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

Summary

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.

Abstract

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.

Introduction

Like any field in solid organ transplantation, lung transplantation suffers from a shortage of donor organs. In order to increase the donor pool, significant research has been dedicated to investigating the potential of allografts that were once thought to be unsuitable for transplantation, i.e., extended criteria donors (ECD). These allografts can be considered ECD for a milieu of reasons, including questionable quality, poor function, infection, trauma, prolonged warm or cold ischemic times, and advanced age^1,2. In certain cases, where these lungs are suitable for immediate transplant³, it is often advantageous to providers and recipients alike to evaluate these lungs for an additional time to determine their suitability for transplantation. Ex vivo lung perfusion (EVLP) is such a technology that allows for extended assessment of potential lung allografts in a closed circuit outside the donor^2,4,5,6,7, affording the transplant provider the ability to determine transplantation suitability. EVLP has shown the ability to adequately assess donor organs^8,9,10,11, decrease the effects of ischemic reperfusion injury (IRI)^12,13 and increase the donor pool^14,15 thus making lung transplantation a more accessible treatment for all.

In general, an EVLP system is a closed system with a ventilatory circuit (achieved by connecting a ventilator to the trachea to introduce air into the system) and a vascular circuit (achieved by connecting the left atrium (LA) to the pulmonary artery (PA) with tubing)⁷. The vascular circuit has perfusate running through the tubing to give the lung vital nutrients and oxygen while limiting the cold ischemic time (CIT)^5,8,16,17. This solution is either blood-based (i.e., via the addition of packed red blood cells (PRBCs))^16,17 or acellular-based (i.e., no PRBCs)^4,5. However, there are several notable disadvantages to using PRBCs. If using PRBCs from donors who died from trauma or brain-dead donors (BDD), these fluids often contain large amounts of inflammatory cytokines, which may increase cellular damage during EVLP as well as increase levels of cell-free hemoglobin (Hb), heme, iron, and cell fragments which deliver additional damage to cells^18,19. Furthermore, as these donors are often multi-organ, the collection of PRBCs prior to procurement could lead to decreasing blood volume in the donor and subsequently increasing ischemia to all organs. If using PRBCs from another source, providers could face blood shortages as this is a scarce material in and of itself^20,21. Finally, PRBCs are prone to mechanical lysis on the EVLP circuit regardless of their source, releasing Hb and other components that contribute to cellular damage.

Thus, for many reasons, it could be advantageous to use an artificial red blood cell substitute, i.e., hemoglobin-based oxygen carriers (HBOCs), as a perfusate supplement. One particularly promising HBOC is polymerized human hemoglobin (PolyhHb). PolyhHb is synthesized from Hb purified from expired PRBCs that were deemed unsuitable for immediate transfusion²². They have been shown to be viable blood substitutes in hemorrhagic shock²³ and transplantation²⁴ and can be produced in large quantities²². However, large-scale adoption of PolyhHb has been unsuccessful due to unforeseen complications such as vasoconstriction, increasing blood pressure, and cardiac arrest^23,25. The reasons behind these findings were likely due to the presence of cell-free Hb or low molecular weight Hb polymers (< 500 kDa) in the PolyhHb solution, as they have a propensity to extravasate into the tissue space, which resulted in decreased nitric oxide availability, subsequent vasoconstriction, systemic hypertension, and ultimately oxidative tissue injury^26,27. To improve upon these issues, the Palmer Laboratory has worked to develop a next-generation PolyhHb that contains minimal low MW species and cell-free Hb, which has demonstrated improved biophysical characteristics and in vivo responses^22,28,29,30. Several transfusion studies in animals have shown that if low molecular weight Hb polymers are eliminated from the HBOC, vasoconstriction, systemic hypertension, and oxidative damage can be mitigated^{28,29,31,32,33,34,35}. Therefore, making this next-generation PolyhHb a promising perfusate candidate.

Here, we describe the application of a next-generation PolyhHb to be used in a perfusate and the protocol by 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 provide protocols to test them in clinically relevant translational transplant models.

Protocol

Sprague-Dawley rats (300 g body weight) were commercially obtained and housed under pathogen-free conditions at The Ohio State University Wexner Medical Center Animal Facility. All procedures were humanely performed according to the NIH and the National Research Council's Guide for the Humane Care and Use of Laboratory Animals and with the approval of The Ohio State University Institutional Animal Care and Use Committee (IACUC Protocol 2023A00000071).

1. PolyhHb synthesis and purification

NOTE: The production and synthesis of the PolyhHb material that was used for the following EVLP experiments were initially published by Cuddington et al. in 2020²². Please refer to this work for in-depth schematics and analysis of the PolyhHb synthesis. The following is a summary of the synthesis and purification of PolyhHb on a pilot scale and its subsequent preparation as a perfusate.

1. RBC wash, lysis, and Hb purification
   1. Procure 18 units of expired human PRBCs and pour them into a 20 L filtration vessel, dilute with 0.9 wt% saline to a final hematocrit of 22% (Figure 1B,C).
   2. Perform six system volume exchanges (diacycles) on a 0.65 µm modified polyethylene sulfone (mPES) tangential flow filtration (TFF) module with 0.9 wt% saline on the RBC solution. NOTE: The purpose of this washing step is to remove damaged RBCs, membrane fragments, and other extracellular materials prior to hemolysis (Figure 1B,C).
   3. Lyse the RBC solution with 10 L of phosphate buffer (PB, 3.75 mM, pH 7.4) for 1 h at 4 °C with constant stirring.
   4. Remove the lysed membrane fragments and other aggregates by filtering the solution over a 500 kDa TFF module and collecting the permeate in the 30 L batch reactor vessel (Figure 1A-C).
   5. Once 480 g of Hb is in the reactor, add a salt charge to convert PB into phosphate buffered saline (PBS).
   6. Recirculate the Hb through a gas contactor fed with nitrogen, as well as maintaining a nitrogen head space in the reactor, to deoxygenate the protein overnight. Chill to 14 °C to limit methemoglobin (metHb) formation.
2. Hb polymerization
   1. Heat the Hb solution to physiological temperature (37 °C) while recirculating the solution on a gas contactor loop.
      NOTE: The goal is to deoxygenate the protein to a pO₂ between 0-10 mmHg to ensure most of the Hb is in the tense quaternary state (Figure 1A).
   2. Add 1 g charge of sodium dithionite, as needed, to ensure effective deoxygenation.
   3. While maintaining the recirculation loop and degassing the Hb solution, add a 30:1 molar ratio of glutaraldehyde (GA) to Hb diluted in 3 L of deoxygenated PBS (pH 7.4).
   4. Add solution to reactor vessel over 3 h with an additional hour of reaction time.
   5. Quench the crosslinking reaction with a 7:1 molar ratio of sodium cyanoborohydride to GA, diluted in 3 L PBS (pH 7.4). Add to the reactor for over 10 min.
   6. Chill reactor at 14 °C overnight.
3. PolyhHb purification
   1. Pump the reactor contents into a 10 L filtration vessel and begin circulation through a 0.2 µm polyethylene sulfone (PES) TFF module (Stage 1). This step will remove large aggregates and undesired contaminants.
   2. Feed the permeate into a secondary 10 L filtration vessel that will circulate over a 500 kDa polysulfone (PS) TFF module (Stage 2) once full. Continue until the reactor is emptied (Figure 1B,D).
   3. Once the reactor is emptied into the purification circuit, start excipient exchange on Stage 1 with a modified lactated Ringer's solution (pH 7.4). After each full volume exchange, measure the protein concentration in the permeate of Stage 1 using UV-visible spectroscopy.
   4. When the Stage 1 permeate has a concentration of less than 1 mg Hb/mL, transfer the modified Ringer's solution to Stage 2. Any hold-up in Stage 1 is a waste and should be disposed of appropriately. In total, ensure that 12 full volume exchanges of the modified Ringer's solution are performed across both stages.
   5. Following the completion of diacycles, concentrate the contents of Stage 2 to at least 10 g/dL over the 500 kDa TFF module.
   6. Package the concentrated solution in 50 mL conical tubes and store at -80 °C until use.

2. Perfusate formulation

1. Prepare the perfusate to a final volume of 165 mL. Dilute PolyhHb to a final concentration of 3.7 g/dL with William's E Medium.
2. Add human serum albumin (HSA) to a final concentration of 3% HSA by weight. Add 1 mL of heparin to final solution.

3. Ex Vivo lung perfusion circuit setup

1. Place PolyhHb perfusate into the EVLP circuit reservoir and turn on the warm water bath to 37 °C. Ensure the perfusate is circulating within the circuit by turning on the roller pumps.
2. Connect de-oxygenation gas (i.e. 6% O₂, 8% CO₂, 84% N₂) to the hollow fiber oxygenator to de-oxygenate the perfusate. This is done to assess the lung's ability to oxygenate the perfusate.
3. Open data acquisition software on a nearby computer. Ensure pulmonary artery pressure, tracheal differential pressure, respiratory flow differential pressure, lung weight, and pump speed transducers are connected to both the circuit and data-converter box.
4. Ensure that no leaks are present throughout the system by carefully examining all tube connections and that warm water is circulating throughout (Figure 2). Press Run on the data-acquisition software to ensure all pressure transducers are functioning. Once the system is properly functioning, turn off the roller pumps.

4. Procurement of donor rat lung block

1. Set up the surgical table and layout the instruments (Figure 3). Autoclave all instruments at 121 °C for 30 min.
2. Prepare 1200 U/kg of heparin, a ketamine/xylazine mixture for anesthetic (60 mg/kg ketamine and 5 mg/kg), as well as 5-10 cm long silk sutures (3-0 or 4-0).
3. Inject ketamine/xylazine solution intraperitoneally into the rat. Wait 5-10 min for the anesthetic plane to develop. To ensure a proper level of anesthesia, toe pinch the rat to elicit a reaction. If there is no reaction, then the proper level of anesthesia has been met.
4. Shave the abdomen of the rat and place the rat in the supine position on the surgical board. Clean the abdomen with povidone-iodine and 70% ethanol. Place ophthalmic ointment under the rat's eyes to prevent dryness.
5. Move the rat to the surgical board and secure the rat in place (Figure 4A). Turn on data acquisition software and begin recording. Turn on the ventilator at 4 mL/kg and ensure positive end expiratory pressure (PEEP) is around 2 cm/H₂O.
   NOTE: These initial settings are experiment specific. It is up to all researchers to determine the best ventilatory strategies for individual experiments.
6. Once proper anesthetic depth is met, perform a midline laparotomy from the xiphoid process to the pubic symphysis using a pair of scissors. Next, perform a medial-lateral visceral rotation and visualize the infra-hepatic inferior vena cava using a blunt instrument (IVC)^36,37,38 (Figure 4B). Inject heparin into the IVC with a 20G needle (Figure 4C).
7. Turn attention to the neck and cut the skin from the sternal notch to just below the angle of the mandible with a pair of scissors. Next, begin to dissect toward the trachea (Figure 5A).
8. In the neck, bluntly dissect away necessary strap muscles to expose the trachea (Figure 5B). Make a transverse incision with a pair of scissors on the anterior trachea between the cartilaginous rings big enough for the endotracheal (ET) tube (several millimeters), but do not cut through the posterior portion of the trachea. Place a 5-0 silk suture around trachea (Figure 5C).
9. Insert the endotracheal tube and secure it in place with the aforementioned 5-0 silk suture (Figure 5D). Connect the ET tube to the ventilator and ensure proper chest rising.
10. Perform a median sternotomy and enter the thoracic cavity again using scissors. Place chest wall retractors to expose the heart and lungs (Figure 6A). Avoid any inadvertent manipulation of the lungs, as they are incredibly friable.
11. Remove the thymus from the anterior mediastinum by a combination of sharp (scissors) and blunt dissection. Be careful not to damage great vessels or lungs.
12. Identify the pulmonary artery (PA; Figure 6B) and place a 5-0 silk suture around it to prepare for cannulation (Figure 6C). Due to the microscopic anatomy of the rat's great vessels, it is often easier to place the suture around the PA and aorta at the same time.
13. Make a 2-3 mm incision in the right ventricular outflow track (RVOT) using a pair of scissors (Figure 6D-E) to place the arterial cannula within the PA and secure it in place with the 5-0 suture described a step earlier (Figure 6F).
14. Make a 5 mm incision in the left ventricle (LV) and well infra-hepatic IVC using a pair of scissors to euthanize the rat. Quickly connect the lung preservation fluid to the arterial cannula to gravity flush the lungs with about 20 mL (Figure 7A-B). Ensure the lung preservation fluid is de-aired prior to connecting it to the arterial cannula as air emboli are very damaging to the lungs.
15. Connect the arterial cannula to the EVLP circuit. Turn on the roller pump and allow a small amount of perfusate to flow through the lung and out of the left ventricle into the thoracic cavity. Once perfusate begins to flow out of the left atrium, turn off the roller pump (Figure 7C). While allowing perfusate to flow, ensure PA pressure does not spike - which would indicate blockage or incorrect placement.
16. Place small forceps in the LV and gently stretch the mitral valve annulus, which will allow for the introduction of the left atrium (LA) cannula (Figure 8A). Place a 5-0 silk tie around the heart and loosely tie (Figure 8B).
17. Insert the LA cannula into the LV and advance the LA cannula until it can be seen within the atrium. Finish securing the LA with the pre-tied 5-0 suture (Figure 8C).
18. Identify the esophagus and clamp it with a hemostat as close to the diaphragm as possible. Cut the esophagus below the hemostat to ensure there is no spillage into the thoracic cavity (Figure 9A).
19. Using the spine as a guide, cut all ligamentous attachments connecting the heart-lung block to the surrounding structures using a pair of scissors (Figure 9B). Once the heart-lung block is freely mobile, dissect the trachea from the neck and finally cut the trachea above the ET tube using a pair of scissors to free the heart-lung block (Figure 9C).
20. Move the heart-lung block to the thoracic jacket within the EVLP circuit and attach the LA cannula to the EVLP circuit (Figure 9D). Turn the roller pump on and connect the ventilator monitor.
21. Check the bubble trap to ensure no air emboli are being introduced into the system.
22. Slowly change ventilation and perfusion settings to desired experimental levels during the initial 15 min^36,37,38. Additionally, during this initial ramp-up phase, increase the perfusion flow rate to the desired rate and/or pressure.
23. At experiment-designated time-points, check perfusate gas levels as well as pulmonary function tests.

Representative Results

The validation of our PolyhHb-based perfusate, and furthermore, the stability of this perfusate over several hours, is demonstrated in Figure 10. Over the first 1 h, all perfusates tested (PolyhHb, Control (Williams Media + 5% HSA), RBC based) showed a slight decrease in LA pO₂ (Post pO₂). However, the RBC-based perfusate showed a significant decrease at 1 h compared to PolyhHb (p < 0.05). When tested over the next several hours, both PolyhHb and Control perfusates had stable LA pO_2, while PolyhHb had a non-significant trend (p > 0.05) of higher pO₂ (Figure 10A). Delta pO₂, i.e., the change in the LA pO₂ from PA pO₂, again significantly decreased at 1 h in the RBC perfusate group (p < 0.05), while it remained stable in the PolyhHb and Control perfusates with a non-significant trend (p > 0.05) of higher pO₂ in the PolyhHb group (Figure 10B). LA pCO₂ was significantly lower in the RBC perfusate and the Control perfusate when compared to the PolyhHb perfusate after the first hour (p < 0.05), and this held true over the next several hours when comparing PolyhHb and Control perfusate (Figure 10C). Finally, delta pCO₂ (i.e., the change in the LA pCO2 from PA pCO2) was significantly increased in the RBC perfusate after 1 h (p < 0.05), and after several hours remained stable in both the PolyhHb and Control perfusate (Figure 10D).

The real-time lung physiological data collected through the acquisition software provides complementary information to perfusate gas levels (Figure 11). Pulmonary vascular resistance (PVR) again showed that the RBC perfusate significantly increased over the first hour (p < 0.05). Over the several remaining hours, both the PolyhHb and Control perfusates had stable and low PVR (Figure 11A). The change in lung weight also significantly increased in the RBC perfusate over the first hour (p < 0.05) and increased in both the PolyhHb and Control perfusate over the remaining hours with a slightly higher weight in the PolyhHb perfusate (Figure 11B). Finally, compliance decreased significantly in the RBC perfusate group within the first hour (p < 0.05), while there was a non-significant decrease in the PolyhHb and Control perfusate (p > 0.05), with PolyhHb having the highest compliance after 4 h (Figure 11C).

In terms of technical success and/or failure (Figure 12), several things are important to draw attention to. In Figure 12A, we can see allograft failure due to right upper lobe necrosis due to a possible clot within the pulmonary vasculature. In Figure 12B, we note severe tissue edema within the right lobe as well, leading to experimental failure. Figure 12C-E show proper tissue preservation and appearance within respective experimental conditions. Finally, in Figure 12F, we can see ideal tissue preservation following flushing with a lung preservation solution.

Figure 1: Synthesis and purification of PolyhHb on a pilot scale. (A) Bioreactor for polymerization. (B) Tangential flow filtration (TFF) processes are set up in a 4 °C fridge. (C) Close-up of parallel TFF set-up for the red blood cell (RBC) wash and hemoglobin (Hb) purification. (D) Close-up of the two-stage series TFF system for PolyhHb purification. Vessels for stages one and two are located to the left and right of the filters, respectively. Please click here to view a larger version of this figure.

Figure 2: Ex vivo lung perfusion (EVLP) circuit overview. (A) Schematic drawing of EVLP circuit. (B) In vivo placement of pulmonary artery cannula and left atrial cannula. Please click here to view a larger version of this figure.

Figure 3: Surgical instruments used for ex vivo lung perfusion. (A) Silk suture. (B) Fine-tipped forceps (medium length). (C) Fine-tipped forceps (long length). (D) Curved fine-tipped forceps. (E) Mayo scissors. (F) Tracheal cannula. (G) Pulmonary artery (PA) cannula. (H) Left atrial (LA) cannula. (I) Rib cage retractors. (J) Spring scissors. (K) DeBakey forceps. (L) Hemostat. (M) Small scissors. (N) Small curved fine-tip forceps. (O) Adson pick-ups. Please click here to view a larger version of this figure.

Figure 4: Surgical positioning and exposing the inferior vena cava (IVC). (A) Rat positioning for lung procurement. (B) Exposing the infra-hepatic IVC. (C) Cannulating the IVC and injecting heparin with a 27G needle. Please click here to view a larger version of this figure.

Figure 5: Cannulating the trachea with the endotracheal (ET) tube. (A) Begin by cutting the skin of the neck area. (B) Dissect strap muscles and connective tissue to expose the trachea. (C) Making a transverse incision on the anterior trachea between the cartilaginous rings big enough for the ET tube. (D) Insert ET tube into trachea and secure in place with silk suture. Please click here to view a larger version of this figure.

Figure 6: Pulmonary artery cannula placement. (A) Exposing the thoracic cavity to visualize the heart and lungs. (B) Identifying the PA and isolating it. (C) Placing suture around PA. (D) Cutting a small hole in the right ventricle outflow tract (RVOT) for the PA cannula. (E) Proper placement of the PA cannula inside of the PA. Please click here to view a larger version of this figure.

Figure 7: Flushing the lungs with preservation solution. (A) Connecting the flush cannula to the pulmonary artery (PA) cannula. (B) Clear fluid should come out of the left atrium (LA). (C) Connecting the PA cannula to the ex vivo lung perfusion circuit to ensure proper flow and placement of the PA cannula. Please click here to view a larger version of this figure.

Figure 8: Placing the left atrial (LA) cannula. (A). Gently dilating the mitral valve annulus with a pair of forceps. (B) Loosely placing a silk suture around the left ventricle (LV). Placing the LA cannula within the left atrium. Please click here to view a larger version of this figure.

Figure 9: Extracting the heart-lung block. (A) Ligating the esophagus below the hemostat. (B) Dissecting frees the heart-lung block from the spine. (C) Dissecting free the trachea. (D) Proper connections and placement of ex vivo lung perfusion (EVLP) cannula. Please click here to view a larger version of this figure.

Figure 10. Perfusate gas levels over time. (A) Post pO₂, i.e. left atrial (LA) pO₂, over a 4 h perfusion. (B) Delta pO₂, i.e. the change in the LA pO₂ from pulmonary artery (PA) pO₂ over a 4 h perfusion. (C) Post pCO₂, i.e. LA pO₂, over a 4 h perfusion. (D) Delta pCO₂, i.e. the change in the LA pO₂ from PA pO₂ over a 4 h perfusion. Blue represents PolyhHb perfusate, black represents Control perfusate (standard William's media) and red represents RBC-based perfusate. N=6 per group. Error bars indicate standard deviation. Significance was tested using a Student's T-test, and is denoted by a *, p < 0.05. Please click here to view a larger version of this figure.

Figure 11. Real-time lung physiological data. (A) Pulmonary vascular resistance (PVR) over 4 h reperfusion. (B) Change (denoted by Δ) in lung weight over time. (C) Compliance over 4 h reperfusion. Blue represents PolyhHb perfusate, black represents Control perfusate (standard William's media) and red represents RBC-based perfusate. N=6 per group. Error bars indicate standard deviation. Significance was tested using a Student's T-test, and is denoted by a *, p < 0.05. Please click here to view a larger version of this figure.

Figure 12: Representative technical results. (A) Failure of graft due to right upper lobe infarction. (B) Failure of graft due to severe right lobe edema. (C) Successful canulation and perfusion of lung allograft with RBC perfusate. (D) Successful canulation and perfusion of lung allograft with PolyhHb perfusate. (E) Successful canulation and perfusion of lung allograft with standard perfusate. (F) Ideal tissue preservation following flushing with lung preservation solution. Please click here to view a larger version of this figure.

Discussion

The development and testing of perfusion solutions is a novel endeavor that many throughout the globe are embarking on. Traditionally, standard perfusates offer the ability to suspend ischemic time and mitigate the associated injuries with ischemia, as well as reperfusion¹⁸. However, the next evolution of EVLP is to improve current perfusate technology as well as incorporate repair and reconditioning therapies^{39,40,41,42,43}.

The PolyhHb described in this work is bracketed between 500 kDa and 0.2 µm to prevent the material from extravasating from the circuit into the lung, which will prevent vasoconstriction and increased PA pressure³⁰. It is critical that throughout the polymerization steps of this synthesis, the partial pressure of oxygen (pO₂) is maintained at the appropriate value for the desired oxygen affinity PolyhHb product. This includes all added solutions throughout the reaction (i.e., crosslinker, quenching solution, etc.) having a matched pO2 to the bioreactor (i.e., degassed with nitrogen, oxygenated, etc.). A major advantage to this synthesis procedure is that the final product has modifiable oxygen equilibria to allow for different applications with different oxygen demands (i.e., low oxygen affinity PolyhHb for transfusion medicine, moderate oxygen affinity for lung perfusion, or high oxygen affinity for targeted oxygen delivery). It is also important to ensure there is a heating mechanism on the bioreactor that does not result in excessive heating to contact points, resulting in the formation of damaged proteins. We found that a copper coil throughout the vessel provided more even and less damaging heating/cooling than an insulated heating jacket on the outside of the vessel (Figure 1A).

While the development of an EVLP rat model is not new^37,38, we have noted several areas that can lead to improved results. Firstly, it is necessary to make small incisions in the IVC upon sacrifice to ensure there is no additional air that could enter the lungs through the circulation. When flushing the lung allograft with the lung preservation solution, a uniform pale white color of the lungs lets the micro-surgeon know that there is technical success for the procurement process. If there is still a pink color lung within the parenchyma, it is sometimes advisable to adjust the PA cannula so that the whole lung is evenly perfused. While the PA cannula is often the easier part of the procedure to complete, introducing the LA cannula is slightly more difficult. It is always necessary to dilate the mitral valve annulus in order to have the LA cannula reach the LA. However, this must be done with extreme caution as it is easy to perforate the ventricle or atria. Once the tip of the cannula is within the atria, it can often become misplaced while securing the suture around the ventricle. It is oftentimes necessary to adjust the table angle (more horizontal) or place a piece of gauze at the bottom of the cannula so it stays in place.

Limitations
There are some limitations to this model. While it is helpful to evaluate the efficacy of perfusates and their ability to improve potential allografts, this is not a transplant model that would be able to tell us in vivo results of differing perfusates and technologies. Additionally, while PolyhHb is an exciting new perfusate technology, its use, efficacy, and potential limitations will have to be further substantiated in additional pre-clinical and clinical perfusion experiments before widespread adoption of this technology can be considered.

Conclusions
Here, we demonstrated the application of a next-generation PolyhHb perfusate and the protocol by which this perfusion solution can be tested in a model of rat EVLP. As perfusate technology advances, it will be advantageous to explore the possibilities of using PolyhHb as a potential substitute for traditional perfusates³⁰. Previous generations of PolyhHb have led to detrimental side effects based on their composition; however, improvements to the synthesis have created a polymer that is less likely to extravasate, lead to edema, and thus cause cellular injury³⁰. With PolyhHb, it is possible to perform EVLP without the need for RBCs while still meeting the metabolic demand of lung allografts. This will undoubtedly allow for better allograft function ex vivo. However, further validation of PolyhHb in both the pre-clinical and clinical settings is needed. We hope this protocol provides 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.

Materials

Name	Company	Catalog Number	Comments
10 cc insulin syringe 29 G x 1/2" needle	B-D	309301
30 L Glass Batch Bioreactor	Ace Glass
30g Needle	Med Needles	BD-305106
Baytril (enrofloxacin) Antibacterial Tablets	Elanco	NA
Calcium Chloride dihydrate (CaCl2.2H2O)	Sigma Aldrich	10035-04-8	For modified Ringer's lactate
CFBA carrier frequency bridge amplifier type 672	Harvard Apparatus	731747
Connect kit D150	Cole-Parmer	VK 73-3763
Dumont #5 Forceps	Fine Science tools	11252-50
Dumont Medical #5/45 Forceps - Angled 45°	Fine Science tools	11253-25
Ecoline Star Edition 003, E100 Water Heater	Lauda	LCK 1879
Expired human leukoreduced, packed RBC units	Wexner Medical Center Canadian Blood Services Zen-Bio Inc
Fiberoxygenator D150	Hugo Sachs Elektronik	PY2 73-3762
Forceps	Fine Science tools	11027-12
Glutaraldehyde (C5H8O2 70 wt%)	Sigma Aldrich	111-30-8 (G7776)
Halsted-Mosquito Hemostat	Roboz Surgical	RS-7112
Heparin 30,000 units per 30 ml	APP Pharmaceuticals
Human Serum Albumin (HSA)	OctaPharma Plasma		Perfusate additive
IL2 Tube set for perfusate	Harvard Apparatus	733842
IPL-2 Basic Lung Perfusion System	Harvard Apparatus
Ketamine 500 mg per 5 ml	JHP Pharmaceuticals
Left Atrium cannula	Harvard Apparatus	730712
Liqui-Cel EXF Series G420 Membrane Contactor	3M	G420	gas contactor
low potassium dextran glucose solution (perfadex)	XVIVO		solution flushing the lung
Masterflex Platinum Coated Tubing(Size: 73,17,16,24)	Cole-Palmer
N-Acetyl-L-cysteine (NALC, C5H9NO3S)	Sigma Aldrich	616-91-1 (A7250)	For modified Ringer's lactate
Nalgene Vessels (10L, 20L)	Nalgene		Filtration vessels
Peristaltic Pump	Ismatec	ISM 827B
PES, 0.65 µm TFF module	Repligen	N02-E65U-07-N
PhysioSuite	Kent Scientific Corporation	PS-MSTAT-RT
polyethersulfone (PES), 0.2 µm TFF module	Repligen	N02-S20U-05-N
Polysulfone (PS), 500 kDa TFF module	Repligen	N02-P500-05-N
Potassium Chloride (KCl)	Fisher Scientific	7447-40-7	For PBS
PowerLab 8/35	ADInstruments	730045
Pulmonary Artery cannula	Harvard Apparatus	730710
Pump Head tubing (Size: 73,17,16,24)	PharMed BPT
Puralube Ophthalmic Ointment	Dechra	NA
Scissors	Fine Science tools	14090-11
SCP Servo controller for perfusion type 704	Harvard Apparatus	732806
Small Animal Ventilator model 683	Harvard Apparatus	55-000
Sodium Chloride (NaCl)	Fisher Scientific	7647-14-5 (S271-10)	For PBS and saline
Sodium cyanoborohydride (NaCNBH3)	Sigma Aldrich	25895-60-7
Sodium Dithionite (Na2S2O4)	Sigma Aldrich	7775-14-6
Sodium Hydroxide (NaOH)	Fisher Scientific	1310-73-2	For modified Ringer's lactate
Sodium Lactate (NaC3H5O3)	Sigma Aldrich	867-56-1	For modified Ringer's lactate
Sodium phosphate dibasic (Na2HPO4)	Fisher Scientific	7558-79-4	For PBS
Sodium phosphate monobasic (NaH2PO4)	Fisher Scientific	7558-80-7	For PBS
SomnoSuite Small Animal Anesthesia System	Kent Scientific Corporation	SS-MVG-Module
Sprague-Dawley rats	Envigo
TAM-A transducer amplifier module type 705/1	Harvard Apparatus	73-0065
TAM-D transducer amplifier type 705/2	Harvard Apparatus	73-1793
TCM time control module type 686	Harvard Apparatus	731750
Tracheal cannula	Harvard Apparatus	733557
Tube set for moist chamber	Harvard Apparatus	73V83157
Tubing Cassette	Cole-Parmer	IS 0649
Tweezer #5 Dumostar	Kent Scientific Corporation	INS500085-A
Tweezer #5 stainless steel, curved	Kent Scientific Corporation	IND500232
Tweezer #7 Titanium	Kent Scientific Corporation	INS600187
Tygon E-3603 Tubing 2.4 mm ID	Harvard Apparatus	721017	perfusate line entering lung
Tygon E-3603 Tubing 3.2 mm ID	Harvard Apparatus	721019	perfusate line leaving lung
Vannas-Tubingen Spring Scissors	Fine Science Tools	15008-08
VCM ventilator control module type 681	Harvard Apparatus	731741
William's E Media	Gibco, ThermoFisher Scientific	A12176-01	Perfusate additive
Xylazine 100 mg per 1 ml	Akorn