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>
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			<td>Tube set for moist chamber</td>
			<td>Harvard Apparatus</td>
			<td>&#160;73V83157</td>
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			<td>Tubing Cassette</td>
			<td>Cole-Parmer</td>
			<td>IS 0649</td>
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			<td>Tweezer #5 Dumostar</td>
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			<td>Tweezer #5 stainless steel, curved</td>
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			<td>Tygon E-3603 Tubing 2.4 mm ID</td>
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			<td>perfusate line entering lung</td>
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			<td>Tygon E-3603 Tubing 3.2 mm ID</td>
			<td>Harvard Apparatus</td>
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			<td>perfusate line leaving lung</td>
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			<td>Vannas-Tubingen Spring Scissors</td>
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			<td>Harvard Apparatus</td>
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			<td>William&#39;s E Media</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.

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

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

Ex vivo lung perfusion or EVLP enables extended evaluation of donor lungs. There are two perfusion solutions, an acellular and cellular. This study details using a polymerized human hemoglobin or a poly HB-based oxygen carrier as a perfusate in the protocol for testing this solution in a rat EVLP model.The recent advances in ex vivo organ perfusion have enabled expanding the donor organ pool with improved function. Working to extend normothermic profusion times with tailored perfusates will allow for further advances in this space. Current perfusion techniques use cellular or acellular perfusates, each with pros and cons.Perfusion composition is crucial for metabolic support, reducing inflammation and maintaining oncotic pressure. However, existing solutions can cause pulmonary edema and damage. Thus, it is necessary to develop novel perfusion solutions that prevent excessive damage while maintaining proper cellular homeostasis.A promising hemoglobin-based oxygen carrier or HBOC is polymerized human hemoglobin synthesized from expired packed red blood cells. Our laboratory developed a next generation poly HB with minimal low molecular weight species and cell-free hemoglobin. This has shown improved biophysical characteristics and has reduced vasoconstriction, hypertension, and oxidative damage in animal studies.These characteristics make it a promising perfusate candidate.

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 ...

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

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

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

Research

JoVE Journal

Medicine

326 Views.  The Ohio State University Wexner Medical Center. Ex vivo lung perfusion or EVLP enables extended evaluation of donor lungs. There are two perfusion solutions, an acellular and cellular. This study details using a polymerized human hemoglobin or a poly HB-based oxygen carrier as a perfusate in the protocol for testing this solution in a rat EVLP model.The recent advances in ex vivo organ perfusion have enabled expanding the donor organ pool with improved function. Working to extend normothermic profusion times with tailored perfusates...

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

Setup of an Ex Vivo Lung Perfusion (EVLP) Circuit with PolyhHb for Lung Perfusion in Rats

To begin, take human serum albumin at a final concentration of 3%Into it, add polymerized hemoglobin, or PolyhHb, diluted to a final concentration of 3.7 grams per deciliter with William's E Medium. Then add one milliliter of heparin solution to the resultant diluted PolyhHb solution. Place PolyhHb perfusate into the EVLP circuit reservoir and set the water bath temperature to 37 degrees Celsius.Then turn on the roller pumps and ensure that perfusate is circulating within the circuit. Connect de-oxygenation gas to the hollow fiber oxygenator to de-oxygenate the perfusate. Open data acquisition software on the computer.Verify that 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. Carefully examine all tube connections to ensure no leaks are present and warm water is circulating throughout. Press Run on the software to check that all pressure transducers are functioning.Once the system is properly functioning, turn off the roller pumps.

Procurement and Cannulation of Donor Rat Lung Block for Ex Vivo Lung Perfusion

To begin, place the autoclaved surgical instruments on the surgical table. Shave the abdomen of the anesthetized rat and place the rat in the supine position on the surgical board. Clean the abdomen with povidone iodine followed by 70%ethanol.Place ophthalmic ointment under the rat's eyes to prevent dryness. Secure the rat in place on the surgical board. Start the data acquisition software and begin recording.Turn on the ventilator at four milliliters per kilogram and ensure positive end expiratory pressure is around two centimeters of water. Using scissors, perform a midline laparotomy from the xiphoid process to the pubic symphysis. Then with the help of a blunt instrument, perform a medial-lateral visceral rotation and visualize the infrahepatic inferior vena cava.Inject heparin into the inferior vena cava with a 20-gauge needle. Using a pair of scissors, cut the skin from the sternal notch to just below the angle of the mandible and begin to dissect toward the trachea. Then, bluntly dissect away necessary strap muscles to expose the trachea.Make a transverse incision on the anterior trachea between the cartilaginous rings, ensuring not to cut through the posterior portion of the trachea. Place a 5-0 silk suture around the trachea. Insert the endotracheal tube into the cartilaginous rings, and secure it with the 5-0 silk suture.Connect the endotracheal tube to the ventilator and ensure proper chest rising. Using scissors, perform a median sternotomy and enter the thoracic cavity. Place chest wall retractors to expose the heart and lungs, avoiding any inadvertent manipulation of the lungs.With the combination of sharp and blunt dissection, remove the thymus from the anterior mediastinum. Identify the pulmonary artery and place a 5-0 silk suture around it to prepare for cannulation. Make a two to three millimeter incision in the right ventricular outflow tract using scissors to place the arterial cannula within the pulmonary artery and secure it with the 5-0 suture.After euthanizing the rat, quickly connect the de-aired lung preservation fluid to the arterial cannula to gravity flush the lungs. 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. Next, place a small forceps in the left ventricle and gently stretch the mitral valve annulus. Place a 5-0 silk tie around the heart and loosely tie.Insert the left atrium cannula into the left ventricle and advance the cannula until it can be seen within the atrium. Secure the left atrium with the pre-tied 5-0 suture. Identify the esophagus and clamp it with a hemostat as close to the diaphragm as possible.Then, cut the esophagus below the hemostat. Using the spine as a guide, cut all ligamentous attachments connecting the heart-lung block to the surrounding structures with scissors. Then dissect the trachea from the neck and cut the trachea above the endotracheal tube to free the heart-lung block.Move the heart-lung block to the thoracic jacket within the EVLP circuit and attach the left atrium cannula to the circuit. Turn the roller pump on and connect the ventilator monitor. Check the bubble trap to ensure no air emboli are being introduced into the system.Slowly change ventilation and perfusion settings to the desired experimental levels during the initial 15 minutes. Additionally, increase the perfusion flow rate to the desired rate and pressure. At designated time points, check perfusate gas levels and pulmonary function tests.All tested perfusates showed a slight decrease in left atrium partial pressure of oxygen with the RBC-based perfusate significantly decreasing at one hour. For the next several hours, both polyhHB and control perfusates had stable left atrium partial pressure of oxygen. The delta partial pressure of oxygen significantly decreased at one hour in the RBC perfusate group while it remained stable in the polyhHB and control perfusates with a non-significant trend.Left atrium partial pressure of carbon dioxide was significantly lower in the RBC and control perfusate compared to polyhHB after the first hour, and this trend continues over the following hours. The delta partial pressure of carbon dioxide was significantly increased in the RBC perfusate after one hour and after that remained stable in both the polyhHB and control perfusate. Real-time lung physiological data demonstrate that the RBC perfusate significantly increased pulmonary vascular resistance within the first hour.While both polyhHB and control perfusates maintained low and stable pulmonary vascular resistance over the period, the change in lung weight was significant in the RBC perfusate initially, with a continued increase in all perfusates, slightly more in polyhHB. Compliance decreased significantly in the RBC perfusate within the first hour, while it decreased non-significantly in other perfusates. PolyhHB had the highest compliance after four hours.