studies reported divergent outcomes in females-reduced reperfusion injury in one and increased inflammation in another48,49. Although our study used males, we previously observed TP benefits in a porcine EVLP model using females29, suggesting relevance across sexes.
Important constraints and drawbacks of our method need to be emphasized. First, to guarantee that the lungs are perfused with the actual desired temperature, the latter must be monitored at the inflow cannula by firmly applying the temperature sensor. Second, it is mandatory that the temperature changes be obtained within very short time periods, which requires the use of a rapid response thermal water bath for heating and the addition of ice to the water bath for rapid cooling. Third, the physiological response to heat stress may differ according to the species used and is highly dependent on the level and duration of the stress. Therefore, it is mandatory to perform pilot experiments using various heating temperatures for various durations in case this protocol is applied to different animal species or human lungs and evaluate specific endpoints pertaining both to protective and to potential detrimental effects of the applied heat stress. Fourth, owing to the kinetic expression of heat-responsive genes, the effects of thermal preconditioning depend on the recovery time following heat stress application. Experiments using different post-heat stress durations (that is, with different durations of EVLP) are required to assess such time-dependent effects of heat application, as shown in our recent publication on this topic30.
Altogether, our current study establishes a detailed, reproducible rat EVLP protocol incorporating thermal preconditioning as a viable strategy to improve marginal donor lung quality. The protocol is scalable and adaptable to larger animal or human EVLP systems, reinforcing its translational relevance. By providing both methodological rigor and physiological fidelity, this model opens new avenues for exploring non-pharmacological interventions aimed at reducing ischemia-reperfusion injury and improving lung graft outcomes after transplantation.

Ex-vivo lung perfusion (EVLP) has represented a major advance in the field of lung transplantation. Its foremost impact lies in expanding the pool of available donor lung grafts by enabling the functional assessment of marginal lungs to allow the use of organs that would otherwise be discarded for transplantation1,2. Marginal (or extended criteria) donor lungs refer to one or more of the following criteria: donor age more than 55, PaO2/FiO2 of less than 300, ischemia time of more than 6 h, positive sputum microbiology, abnormal radiography3. In addition to physiological evaluation, EVLP may serve as a platform for the application of various therapeutic interventions aimed at improving graft quality prior to transplantation (concept of therapeutic reconditioning)4. As such, EVLP provides the unique opportunity to rehabilitate damaged (marginal) lungs and reduce the risk of primary graft dysfunction (PGD), a major complication impairing both early and long-term outcomes after lung transplantation5,6. PGD is the clinical expression of the process of ischemia-reperfusion injury (IRI), whose pathophysiology relies on oxidative stress, inflammation, the activation of the complement and coagulation cascades, and the induction of cell death processes7,8,9. Ultimately, these mechanisms contribute to disrupting normal functions of the lung endothelium and epithelium, leading to non-cardiogenic pulmonary edema with reduced oxygenation capacity, the two key hallmarks of PGD6,10,11,12.
A growing number of experimental studies, both preclinical and clinical, have demonstrated the efficacy of various ex-vivo therapeutic strategies to alleviate IRI during lung transplantation. Examples include pharmacological agents targeting oxidants and free radicals, cell death pathways, inflammatory mediators, as well as cellular-based and gene therapies, to name only a few13,14,15,16,17,18,19,20. Beyond such therapeutic approaches, EVLP may also serve as a platform for the application of physiological stress directly to the lung graft, to elicit intrinsic protective mechanisms providing an adaptive tolerance to subsequent pathophysiological insults. One such strategy involves the application of transient, non-lethal heat stress to the lung graft during EVLP. Indeed, it has been known for a long time that conditions of heat stress promote an adaptive heat shock response, resulting from the expression of a series of heat shock proteins (HSPs) acting as molecular chaperones and conferring resistance to further exposure to various stressful stimuli21. Besides HSP induction, some additional cellular adaptations have been identified following heat exposure, including (a) the NF-E2 related factor 2 (NRF2) antioxidant and cytoprotective pathway, whose activation upon heat stress notably upregulates the cytoprotective gene heme oxygenase22,23; (b) the unfolded protein response (UPR), dealing with proteotoxic stress in the endoplasmic reticulum (ER stress), and whose activation by heat may be either protective or detrimental, depending on the intensity of the thermal stress24,25,26; (c) autophagy, a cytoprotective process promoting lysosomal degradation of damaged organelles and macromolecules, which can be activated or inhibited by heat in a temperature and time-dependent manner27,28.
We have demonstrated that such &#34;thermal preconditioning&#34; provides major benefits in terms of reduced oxidative stress, inflammation, cell death, and endothelial dysfunction, leading to significant protection against lung IRI. Therefore, we proposed that thermal preconditioning may represent a novel, non-pharmacological method for ex-vivo therapeutic rehabilitation of damaged lung grafts prior to transplantation29,30,31. Thermal preconditioning may offer several advantages over existing pharmacological and non-pharmacological strategies. By eliciting an integrated cellular endogenous response, it may simultaneously provide protection against distinct pathophysiological pathways pertaining to IRI, whereas other strategies generally target a single pathway. Further, it avoids the risk of potential side effects associated with the use of pharmacological compounds. The aim of the present article is to present a detailed step-by-step protocol of thermal preconditioning during ex-vivo lung perfusion of damaged rat lungs, which could be applied across diverse settings, notably to larger animal models or human lungs for research purposes.
We utilize the rat isolated perfused lung system IPL-2 for EVLP. The setup is described in Figure 1 and includes the following: Perfusate reservoir (Figure 1-1); two roller pumps, for perfusate circulation (Figure 1-2A) and perfusate sampling (Figure 1-2B); a thermostatic water bath (Figure 1-3) for the maintenance of perfusate temperature; a membrane gas exchanger (Figure 1-4) connected to a gas tank (not shown) containing a mixture of 6% O2, 10% CO2, and 84% N2 to deoxygenate and carboxylate the inflow perfusate; two pressure sensors (Figure 1-5) to monitor left atrial (LA) and pulmonary artery (PA) pressures (the PA pressure sensor is not visible on the figure); a heated and sealed chamber (Figure 1-6) housing the heart-lung block with dedicated connections for PA, LA, and tracheal cannulas; a ventilator (Figure 1-7) connected to the tracheal cannula for lung ventilation and for the measurement of airway pressure and static lung compliance; an electronic thermometer (Figure 1-8) to display the perfusate temperature at the level of the PA inflow. A connection platform (Figure 1-9) whose elements are displayed in detail in Figure 2: Connection for the PA (Figure 2-1), the LA (Figure 2-2) and tracheal (Figure 2-3) cannulas; connection to a weight module (Figure 2-4) for the continuous monitoring of the weight of the heart-lung block; A temperature sensor (Figure 2-5) for the monitoring of the perfusate temperature at the PA inflow. A detailed schematic description of the circuit can be found elsewhere18.

<table><tbody><tr><td>&lt;strong&gt;General equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aneurysm clip&amp;nbsp;</td><td>B. Braun</td><td>FE762K</td><td></td></tr><tr><td>Air/surface bead thermal sensor</td><td>TES Electrical Electronic Corp/Distrelec AG</td><td>TP01/176-67-162</td><td></td></tr><tr><td>Blunt Retractors</td><td>Fine Science Tools</td><td>18200-09</td><td></td></tr><tr><td>Bulldog Serafines</td><td>Fine Science Tool</td><td>18051-50</td><td></td></tr><tr><td>Gaze Compresses&amp;nbsp;</td><td>Promedical</td><td>25404</td><td></td></tr><tr><td>Connecting kit for anesthesia</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3076</td><td></td></tr><tr><td>Cotton Swabs</td><td>Applimed SA</td><td>6001109</td><td></td></tr><tr><td>Dissecting Scissors</td><td>B. Braun&amp;nbsp;</td><td>BC165R</td><td></td></tr><tr><td>Facial mask for rat</td><td>Tem Sega</td><td>PFM0006</td><td></td></tr><tr><td>Heparin 5000 U/mL</td><td>B. Braun</td><td>3522430</td><td></td></tr><tr><td>Induction chamber for anesthesia</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>50-0116</td><td></td></tr><tr><td>Iris Dissecting Forceps Full Curved</td><td>Aesculap</td><td>&amp;nbsp;OC022R</td><td></td></tr><tr><td>Isofluran</td><td>Provet AG</td><td>2222</td><td></td></tr><tr><td>Isofluran vapor 19.3</td><td>Rothacher medical</td><td>CV30-310 O&lt;sub&gt;2&lt;/sub&gt;/Air</td><td></td></tr><tr><td>iSTAT4 cartridge</td><td>Axon lab AG</td><td>03P85-25</td><td></td></tr><tr><td>Ketasol 100 (100 mg/mL)</td><td>Dr. E. Graeub AG</td><td>QN01AX03</td><td></td></tr><tr><td>Lister scissors&amp;nbsp;</td><td>B. Braun</td><td>BC876R</td><td></td></tr><tr><td>Micro Scissors, Curved Blunt/Blunt</td><td>Aesculap&amp;nbsp;</td><td>FM011R</td><td></td></tr><tr><td>Micro-Adson Forceps</td><td>Fine Science Tool</td><td>11018-12</td><td></td></tr><tr><td>Micro-Mosquito Hemostats</td><td>Fine Science Tool</td><td>13011-12</td><td></td></tr><tr><td>Microscop OPMI MDU</td><td>Zeiss</td><td>267067</td><td></td></tr><tr><td>NaCl 0.9 %&amp;nbsp;</td><td>B. Braun</td><td>534534</td><td></td></tr><tr><td>Needle 23 G x 25 mm</td><td>BD Microlance 4</td><td>300800</td><td></td></tr><tr><td>Needle 25 G x 25 mm</td><td>BD Microlance 3</td><td>300400</td><td></td></tr><tr><td>Perfadex (preservation solution)</td><td>Xvivo perfusion AB</td><td>19811</td><td></td></tr><tr><td>Perfusion syringe 60 ml luer lock</td><td>BD plastik</td><td>300865</td><td></td></tr><tr><td>Rasor</td><td>Aesculap&amp;nbsp;</td><td>GT-421</td><td></td></tr><tr><td>Scale</td><td>Mettler toledo</td><td>PB602-L</td><td></td></tr><tr><td>Silk suture 4-0</td><td>B. Braun</td><td>F1134035</td><td></td></tr><tr><td>Silk suture 5-0</td><td>B. Braun</td><td>F1134027</td><td></td></tr><tr><td>Steen (perfusion solution)</td><td>Xvivo perfusion AB</td><td>19004</td><td></td></tr><tr><td>Surgical gloves</td><td>Ansell</td><td>93-843</td><td></td></tr><tr><td>Syringe 1 mL, Omnifix-F</td><td>B. Braun</td><td>9161406V</td><td></td></tr><tr><td>Tape</td><td>Leukofix</td><td>02136-00</td><td></td></tr><tr><td>Thermometer</td><td>TES Electrical Electronic Corp</td><td>1303</td><td></td></tr><tr><td>Tubes containing Formol&amp;nbsp;</td><td>Biosystems Switzerland AG</td><td>87-0960-00-10113</td><td></td></tr><tr><td>Ventilator (EVLP) (Set tidal volume according to EVLP protocol)</td><td>Flexivent</td><td>FX3</td><td></td></tr><tr><td>Ventilator (lung procurement)&amp;nbsp; (Set tidal volume as: 7.69*(body weight (kg))^1.04)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>683</td><td></td></tr><tr><td>WEITLANER Retractor</td><td>B. Braun</td><td>BV073R</td><td></td></tr><tr><td>Xylasol (1 mg/mL)</td><td>Dr. E. Graeub AG</td><td>QN05CM92</td><td></td></tr><tr><td>Yasargil applier</td><td>B. Braun</td><td>FE502T</td><td></td></tr><tr><td>&lt;strong&gt;IPL-2 EVLP system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adapter for positive pressure</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3635</td><td></td></tr><tr><td>AME 14- SC0062 pump tubing (tygon) 3-stop collared, pack of 12</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0126</td><td></td></tr><tr><td>AME 24- SC0072 pump tubing (tygon) 3-stop collared, pack of 12</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0155</td><td></td></tr><tr><td>Basic unit for the isolated perfused lung size 2 (IPL-2) type 829/2</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-2266</td><td></td></tr><tr><td>Connection kit for fiber oxygenator D150</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3765</td><td></td></tr><tr><td>EBM Edema Balance Module (EBM) with Sensor</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-4626</td><td></td></tr><tr><td>Fiber oxygenator type D150, pack of 5</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3762</td><td></td></tr><tr><td>Holder for clamping aortic or tracheal cannula for OP-tables size 5</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3855</td><td></td></tr><tr><td>Holder for oxygenators</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3061</td><td></td></tr><tr><td>HSE Weight Measurement System</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-4604</td><td></td></tr><tr><td>HSE-BDAS basic data acquisition software for windows 2000, XP, windows 7</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-1712</td><td></td></tr><tr><td>HSE-USB data acquisition hardware, stand alone USB box for windows 10 or 11</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3330</td><td></td></tr><tr><td>ISM 827/230 V roller pump reglo analogue, 4 channels, 0.003-35 ml/min (2 x)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0114</td><td></td></tr><tr><td>Left atrium cannula (DD 4.0 mm)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0712</td><td></td></tr><tr><td>Mini flow-thru oxygen electrode with 1/16&quot; fittings (Optional)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-4189</td><td></td></tr><tr><td>Mounting kit for fiber oxygenator type D150 on holders</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3759</td><td></td></tr><tr><td>One-way stopcock type 9500 (one for PA, one for LA)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0097</td><td></td></tr><tr><td>OPPM plugsys oxygen partial pressure module type 697&amp;nbsp; (Optional)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0210</td><td></td></tr><tr><td>Plugsys basic system case type 603</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0045</td><td></td></tr><tr><td>pO&lt;sub&gt;2&lt;/sub&gt; zero solution 20 packages of 20 ml&amp;nbsp; (Optional)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3812</td><td></td></tr><tr><td>Pressure transducer P75 type 379/HSE, range -75 to + 75 mm Hg (PA, LA)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0020</td><td></td></tr><tr><td>Pulmonary artery cannula (OD 2.0 mm, Head Diameter, 2.5 mm)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0711</td><td></td></tr><tr><td>Reservoir (jacketed) for buffer solution</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3496</td><td></td></tr><tr><td>SCP plugsys servo controller for perfusion type 704</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-2806</td><td></td></tr><tr><td>Shielding case for one pO&lt;sub&gt;2&lt;/sub&gt; or pCO&lt;sub&gt;2&lt;/sub&gt; mini sensor&amp;nbsp; (Optional)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-4196</td><td></td></tr><tr><td>Small mounting plate for a single O&lt;sub&gt;2&lt;/sub&gt; or CO&lt;sub&gt;2&lt;/sub&gt; sensor&amp;nbsp; (Optional)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3000</td><td></td></tr><tr><td>TAM-A plugsys transducer amplifier module type 705/1 (one for PA, one for LA)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0065</td><td></td></tr><tr><td>Thermostatic circulator E 103 (3 L)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0125</td><td></td></tr><tr><td>Three-way stopcock type 9560 R (one for PA, one for LA)</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-0096</td><td></td></tr><tr><td>Tube set for jacked buffer reservoir with fluid line shutoff valves</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3456</td><td></td></tr><tr><td>Ventilation cannula (tracheal) OD 2.0 mm, L 14 mm</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>73-3384</td><td></td></tr><tr><td>&lt;strong&gt;Computer softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HSE-BDAS basic data acquisition software for windows 2000, XP, windows 7</td><td>Hugo Sachs Elektronik- Harvard Apparatus</td><td>V2.0</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td>2502, Microsoft 365</td><td></td></tr><tr><td>Flexiware</td><td>Scireq</td><td>8</td><td></td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software inc.</td><td>10.0.1</td><td></td></tr><tr><td>Sprague-Dawley rats&amp;nbsp;</td><td>Charles River Laboratories (Saint-Germain-Nuelles, France)&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

thermal preconditioning during ex-vivo lung perfusion for the rehabilitation of damaged lung grafts before transplantation

Thermal Preconditioning During Ex-vivo Lung Perfusion for the Rehabilitation of Damaged Lung Grafts before Transplantation

Ex-vivo lung perfusion (EVLP) has emerged as a breakthrough in lung transplantation, enabling both functional assessment of donor lungs and therapeutic interventions to rehabilitate marginal grafts. Reconditioning strategies include pharmacological and non-pharmacological approaches. Among the latter, we demonstrated that transient, controlled heat stress ("Thermal Preconditioning," TP) is an effective means of restoring damaged lungs by inducing an endogenous protective heat shock response. Here, we present a step-by-step protocol for a rat EVLP model that incorporates TP to recondition lungs subjected to warm ischemia, simulating donation after circulatory death (DCD). The protocol details lung preparation, cannulation, perfusion, and induction of the heat shock response. TP consists of a 30 min increase in perfusate temperature to 41.5 &#176;C early in EVLP, followed by rapid return to 37 &#176;C for the remainder of perfusion. In a 6 h EVLP model, TP-treated lungs (n = 30) showed increased heat shock protein 70 expression, improved static pulmonary compliance, reduced edema, lower release of von Willebrand factor (an endothelial injury biomarker), and attenuated histological damage compared to controls (n = 30). The protocol enables real-time monitoring of vascular resistance, airway pressures, pulmonary compliance, and oxygenation, and supports molecular and morphological analyses in both perfusate and tissue. This reproducible, scalable protocol is adaptable to larger animal or human EVLP systems, providing a practical translational platform for studying non-pharmacological interventions aimed at improving transplant outcomes.

Lung physiological variables are continuously monitored throughout the procedure, including pulmonary artery pressure (PAP), calculated pulmonary vascular resistance, and ventilatory parameters, including static pulmonary compliance (SPC) and peak airway pressure (Pmax). Additionally, the weight module enables the evaluation of edema formation by providing an indirect measure of fluid accumulation in the lung. Benefits of thermal preconditioning (TP) with respect to control lungs (Ctrl) include an improvement of SPC (Figure 9A, expressed as the ratio of SPC at each time point to the initial SPC value, n = 5/group) and a reduction of lung weight gain (Figure 9B, n = 5/group) during EVLP. The data are presented as means ± SEM.
Perfusate fluid can be assayed for various biomarkers. For example, thermal preconditioning is associated with a significantly reduced release of von Willebrand (vWF), an endothelial cell biomarker at the end of EVLP (Figure 9C, n = 5/group, means ± SEM).
Morphological analyses of the lung tissue obtained at the end of EVLP include histology and immunohistochemistry. Examples of hematoxylin and eosin (H &amp; E)-stained sections are shown in Figure 9D, showing reduced histological damage in lungs exposed to thermal preconditioning, expressed as a lung injury score (Figure 9E, n = 5/group). The score was established by quantifying perivascular edema in 10 fields/section (see protocol steps 4.6.1 and 4.6.2). The sum of A + B in the 10 sections examined was used to calculate the score31.
Molecular analyses in the lung tissue at the end of EVLP include RNA or protein expression profiling. As an example, the protein expression levels of the inducible heat shock protein HSP70 can be measured by ELISA, to show the development of the heat shock response in lungs exposed to thermal preconditioning (Figure 9F, showing a time-course experiment with different durations of EVLP, n = 5/group at each time-point). The expression of HSP70 in the TP group peaked after 3h EVLP, that is, 90 min after the end of TP, and remained stable thereafter until the end of EVLP. The data are presented as means ± SEM.
<img alt="Bioreactor process diagram, bioprocess setup with fermenter, chromatography columns, peristaltic pumps." class="xfigimg" src="/files/ftp_upload/69084/69084fig1.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Description of the EVLP platform. (1) Double-walled reservoir for perfusate. (2A) Roller pump for perfusate circulation. (2B) Roller pump for perfusate sampling (biochemical measurements and gas analyses). (3) Thermostatic water bath. (4) Membrane gas exchanger connected to a gas tank (not shown). (5) Left Atrial Pressure sensor. (6) Heated and sealed chamber. (7) EVLP ventilator. (8) Temperature probe and thermometer. (9) Connection platform for the heart-lung block, whose elements are detailed in Figure 2. (Note: the PA pressure sensor is not visible). <a href="https://www.jove.com/files/ftp_upload/69084/69084fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Static equilibrium in perfusion setup; inflow/outflow system; diagram; fluid dynamics in organ study." class="xfigimg" src="/files/ftp_upload/69084/69084fig2.jpg" data-altupdatedat="1783396841000" data-alt="Figure 2"> 
Figure 2: Connection platform for the heart-lung block. (1) Inflow cannula to the pulmonary artery. (2) Outflow cannula from the left atrium. (3) Connection for the tracheal cannula. (4) Weight module. (5) In-house designed temperature probe firmly attached against the inflow cannula. <a href="https://www.jove.com/files/ftp_upload/69084/69084fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Spectroscopy analysis setup; optical excitation measurement using connected spectroscopy system and laptop." class="xfigimg" src="/files/ftp_upload/69084/69084fig3.jpg" data-altupdatedat="1783396841000" data-alt="Figure 3"> 
Figure 3: Signal transducers/amplifiers and Analog/Digital converter box. (A). Pulmonary artery pressure transducer/amplifier. (B). Pump speed flow controller. (C). Left atrial pressure transducer/amplifier. (D). Lung weight module. (E). Analog/Digital converter. (F). Personal computer for data display and recording. <a href="https://www.jove.com/files/ftp_upload/69084/69084fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Organ perfusion system diagram; temperature control setup for organ preservation study." class="xfigimg" src="/files/ftp_upload/69084/69084fig4.jpg" data-altupdatedat="1783396841000" data-alt="Figure 4"> 
Figure 4: Temperature maintenance of the perfusate during EVLP. (1) Thermostatic water bath. (2) Perfusate reservoir with double-walled glass container where water from the thermostatic water bath circulates. (3) Heating coil tubing inside a heated water container connected to the thermostatic water bath. (4) Organ chamber with double-walled glass container for warm water circulation from the water bath. (5) Electronic thermometer and in-house designed temperature probe firmly attached against the inflow cannula. <a href="https://www.jove.com/files/ftp_upload/69084/69084fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Thermal stress impact on proteins; chart and table; temperature effects on cellular processes." class="xfigimg" src="/files/ftp_upload/69084/69084fig5.jpg" data-altupdatedat="1783396841000" data-alt="Figure 5"> 
Figure 5: Identification of the optimal temperature for thermal preconditioning. (A) EVLP Protocol for the evaluation of different heating temperatures. From 60 min to 90 min of EVLP, the perfusate temperature was increased at the indicated temperatures. At 90 min of EVLP, temperature was returned to 37 °C for 90 min until the end of EVLP, where various physiological and molecular measurements were performed. (B) Result summary of the effects of the different heating temperatures on physiological and molecular parameters (green indicates favorable effects; red indicates unfavorable effects; and orange indicates mixed effects). The heating temperature of 41.5 °C provided the most protective effects. Abbreviations: EVLP = ex-vivo lung perfusion; TP = thermal preconditioning. This figure was modified from Ojanguren et al.21. <a href="https://www.jove.com/files/ftp_upload/69084/69084fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical setup for cardiovascular procedures with cannulas, scissors, forceps; equipment for infusion measurements." class="xfigimg" src="/files/ftp_upload/69084/69084fig6.jpg" data-altupdatedat="1783396841000" data-alt="Figure 6"> 
Figure 6: Surgical material. (A) Instruments for the extraction of the heart-lung block: Aneurysm clip holder and aneurysm microclip (Yasargil system), bulldog clamp, scissors, microscissors, retractors attached to elastic bands. (B) EVLP cannulas for the pulmonary artery (PA), the left atrium (LA) and the trachea. (C) Home-made perfusion system for cold perfusate flush of the lungs. Distance between the level of the PA in the animal and the bottom of the syringe is set at 20 cm. (C) Home-made holder. (Not shown: surgical microscope, ventilator, and anesthesia setup). <a href="https://www.jove.com/files/ftp_upload/69084/69084fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical procedure images showing steps of heart cannulation in a thoracic surgery experiment." class="xfigimg" src="/files/ftp_upload/69084/69084fig7.jpg" data-altupdatedat="1783396841000" data-alt="Figure 7"> 
Figure 7: Representative pictures of the surgical preparation (A) Pre-tied knot around the pulmonary artery. (B) Pre-tied knot around the tip of the heart. (C) Bulldog clamp (blue circle) on the PA cannula and aneurysm clip (red circle) on the trachea. Abbreviation: PA = pulmonary artery. <a href="https://www.jove.com/files/ftp_upload/69084/69084fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Lung ischemia-reperfusion timeline, EVLP process; lung recruitment, tissue preservation, sampling." class="xfigimg" src="/files/ftp_upload/69084/69084fig8.jpg" data-altupdatedat="1783396841000" data-alt="Figure 8"> 
Figure 8: Flowchart. Major steps to guide the progression through the different stages of the protocol. The period between 1 h and 1.5 h EVLP is magnified to indicate the treatment protocol according to the group assignment (control vs thermal preconditioning). Abbreviations: CI = cold ischemia; EVLP = ex-vivo lung perfusion; PA = pulmonary artery; SPC = static pulmonary compliance; TP = thermal preconditioning; WI = warm ischemia. <a href="https://www.jove.com/files/ftp_upload/69084/69084fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Static equilibrium study; multiple graphs and microscopy images; SPC ratio, weight, vWF data analysis." class="xfigimg" src="/files/ftp_upload/69084/69084fig9.jpg" data-altupdatedat="1783396841000" data-alt="Figure 9"> 
Figure 9: Representative results of the effects of thermal preconditioning in damaged rat lungs. Lungs damaged by 1 h of warm ischemia were perfused in an EVLP system for 6 h. From 60 to 90 min of EVLP, a group of lungs was subjected to thermal preconditioning at 41.5 °C (TP group). A control group of lungs was maintained at 37 °C throughout the EVLP. (A) Static pulmonary compliance (SPC), expressed as the ratio of the initial value, in control (Ctrl, n = 5) and TP lungs (n = 5). SPC was better preserved in TP lungs. (B) Weight gain of the lungs during EVLP in control (n = 5) and TP lungs (n = 5). There was a significant reduction in edema formation in the TP group. (C) The release in the perfusate of the endothelial cell biomarker von Willebrand Factor was suppressed in the TP group (n = 5) compared to controls (n = 5). (D) Lung macroscopic and microscopic (hematoxylin-eosin staining) appearance at the end of EVLP in control (n = 5) and TP lungs (n = 5). (E) Lung injury score (for the quantification of the histological damage) was lower in the TP group (n = 5) compared to the control group (n = 5). (F) Protein levels of the inducible heat shock protein HSP70 in lung tissue extracts measured after 1, 2, 3, 4.5, and 6 h EVLP in control and TP groups (n = 5/group at each time point), showing marked heat shock response in the TP group. The expression of HSP70 in the TP group peaked after 3 h EVLP (90 min after the end of TP) and remained stable thereafter until the end of EVLP. 
All graphs show means ± SEM. For time course experiments (A-C,F), statistical comparisons were performed using two-way ANOVA followed by Sidak's test for the group effect and Bonferroni's adjustments for the effect of time (with time 1 h as the reference). For lung injury score (E), statistical comparisons were done using Mann-Whitney test. * p &lt; 0.05 (intergroup differences), † p &lt; 0.05 vs 1 h EVLP. Abbreviations: Ctrl = control; EVLP = ex-vivo lung perfusion; HSP70 = heat shock protein 70; SPC = static pulmonary compliance; TP = thermal preconditioning. Modified from Parapanov et al.23. <a href="https://www.jove.com/files/ftp_upload/69084/69084fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of the solutions used in the protocol. <a href="https://www.jove.com/files/ftp_upload/69084/Table-1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: EVLP settings for perfusion flow, inflow temperature (at the level of the pulmonary artery cannula) and ventilation over time. <a href="https://www.jove.com/files/ftp_upload/69084/Table-2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Troubleshooting. <a href="https://www.jove.com/files/ftp_upload/69084/Table%203.docx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: Protocol detailing the major steps for signal calibration in the ex vivo lung perfusion system, including calibration of pulmonary artery pressure, weight, and flow signals. <a href="https://www.jove.com/files/ftp_upload/69084/Supplementary%20File%201.docx" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Demonstration of the signal calibration procedure for pulmonary artery pressure, weight, and flow during ex vivo lung perfusion. <a href="https://www.jove.com/files/ftp_upload/69084/Supplementary%20File%202.mp4" target="_blank">Please click here to download this File.</a>

This protocol presents a rat ex-vivo lung perfusion model incorporating a transient exposure to a calibrated thermal stress, promoting endogenous protective mechanisms for the therapeutic reconditioning of damaged lung grafts. The approach is scalable and reproducible and supports the development of non-drug therapies to improve lung transplant success.

Ex-vivo lung perfusion (EVLP) has emerged as a breakthrough in lung transplantation, enabling both functional assessment of donor lungs and therapeutic ...

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Research

JoVE Journal

Medicine

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

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.

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

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to  transplantation.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

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

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

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.

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

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.

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

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.

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

Donor heart preservation is a major determinant of success in heart transplantation. Technological advances have led to optimizing the conditions of preservation, along with a more accurate assessment of the donor heart before transplantation. We herein describe two innovative technologies currently applied for cardiac preservation: uniform cooling preservation and normothermic ex-situ heart perfusion.

Innovative Strategies for Organ Preservation in Heart Transplantation: Uniform Cooling Preservation and Ex-situ Normothermic Perfusion

Learn thermal preconditioning for lung graft rehabilitation using an ex-vivo rat perfusion model. This method activates protective mechanisms to improve transplant success without drugs.

Thermal Preconditioning During Ex-vivo Lung Perfusion for the Rehabilitation of Damaged Lung Grafts before Transplantation

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