This research was generously supported by the Jewel and Frank Benson Family Endowment and the Jewel and Frank Benson Research Professorship. B.A.W. is partially supported by National Institutes of Health (NIH) grant R01HL143000. A.F.P. is supported by NIH grants R01HL126945, R01EB021926, R01HL131720, and R01HL138116 and US Army Medical Research and Materiel Command grant W81XWH1810059. S.M.B. is supported by the NIH R01 DK123475.

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&#39;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.
<ol>
	<li>RBC wash, lysis, and Hb purification
	<ol>
		<li>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).</li>
		<li>Perform six system volume exchanges (diacycles) on a 0.65 &#181;m 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).</li>
		<li>Lyse the RBC solution with 10 L of phosphate buffer (PB, 3.75 mM, pH 7.4) for 1 h at 4&#160;&#176;C with constant stirring.</li>
		<li>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).</li>
		<li>Once 480 g of Hb is in the reactor, add a salt charge to convert PB into phosphate buffered saline (PBS).</li>
		<li>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 &#176;C to limit methemoglobin (metHb) formation.</li>
	</ol>
	</li>
	<li>Hb polymerization
	<ol>
		<li>Heat the Hb solution to physiological temperature (37&#160;&#176;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).</li>
		<li>Add 1 g charge of sodium dithionite, as needed, to ensure effective deoxygenation.</li>
		<li>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).</li>
		<li>Add solution to reactor vessel over 3 h with an additional hour of reaction time.</li>
		<li>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.</li>
		<li>Chill reactor at 14&#160;&#176;C overnight.</li>
	</ol>
	</li>
	<li>PolyhHb purification
	<ol>
		<li>Pump the reactor contents into a 10 L filtration vessel and begin circulation through a 0.2 &#181;m polyethylene sulfone (PES) TFF module (Stage 1). This step will remove large aggregates and undesired contaminants.</li>
		<li>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).</li>
		<li>Once the reactor is emptied into the purification circuit, start excipient exchange on Stage 1 with a modified lactated Ringer&#39;s solution (pH 7.4). After each full volume exchange, measure the protein concentration in the permeate of Stage 1 using UV-visible spectroscopy.</li>
		<li>When the Stage 1 permeate has a concentration of less than 1 mg Hb/mL, transfer the modified Ringer&#39;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&#39;s solution are performed across both stages.</li>
		<li>Following the completion of diacycles, concentrate the contents of Stage 2 to at least 10 g/dL over the 500 kDa TFF module.</li>
		<li>Package the concentrated solution in 50 mL conical tubes and store at -80&#160;&#176;C until use.</li>
	</ol>
	</li>
</ol>
2. Perfusate formulation
<ol>
	<li>Prepare the perfusate to a final volume of 165 mL. Dilute PolyhHb to a final concentration of 3.7 g/dL with William&#39;s E Medium.</li>
	<li>Add human serum albumin (HSA) to a final concentration of 3% HSA by weight. Add 1 mL of heparin to final solution.</li>
</ol>
3. Ex Vivo lung perfusion circuit setup
<ol>
	<li>Place PolyhHb perfusate into the EVLP circuit reservoir and turn on the warm water bath to 37 &#176;C. Ensure the perfusate is circulating within the circuit by turning on the roller pumps.</li>
	<li>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&#39;s ability to oxygenate the perfusate.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Procurement of donor rat lung block
<ol>
	<li>Set up the surgical table and layout the instruments (Figure 3). Autoclave all instruments at 121 &#176;C for 30 min.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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&#39;s eyes to prevent dryness.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39;s great vessels, it is often easier to place the suture around the PA and aorta at the same time.</li>
	<li>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).</li>
	<li>Make a 5 mm incision in the left ventricle (LV) as well as 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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>Check the bubble trap to ensure no air emboli are being introduced into the system.</li>
	<li>Slowly change ventilation and perfusion settings to desired experimental levels during the initial 15 min36,37,38. Additionally, during this initial ramp-up phase, increase the perfusion flow rate to the desired rate and/or pressure.</li>
	<li>At experiment-designated time-points, check perfusate gas levels as well as pulmonary function tests.</li>
</ol>

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

For the material presented in this work, A.F.P., A.G., and C.C. are inventors on the US patent application PCT/US2022/041743. A.F.P., C.C., B.A.W., and S.M.B. are inventors on US patent application PCT/US2023/017765.

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 therapies39,40,<sup class="x...

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 age1,2. 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 donor2,4,5,6,7, affording the transplant provider the ability to determine transplantation suitability. EVLP has shown the ability to adequately assess donor organs8,9,10,11, decrease the effects of ischemic reperfusion injury (IRI)12,13 and increase the donor pool14,15 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 cells18,19. 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 itself20,21. 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 arrest23,25. The reasons behind these findings were likely due to the presence of cell-free Hb or low molecular weight Hb polymers (&#60; 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 injury26,27. 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 responses22,28,29,30. 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 mitigated28,29,31,32,33,34,35. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cc insulin syringe 29 G x 1/2&#34; needle</td>
			<td>B-D</td>
			<td>309301</td>
			<td></td>
		</tr>
		<tr>
			<td>30 L Glass Batch Bioreactor</td>
			<td>Ace Glass</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30g Needle</td>
			<td>Med Needles</td>
			<td>BD-305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Baytril (enrofloxacin) Antibacterial Tablets</td>
			<td>Elanco</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate (CaCl2.2H2O)</td>
			<td>Sigma Aldrich</td>
			<td>10035-04-8</td>
			<td>For modified Ringer&#39;s lactate</td>
		</tr>
		<tr>
			<td>CFBA carrier frequency bridge amplifier type 672</td>
			<td>Harvard Apparatus</td>
			<td>731747</td>
			<td></td>
		</tr>
		<tr>
			<td>Connect kit D150</td>
			<td>Cole-Parmer</td>
			<td>&#160;VK 73-3763</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science tools</td>
			<td>11252-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Medical #5/45 Forceps - Angled 45&#176;</td>
			<td>Fine Science tools</td>
			<td>11253-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ecoline Star Edition 003, E100 Water Heater</td>
			<td>Lauda</td>
			<td>LCK 1879</td>
			<td></td>
		</tr>
		<tr>
			<td>Expired human leukoreduced, packed RBC units</td>
			<td>Wexner Medical Center 
			Canadian Blood Services 
			Zen-Bio Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiberoxygenator D150</td>
			<td>Hugo Sachs Elektronik</td>
			<td>PY2 73-3762</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (C5H8O2 70 wt%)</td>
			<td>Sigma Aldrich</td>
			<td>111-30-8 (G7776)</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsted-Mosquito Hemostat</td>
			<td>Roboz Surgical</td>
			<td>RS-7112</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin 30,000 units per 30 ml</td>
			<td>APP Pharmaceuticals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin (HSA)</td>
			<td>OctaPharma Plasma</td>
			<td></td>
			<td>Perfusate additive</td>
		</tr>
		<tr>
			<td>IL2 Tube set for perfusate</td>
			<td>Harvard Apparatus</td>
			<td>733842</td>
			<td></td>
		</tr>
		<tr>
			<td>IPL-2 Basic Lung Perfusion System</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 500 mg per 5 ml</td>
			<td>JHP Pharmaceuticals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Left Atrium cannula</td>
			<td>Harvard Apparatus</td>
			<td>730712</td>
			<td></td>
		</tr>
		<tr>
			<td>Liqui-Cel EXF Series G420 Membrane Contactor</td>
			<td>3M</td>
			<td>G420</td>
			<td>gas contactor</td>
		</tr>
		<tr>
			<td>low potassium dextran glucose solution (perfadex)</td>
			<td>XVIVO</td>
			<td></td>
			<td>solution flushing the lung</td>
		</tr>
		<tr>
			<td>Masterflex Platinum Coated Tubing(Size: 73,17,16,24)</td>
			<td>Cole-Palmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine (NALC, C5H9NO3S)</td>
			<td>Sigma Aldrich</td>
			<td>616-91-1 (A7250)</td>
			<td>For modified Ringer&#39;s lactate</td>
		</tr>
		<tr>
			<td>Nalgene Vessels (10L, 20L)</td>
			<td>Nalgene</td>
			<td></td>
			<td>Filtration vessels</td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>&#160;Ismatec</td>
			<td>&#160;ISM 827B</td>
			<td></td>
		</tr>
		<tr>
			<td>PES, 0.65 &#181;m TFF module</td>
			<td>Repligen</td>
			<td>N02-E65U-07-N</td>
			<td></td>
		</tr>
		<tr>
			<td>PhysioSuite</td>
			<td>Kent Scientific Corporation</td>
			<td>PS-MSTAT-RT</td>
			<td></td>
		</tr>
		<tr>
			<td>polyethersulfone (PES), 0.2 &#181;m TFF module</td>
			<td>Repligen</td>
			<td>N02-S20U-05-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysulfone (PS), 500 kDa TFF module</td>
			<td>Repligen</td>
			<td>N02-P500-05-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>7447-40-7</td>
			<td>For PBS</td>
		</tr>
		<tr>
			<td>PowerLab 8/35</td>
			<td>&#160;ADInstruments</td>
			<td>730045</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulmonary Artery cannula</td>
			<td>Harvard Apparatus</td>
			<td>730710</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump Head tubing (Size: 73,17,16,24)</td>
			<td>PharMed BPT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube&#160;Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science tools</td>
			<td>14090-11</td>
			<td></td>
		</tr>
		<tr>
			<td>SCP Servo controller for perfusion type 704</td>
			<td>Harvard Apparatus</td>
			<td>732806</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Ventilator model 683</td>
			<td>Harvard Apparatus</td>
			<td>55-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5 (S271-10)</td>
			<td>For PBS and saline</td>
		</tr>
		<tr>
			<td>Sodium cyanoborohydride (NaCNBH3)</td>
			<td>Sigma Aldrich</td>
			<td>25895-60-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dithionite (Na2S2O4)</td>
			<td>Sigma Aldrich</td>
			<td>7775-14-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>1310-73-2</td>
			<td>For modified Ringer&#39;s lactate</td>
		</tr>
		<tr>
			<td>Sodium Lactate (NaC3H5O3)</td>
			<td>Sigma Aldrich</td>
			<td>867-56-1</td>
			<td>For modified Ringer&#39;s lactate</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>7558-79-4</td>
			<td>For PBS</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (NaH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>7558-80-7</td>
			<td>For PBS</td>
		</tr>
		<tr>
			<td>SomnoSuite Small Animal Anesthesia System</td>
			<td>Kent Scientific Corporation</td>
			<td>SS-MVG-Module</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-Dawley rats</td>
			<td>Envigo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TAM-A transducer amplifier module type 705/1</td>
			<td>Harvard Apparatus</td>
			<td>73-0065</td>
			<td></td>
		</tr>
		<tr>
			<td>TAM-D transducer amplifier type 705/2</td>
			<td>Harvard Apparatus</td>
			<td>&#160;73-1793</td>
			<td></td>
		</tr>
		<tr>
			<td>TCM time control module type 686</td>
			<td>Harvard Apparatus</td>
			<td>731750</td>
			<td></td>
		</tr>
		<tr>
			<td>Tracheal cannula</td>
			<td>Harvard Apparatus</td>
			<td>733557</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube set for moist chamber</td>
			<td>Harvard Apparatus</td>
			<td>&#160;73V83157</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Cassette</td>
			<td>Cole-Parmer</td>
			<td>IS 0649</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer #5 Dumostar</td>
			<td>Kent Scientific Corporation</td>
			<td>&#160;INS500085-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer #5 stainless steel, curved</td>
			<td>Kent Scientific Corporation</td>
			<td>IND500232</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer #7 Titanium</td>
			<td>Kent Scientific Corporation</td>
			<td>&#160;INS600187</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon E-3603 Tubing 2.4 mm ID</td>
			<td>Harvard Apparatus</td>
			<td>721017</td>
			<td>perfusate line entering lung</td>
		</tr>
		<tr>
			<td>Tygon E-3603 Tubing 3.2 mm ID</td>
			<td>Harvard Apparatus</td>
			<td>721019</td>
			<td>perfusate line leaving lung</td>
		</tr>
		<tr>
			<td>Vannas-Tubingen Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15008-08</td>
			<td></td>
		</tr>
		<tr>
			<td>VCM ventilator control module type 681</td>
			<td>Harvard Apparatus</td>
			<td>731741</td>
			<td></td>
		</tr>
		<tr>
			<td>William&#39;s E Media</td>
			<td>Gibco, ThermoFisher Scientific</td>
			<td>A12176-01</td>
			<td>Perfusate additive</td>
		</tr>
		<tr>
			<td>Xylazine 100 mg per 1 ml</td>
			<td>Akorn</td>
			<td></td>
			<td></td>
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exploring alternative perfusion solutions using next-generation polymerized hemoglobin-based oxygen carriers in a model of rat ex vivo lung perfusion

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

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 &#60; 0.05). When tested over the next several hours, both PolyhHb and Control perfusates ...

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

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

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

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

exploring-alternative-perfusion-solutions-using-next-generation

Research

JoVE Journal

Medicine

356 Views.  The Ohio State University Wexner Medical Center. 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.

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

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

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

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

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

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

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

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

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

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.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

Isolated Lung Perfusion System in the Rabbit Model

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

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

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

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

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

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

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

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

Surgical Technique for Jugular Vein Thermodilution Catheter Placement in Pigs

Determination of Cardiac Output in a Porcine Model for Ex Vivo Pulmonary Perfusion

Due to their physiological similarities to humans, pigs are used as experimental models for ex vivo lung perfusion (EVLP). EVLP is a technique that perfuses lungs that are not suitable for transplantation via an extracorporeal circulation pump to improve their function and increase their viability. Existing EVLP protocols are differentiated by the type of perfusion solution and perfusion flow, which varies from 40%-100% of the estimated cardiac output (CO) according to the body surface area (BSA). Devices for measuring CO use simple physical principles and other mathematical models. Thermodilution in animal models continues to be the reference standard for estimating CO because of its simplicity and ease of reproduction. Therefore, the objective of this study was to reproduce the measurement of CO by thermodilution in pigs and compare its precision and accuracy with those obtained by the BSA, weight, and Fick's method, to establish perfusion flow during EVLP. In 23 pigs, a thermodilution catheter was placed in the right jugular vein, and the carotid artery on the same side was cannulated. Blood samples were obtained for gasometry, and CO was estimated by thermodilution, adjusted body surface area, Fick's principle, and per body weight. The CO obtained by the BSA was greater (p = 0.0001, ANOVA, Tukey) than that obtained by the other methods. We conclude that although the methods used in this study to estimate CO are reliable, there are significant differences between them; therefore, each method must be evaluated by the investigator to determine which meets the needs of the protocol.

Author Spotlight: Assessment of Cardiac Output Calculation by Thermodilution in Pigs for Effective Perfusion Flow During EVLP

This protocol describes the surgical technique used for the placement of a thermodilution catheter through the jugular vein in pigs to estimate cardiac output and ensure adequate lung perfusion during ex vivo lung perfusion (EVLP).

Due to their physiological similarities to humans, pigs are used as experimental models for ex vivo lung perfusion (EVLP). EVLP is a technique that ...

Coronary Artery Assessment During Ex-Situ Heart Perfusion

Coronary Angiography During Ex-Situ Heart Perfusion in a Porcine Model

Heart transplantation is the gold standard treatment for advanced heart failure. The procurement of extended criteria donors (ECD) increases due to the current organ shortage. Coronary angiography is recommended in ECD at risk for coronary artery disease but is not systematically performed. These hearts are, therefore, either declined for transplant or procured without screening for coronary artery disease. Coronary angiography during normothermic ex-situ heart perfusion (NESP) could be an interesting approach to enhance the rate of ECD procurement and to reduce the risk of primary graft failure in the absence of coronary angiography in ECD. The present protocol aims to provide material details along with optimal imaging views for coronary angiography during NESP. Reproducible angiographic views were observed, including one dedicated to the right coronary artery, two for the left anterior descending artery, two for the circumflex artery, and a spider view. Continuous lactate extraction was observed in all procedures with a final median concentration of 1.10 mmol/L (0.61-1.75 mmol/L) two hours after coronary angiography, consistent with myocardial viability. The median contrast agent volume used for ex-situ imaging of the isolated perfused heart was 48 mL (38-108 mL). This protocol was reproducible for coronary artery imaging and did not impair myocardial viability during NESP.

Author Spotlight: Innovative Technique for Coronary Angiography in Marginal Donors

Screening for coronary artery disease could increase the procurement of donor hearts with extended criteria. A protocol for performing reproducible coronary angiography during ex-situ heart perfusion is described herein in a porcine model.

Heart transplantation is the gold standard treatment for advanced heart failure. The procurement of extended criteria donors (ECD) increases due to the ...

Optimization of Donor Heart Preservation Using a Working Rat Heart Perfusion Model

Using an Isolated Working Rat Heart Model to Test Donor Heart Preservation Strategies

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, transportation, and transplantation. Previous work from our laboratory showed that the addition of glyceryl trinitrate and erythropoietin to donor heart preservation solutions could significantly improve cardiac functional recovery after prolonged cold storage and in donation after circulatory death hearts. This supplementation protocol has been implemented in clinical use in transplant units around Australia, Belgium, and the United Kingdom. Here, we outline a protocol for testing supplementation strategies using an ex vivo isolated working rat heart (IWRH) perfusion circuit. Using this methodology, supplementation strategies can be tested in the context of prolonged cold static storage, donation after brain death, and donation after circulatory death donor heart preservation. Cardiac functional recovery, measured by aortic flow, coronary flow, cardiac output, pulse pressure, and heart rate, can be used to determine whether a particular preservation strategy can minimize ischemia-reperfusion injury of the donor heart.

Author Spotlight: Enhancing Donor Heart Preservation Through Isolated Rat Heart Perfusion Studies

We describe an ex vivo isolated working rat heart protocol to test donor heart preservation strategies. This paper describes the protocol for use in static cold storage of rodent donor hearts; however, the protocol can also be used for donor hearts obtained after donation after circulatory death and brain death.

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, ...