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.
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.
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'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's initiation, management, and termination are also described. The strategy for tissue collection and the management of the samples is also explained.
The procedures performed in this manuscript comply with the guidelines of the Canadian Council on Animal Care and the guide for the care and use of laboratory animals. The institutional animal care committee of the University of Alberta approved the protocols. Female juvenile Yorkshire pigs between 35-50 kg were used exclusively. Proper biosafety training was required by all individuals involved in ESLP procedures. A schematic overview of the entire NPV-ESLP experiment is represented in Figure 1.
1. Pre-surgical preparations
2. ESLP software initialization, adjustments, and de-airing circuit
3. Preparations for anesthesia
4. Lung biopsy, exsanguination, and blood collection
5. Cardiectomy
6. Pneumonectomy
7. Placement of the lungs onto the ESLP apparatus
8. Initiation of perfusion and ventilation
9. Metabolic Support of the Lung
10. Heparin, anti-microbial, and anti-inflammatory agents
11. Assessment of lung function
12. Metabolic assessment of the ex situ perfused lungs
13. Terminating perfusion, ventilation, and disconnection of the lungs from ESLP device
At the beginning of lung perfusion and ventilation (preservation mode), the lungs will generally have a low pulmonary artery pressure (< 10 mmHg) and low dynamic compliance (< 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 may continue to increase but naturally stabilize in the 6-8 mL/kg range. TVi will always exceed TVe by 100-200 mL. Likewise, dynamic compliance will begin at 0-10 mL/mmHg within the first hour and occasionally be higher. Between 3-6 h, the dynamic compliance is 10-20 mL/mmHg and stabilizes with the TVe, which are interrelated parameters. The PAP will rise progressively as pulmonary artery flow gradually increases from 10 to 30 % of the cardiac output. Within the first hour, this is typically 10±2 mmHg and rises slightly throughout the 12 h run to a range of 12±2 mmHg. During an evaluation with flows of 50% of cardiac output, PAP can be much higher at 15-20 mmHg. Pulmonary vascular resistance (PVR) will rise gradually throughout ESLP. Figure 6 displays trends in PAP, dynamic compliance, and PVR over 12 h of perfusion and ventilation. All these parameters can be affected by the specific ESLP experimental protocol employed.
During the evaluation mode of ESLP, which occurs at 3, 5, 7, 9, 11 h during a 12 h run, an upward trend in LA PaO2 is observed (Table 4). The evaluation mode lasts for 5 min. It consists of dropping PEEP to 5 cm H2O while maintaining peak pressures by increasing EIP in compensation. Flows are increased to 50% of cardiac output, and mixed sweep gas is added via the deoxygenator at a flow rate of 0.125 L/min to simulate systemic oxygenation consumption. Generally, PaO2 from the PA is in the range of 50-60 mmHg, and LA PaO2 can range from 60-120 mmHg, depending on how well the lungs have responded to the preservation and reconditioning. The absolute step-up value in PaO2 between pre-and-post-deoxygenator is a better indicator of oxygenation capacity of the lungs, and thereby lung function; however, by convention, PF ratios remain a commonly reported parameter to predict successful transplantation. PF ratio is the LA (pre-deoxygenator) PaO2/FiO2 and should be > 300, which is the transplantation cut-off for humans. The FiO2 is 21% (room air); therefore, the minimum LA PaO2 required during ESLP is 63 mmHg. Figure 6 demonstrates a typical trend for the PF ratio at the evaluation time points of 5 and 11 h throughout NPV-ESLP.
Both modes of ESLP benefit from various metabolic assessments, including frequent blood gas analysis, repeat perfusate composition sampling, and tissue biopsies. Perfusate acts as a surrogate indicator of overall lung status; therefore, blood gas analysis of the perfusate provides extensive information on the metabolic state of the lungs (Table 4). Before each evaluation, a 10 mL perfusate sample is drawn to be centrifuged and analyzed via ELISA for various biomarkers of inflammation, including TNF-alpha, IL-6, and IL-8. These values are informative of the inflammatory state of the lungs and the effects of experimental protocols; however, they need to be interpreted in the context of ESLP as a closed circuit without perfusate replacement/exchange. Thus, these biomarker levels do not benefit from the supportive function of natural metabolizers and physiologic clearance as performed by the liver or kidneys. For this reason, a continual increase in these markers over time with ESLP is observed. The tissue biopsies are likewise helpful for biomarker labeling and visualization and histologic assessment of tissue integrity. Edema formation is another important index of inflammation associated with endothelial permeability. Figure 6 demonstrates a typical weight gain of 30% at the end of 12 h of NPV-ESLP. Recently, in vitro functional assessment of lungs on NPV-ESLP has been supplemented with confirmatory in vivo left lung transplantation into 35-50 kg Yorkshire pigs. In-vivo transplanted lung assessment occurs over a 4 h duration before euthanasia via exsanguination. The transplantation protocol adopted for in vivo assessment using this custom NPV-ESLP device can be found in this Reference19.
The P:F ratio is the main functional assessment parameter of ESLP and human lung transplantation. This NPV-ESLP technology was successfully employed in a clinical trial with 100% 30 days and 1 year survival17. Twelve extended criteria human lungs were successfully preserved and reconditioned on ESLP with subsequent transplantation. There were no incidences of PGD grade 3 and no early mortality. Long-term follow-up is ongoing. Although P:F ratio is the gold-standard functional assessment parameter for transplantation and ESLP, NPV-ESLP also measures PAP, pulmonary vascular resistance, edema formation, and compliance as additional functional outcome measures to help guide preservation and reconditioning of lungs. NPV-ESLP provides comprehensive metabolic and functional evaluations of donor lungs. This technology has proven to be clinically beneficial in the context of extended criteria lungs. The software has been designed to require minimal manual adjustments and has minimal inter-and intra-operator variability.
Figure 1: NPV-ESLP Protocol. Schematic representation of lung procurement and 12 h NPV-ESLP run. Please click here to view a larger version of this figure.
Figure 2: Silicone support membrane for the lungs suspended in hard-shell ESLP reservoir. Support membrane pictured with an endotracheal tube (centre) and pulmonary artery cannula (left). Please click here to view a larger version of this figure.
Figure 3: NPV-ESLP circuit. (A) Schematic representation of the circuit with an accompanying legend (left). (B) Photo of NPV-ESLP circuit (right). Please click here to view a larger version of this figure.
Figure 4: Screenshots from NPV-ESLP software program. (A) "Main" Screen. (B) "Flow-Loops" Screen. (C) "Settings" Screen. Please click here to view a larger version of this figure.
Figure 5: Lungs connected to NPV-ESLP circuit. (A) Anterior Donor Lungs Pre-ESLP. (B) Posterior Donor Lungs Post-ESLP. (C, D) Tissue biopsy of right middle lung lobe. (E) Lungs connected to ESLP circuit. (F) Demonstrated positioning of lungs on silicone support. (G) Front view of ESLP device illustrating starting fluid level and lung positioning. (H) Lungs connected to the device demonstrating open left atrial drainage. (I, J, K) Lid secured on the device chamber. (L) The device and lungs are fully connected and functioning in NPV mode. Please click here to view a larger version of this figure.
Figure 6: Functional parameters during evaluation modes over 12 h of NPV-ESLP. (A) P:F ratio, PaO2:FiO2 ratio. (B) Compliance. (C) PAP, pulmonary artery pressure. (D) PVR, pulmonary vascular resistance. (E) Weight Gain. Please click here to view a larger version of this figure.
Table 1: Recorded monitoring chart parameters. Please click here to download this File.
Table 2: Initiation of 12 h NPV-ESLP Protocol. CO, cardiac output; PA, pulmonary artery; PPV, positive pressure ventilation; NPV, negative pressure ventilation. For preservation mode, ventilation parameters, see Table 3. Beginning at T3, evaluation was conducted serially every 2 h for 5 min, with PA flow set to 50% CO, medical gas set to 89% N2, 8% CO2, 3% O2, and preservation settings as per the parameters provided in Table 3. Please click here to download this File.
Table 3: Modes of NPV-ESLP: Preservation vs. Evaluation. CO, cardiac output; FiO2, fraction inspired of oxygen; LAP, left atrial pressure; NPV, negative pressure ventilation; PAP, mean pulmonary artery pressure; PAWP, peak airway pressure; PEEP, positive end-expiratory pressure; PCO2, partial pressure of carbon dioxide in the pulmonary arterial circulation. Please click here to download this File.
Table 4: Blood gas analysis performed during 12 h of ESLP. Ca+, calcium ion; Cl–, chloride ion; Hb, hemoglobin; HCO3–, bicarbonate ion; K+, potassium ion; Na+, sodium ion; Osm, osmolarity; paCO2, arterial partial pressure of carbon dioxide; paO2, arterial partial pressure of oxygen; sO2, oxygen saturation; P/F ratio, PaO2/FiO2 ratio. Please click here to download this File.
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 "no-touch" technique to avoid causing trauma and atelectasis when dissecting out the lungs. "No-touch" means using the bare minimum amount of manual manipulation of the lungs during procurement. Recruitment maneuvers while on the ventilator during surgery are far less effective in porcine lungs than human lungs. It is ill-advised to redirect air manually through the alveoli as is often performed with human lungs because this will cause irreparable injury to juvenile porcine lungs. It is critical to clamp the trachea at tidal volumes that match the tidal induction volumes to maximize the probability of a successful NPV-ESLP run. Any lost compliance during procurement is challenging to regain on NPV-ESLP when working with porcine lungs; humans' lungs using NPV-ESLP are more forgiving in this regard. Ideally, clamping the lungs at tidal induction volumes is performed without the need for increased peak pressure; however, compliance does start to drop shortly after warm ischemia, and sometimes higher pressures are needed to maintain recruitment. It is helpful to switch to an I:E ratio of 2:1 after the cardiectomy to maintain and even increase alveolar recruitment slightly with TVe above 10ml/kg prior to initiating the pneumonectomy. Do not flip the lungs medially to dissect the posterior pleural attachments from the esophagus as is commonly performed in human lung retrievals. The posterior pleural attachments must be bluntly dissected using a blind approach, teasing the tissue away from the lungs using a freehand while simultaneously lifting upward from the clamped trachea to provide counter traction. Juvenile porcine lungs that have lost significant compliance at the time of tracheal clamping will struggle to recover on ESLP. If the lungs have 0 dynamic compliance initially during NPV-ESLP and do not develop any dynamic compliance improvement as measured by the software within the first hour, it is doubtful that these lungs will recover their function. This is almost certainly an issue with the surgical explant technique. If insufficient PA length has been procured, descending Aorta can lengthen the PA via end-to-end anastomosis.
Several critical steps and troubleshooting methods are needed during the operation of the NPV-ESLP apparatus to achieve successful perfusion. The procurement process, mounting the lungs on the NPV-ESLP apparatus, and initiating perfusion/ventilation should not exceed 20-30 min. Extended periods of ischemia decrease the probability of a successful run. The lungs must be positioned on the silicone support membrane such that neither the PA cannula nor the ET tube interferes with the movement of the upper lobes during ventilation. The lungs must be elevated off the hard-shell chamber using the silicone support membrane; however, the lungs should not be so elevated that open LA drainage of blood will result in hemolysis from the force of falling onto the hard-shell reservoir. Any tears in the lung parenchyma must be identified and oversewn with 6-0 prolene to prevent an air leak. Scrap pleura or pericardium can be helpful to perform a patch repair. Likewise, blood-soaked gauze can also serve to plug tears that cannot be surgically repaired. It is better to avoid an injury than to repair the lung parenchyma as the lung is difficult to sew without causing further damage. The lungs must remain inflated when initiating ventilation, so CPAP must begin at 20 cm H2O before unclamping the trachea or ventilation tubing. If the lungs deflate, they will struggle. Any lost alveolar recruitment before initiation of ventilation will be tough to regain during NPV-ESLP, resulting in a slower recovery. When initiating perfusion, the pressure transducer must be zeroed correctly. The PA clamp is removed slowly to avoid the undesirable effect of pulmonary over-circulation from excessively high pressures and flow. The main PA must not be kinked in its position as this will produce falsely elevated pressure readings. The PA adapter must not abut the PA bifurcation for this same reason. Both situations can interfere with the perfusion of lung tissue. It is critical to maintain PEEP above 12 for the first hour of ventilation and not drop PEEP below 8 except for evaluation, where a PEEP of 5 is desirable. Peak pressures should match those used at the time of procurement as they are informative regarding the state of lung compliance. For example, if the lungs required a peak pressure of 25 cm H2O at the time of procurement to achieve TVe of 10 mL/kg, anything less than 25 cm H2O will unlikely sustain the same amount of alveolar recruitment once on the machine.
There are a few limitations of this method that are worth considering. As previously mentioned, the convention in ESLP literature is only to report the PaO2 when calculating P:F ratios8,9,10,11,15,17,18; however, the PA PaO2 is informative because it clarifies the oxygen step-up occurring due to lung oxygenation. This is a better descriptor than P:F ratio alone. When the sweep gas is not running, the machine essentially acts as one large shunt that recirculates blood through the lungs for repeated laps of oxygenation. For this reason, preservation mode ABGs are not particularly informative for the oxygenation capacity of the lungs but are very valuable for the metabolic profile. This is why mixed-gas sweep during evaluation is so important and why demonstrated deoxygenation of the post deoxygenator perfusate is critical. Another limitation is the necessity of an in vivo model for accurate assessment of lung function post-ESLP. In vivo transplantation is surgically demanding compared to the organ procurement operation, with many possible complications resulting in the loss of the transplanted lung. As such, both ESLP and subsequent transplantation are expensive resource endeavors and possess steep learning curves.
There are several advantages of this NPV-ESLP technology compared to currently available models. Pre-clinical studies comparing NPV-ESLP to PPV-ESLP have shown that NPV is a superior form of ventilation15. This is most likely because NPV is a more physiologic method for ESLP. NPV replicates the negative intrathoracic pressure environment of the thorax to induce lung expansion by evenly distributing the force across the pleural surface. PPV induces greater barotrauma as it forces the lungs to open through higher pressures directed down the airways. One of the other significant advantages of this NPV-ESLP device is that it is designed to be entirely portable. Portability allows for the virtual elimination of warm ischemic time as the device can accompany transplant teams to the donor center. Ischemic time is directly related to the extent of lung ischemic reperfusion injury (LIRI) and subsequent development of primary graft dysfunction (PGD), the major cause of death and morbidity post lung transplantation. Therefore, any effort to decrease ischemia should translate into improved post-transplantation outcomes. Reducing ischemic time also allows for the procurement of lungs from distant geographic locations. This is because transport time becomes less of a concern for the development of LIRI and PGD, thereby increasing the availability of donor organs that otherwise would have been rejected.
This device and the described methods have useful clinical and research applications. As previously mentioned, the prototype of this device has already been used for a successful clinical trial of extended criteria donor lungs for transplantation with 100% 30 days and 1-year survival and zero incidences of PGD grade 317. A multi-center trial is a next step for this device as it moves towards commercial development. Regarding research applications, there is pre-clinical evidence that NPV-ESLP is superior to PPV-ESLP15. NPV-ESLP holds the promise of becoming the exemplary device, which will drive further research using this technology. The application of ESLP in the lab setting has the advantage of continuous monitoring of organ function, immediate feedback upon the introduction of novel treatment modalities, isolation of the lungs from other organ systems for testing therapeutics, and a vehicle for the delivery of therapies that previously lacked a route of administration to donor lungs. In this sense, its application in translational research for lung transplantation is unparalleled. This particular device with an automated ESLP software program is easy to use, results in minimal inter-and intra-operator variability in functional parameters, and is designed to require minimal manual adjustments.
The authors have nothing to disclose.
This research was funded on behalf of The Hospital Research Foundation.
0 ETHIBOND Green 1 x 36" Endo Loop 0 | ETHICON | D8573 | |
2-0 SILK Black 12" x 18" Strands | ETHICON | SA77G | |
ABL 800 FLEX Blood Gas Analyzer | Radiometer | 989-963 | |
Adult-Pediatric Electrostatic Filter HME – Small | Covidien | 352/5877 | |
Arterial Filter | SORIN GROUP | 01706/03 | |
Backhaus Towel Clamp | Pilling | 454300 | |
Biomedicus Pump | Maquet | BPX-80 | |
Cable Ties – White 12” | HUASU International | HS4830001 | |
Calcium Chloride | Fisher Scientific | C69-500G | |
Cooley Sternal Retractor | Pilling | 341162 | |
CUSHING Gutschdressing Forceps | Pilling | 466200 | |
D-glucose | Sigma-Aldrich | G5767-500G | |
Deep Deaver Retractor | Pilling | 481826 | |
Debakey Straight Vascular Tissue Forceps | Pilling | 351808 | |
Debakey-Metzenbaum Dissecting | Pilling | 342202 | |
Scissors | Pilling | 342202 | |
Endotracheal Tube 9.0mm CUFD | Mallinckrodt | 9590E | Cuff removed for ESLP apparatus |
Flow Transducer | BIO-PROBE | TX 40 | |
Human Albumin Serum | Grifols Therapeutics | 2223708 | |
Infusion Pump | Baxter | AS50 | |
Inspire 7 M Hollow Fiber Membrane Oxygenator | SORIN GROUP | K190690 | |
Intercept Tubing 1/4" x 1/16" x 8' | Medtronic | 3108 | |
Intercept Tubing 3/8" x 3/32" x 6' | Medtronic | 3506 | |
Intercept Tubing Connector 3/8" x 1/2" | Medtronic | 6013 | |
MAYO Dissecting Scissors | Pilling | 460420 | |
Medical Carbon Dioxide Tank | Praxair | 5823115 | |
Medical Nitrogen Tank | Praxair | NI M-K | |
Medical Oxygen Tank | Praxair | 2014408 | |
Organ Chamber | Tevosol | ||
PlasmaLyte A | Baxter | TB2544 | |
Poole Suction Tube | Pilling | 162212 | |
Potassium Phosphate | Fischer Scientific | P285-500G | |
Scale | TANITA | KD4063611 | |
Silicon Support Membrane | Tevosol | ||
Sodium Bicarbonate | Sigma-Aldrich | 792519-1KG | |
Sodium Chloride 0.9% | Baxter | JB1324 | |
Sorin XTRA Cell Saver | SORIN GROUP | 75221 | |
Sternal Saw | Stryker | 6207 | |
Surgical Electrocautery Device | Kls Martin | ME411 | |
Temperature Sensor probe | Omniacell Tertia Srl | 1777288F | |
THAM Buffer | Thermo Fisher Scientific | 15504020 | made from UltraPureTM Tris |
TruWave Pressure Transducer | Edwards | VSYPX272 | |
Two-Lumen Central Venous Catheter 7fr | Arrowg+ard | CS-12702-E | |
Vorse Tubing Clamp | Pilling | 351377 | |
Willauer-Deaver Retractor | Pilling | 341720 | |
Yankauer Suction Tube | Pilling | 162300 |