Name: Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion (Video) | JoVE Journal
Uploaded: 2024-06-14T14:10:00.000+00:00
Duration: 9 min 47 s
Description: Explore polymerized hemoglobin-based oxygen carriers for ex vivo lung perfusion using a rat EVLP model. This method improves lung function metrics and extends preservation times for donor organ evaluation.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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

<ol><li>Valapour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OPTN%2FSRTR+2021+annual+data+report%3A+Lung%5BTitle%5D)+AND+%22Am+J+Transplant%22%5BJournal%5D)">OPTN/SRTR 2021 annual data report: Lung.</a> Am J Transplant. 23, 2 Suppl 1 S379-S442 (2023).</li>
<li>Gouchoe, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion+in+donation+after+circulatory+death%3A+A+post+hoc+analysis+of+the+normothermic+Ex+Vivo+lung+perfusion+as+an+assessment+of+extended%2Fmarginal+donors+lungs+trial%5BTitle%5D)+AND+%22J+Thorac+Cardiovasc+Surg%22%5BJournal%5D)">Ex vivo lung perfusion in donation after circulatory death: A post hoc analysis of the normothermic Ex Vivo lung perfusion as an assessment of extended/marginal donors lungs trial.</a> J Thorac Cardiovasc Surg. , 10.1016/j.jtcvs.2024.03.011 (2024).</li>
<li>Bobba, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trends+in+donation+after+circulatory+death+in+lung+transplantation+in+the+United+States%3A+Impact+of+era%5BTitle%5D)+AND+%22Transpl+Int%22%5BJournal%5D)">Trends in donation after circulatory death in lung transplantation in the United States: Impact of era.</a> Transpl Int. 35, 10172(2022).</li>
<li>Steen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+lungs+from+a+non-heart-beating+donor%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Transplantation of lungs from a non-heart-beating donor.</a> Lancet. 357 (9259), 825-829 (2001).</li>
<li>Cypel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normothermic+ex+vivo+lung+perfusion+in+clinical+lung+transplantation%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">Normothermic ex vivo lung perfusion in clinical lung transplantation.</a> N Engl J Med. 364 (15), 1431-1440 (2011).</li>
<li>Cypel, M., Neyrinck, A., Machuca, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+perfusion+techniques%3A+state+of+the+art+and+potential+applications%5BTitle%5D)+AND+%22Intens+Care+Med%22%5BJournal%5D)">Ex vivo perfusion techniques: state of the art and potential applications.</a> Intens Care Med. 45 (3), 354-356 (2019).</li>
<li>Gouchoe, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((XPS%E2%84%A2+Jensen+lung+as+a+low-cost%2C+high-fidelity+training+adjunct+to+ex-vivo+lung+perfusion%5BTitle%5D)+AND+%22Artif+Organs%22%5BJournal%5D)">XPS™ Jensen lung as a low-cost, high-fidelity training adjunct to ex-vivo lung perfusion.</a> Artif Organs. , 10.1111/aor.14623 (2023).</li>
<li>Van Raemdonck, D., Rega, F., Rex, S., Neyrinck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Machine+perfusion+of+thoracic+organs%5BTitle%5D)+AND+%22J+Thorac+Dis%22%5BJournal%5D)">Machine perfusion of thoracic organs.</a> J Thorac Dis. 10, Suppl 8 S910-S923 (2018).</li>
<li>Andreasson, A. S., Dark, J. H., Fisher, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion+in+clinical+lung+transplantation--state+of+the+art%5BTitle%5D)+AND+%22Eur+J+Cardiothorac+Surg%22%5BJournal%5D)">Ex vivo lung perfusion in clinical lung transplantation--state of the art.</a> Eur J Cardiothorac Surg. 46 (5), 779-788 (2014).</li>
<li>Ahmad, K., Pluhacek, J. L., Brown, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion%3A+A+review+of+current+and+future+application+in+lung+transplantation%5BTitle%5D)+AND+%22Pulm+Ther%22%5BJournal%5D)">Ex vivo lung perfusion: A review of current and future application in lung transplantation.</a> Pulm Ther. 8 (2), 149-165 (2022).</li>
<li>Kim, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biometric+profiling+to+quantify+lung+injury+through+ex+vivo+lung+perfusion+following+warm+ischemia%5BTitle%5D)+AND+%22Asaio+j%22%5BJournal%5D)">Biometric profiling to quantify lung injury through ex vivo lung perfusion following warm ischemia.</a> Asaio j. 69 (8), e368-e375 (2023).</li>
<li>Jeon, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acellular+ex+vivo+lung+perfusate+silences+pro-inflammatory+signaling+in+human+lung+endothelial+and+epithelial+cells%5BTitle%5D)+AND+%22J+Transl+Med%22%5BJournal%5D)">Acellular ex vivo lung perfusate silences pro-inflammatory signaling in human lung endothelial and epithelial cells.</a> J Transl Med. 21 (1), 729(2023).</li>
<li>Baciu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+purine+metabolism+at+reperfusion+affects+clinical+outcome+in+lung+transplantation%5BTitle%5D)+AND+%22Thorax%22%5BJournal%5D)">Altered purine metabolism at reperfusion affects clinical outcome in lung transplantation.</a> Thorax. 78 (3), 249-257 (2023).</li>
<li>Peel, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+the+impact+of+ex+vivo+lung+perfusion+on+organ+transplantation%3A+A+retrospective+cohort+study%5BTitle%5D)+AND+%22Ann+Surg%22%5BJournal%5D)">Evaluating the impact of ex vivo lung perfusion on organ transplantation: A retrospective cohort study.</a> Ann Surg. 278 (2), 288-296 (2023).</li>
<li>Peel, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determining+the+impact+of+ex+vivo+lung+perfusion+on+hospital+costs+for+lung+transplantation%3A+A+retrospective+cohort+study%5BTitle%5D)+AND+%22J+Heart+Lung+Transpl%22%5BJournal%5D)">Determining the impact of ex vivo lung perfusion on hospital costs for lung transplantation: A retrospective cohort study.</a> J Heart Lung Transpl. 42 (3), 356-367 (2023).</li>
<li>Warnecke, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normothermic+ex+vivo+preservation+with+the+portable+Organ+Care+System+Lung+device+for+bilateral+lung+transplantation+%28INSPIRE%29%3A+a+randomised%2C+open-label%2C+non-inferiority%2C+phase+3+study%5BTitle%5D)+AND+%22Lancet+Respir+Med%22%5BJournal%5D)">Normothermic ex vivo preservation with the portable Organ Care System Lung device for bilateral lung transplantation (INSPIRE): a randomised, open-label, non-inferiority, phase 3 study.</a> Lancet Respir Med. 6 (5), 357-367 (2018).</li>
<li>Loor, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Portable+normothermic+ex+vivo+lung+perfusion%2C+ventilation%2C+and+functional+assessment+with+the+Organ+Care+System+on+donor+lung+use+for+transplantation+from+extended-criteria+donors+%28EXPAND%29%3A+a+single-arm%2C+pivotal+trial%5BTitle%5D)+AND+%22Lancet+Resp+Med%22%5BJournal%5D)">Portable normothermic ex vivo lung perfusion, ventilation, and functional assessment with the Organ Care System on donor lung use for transplantation from extended-criteria donors (EXPAND): a single-arm, pivotal trial.</a> Lancet Resp Med. 7 (11), 975-984 (2019).</li>
<li>Loor, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prolonged+EVLP+using+OCS+lung%3A+Cellular+and+acellular+perfusates%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Prolonged EVLP using OCS lung: Cellular and acellular perfusates.</a> Transplantation. 101 (10), 2303-2311 (2017).</li>
<li>Bansal, S., Biswas, G., Avadhani, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria-targeted+heme+oxygenase-1+induces+oxidative+stress+and+mitochondrial+dysfunction+in+macrophages%2C+kidney+fibroblasts+and+in+chronic+alcohol+hepatotoxicity%5BTitle%5D)+AND+%22Redox+Biol%22%5BJournal%5D)">Mitochondria-targeted heme oxygenase-1 induces oxidative stress and mitochondrial dysfunction in macrophages, kidney fibroblasts and in chronic alcohol hepatotoxicity.</a> Redox Biol. 2, 273-283 (2014).</li>
<li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initial+investigation+on+the+feasibility+of+porcine+red+blood+cells+from+genetically+modified+pigs+as+an+alternative+to+human+red+blood+cells+for+transfusion%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as an alternative to human red blood cells for transfusion.</a> Front Immunol. 14, 1298035(2023).</li>
<li>Ellingson, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continued+decline+in+blood+collection+and+transfusion+in+the+United+States-2015%5BTitle%5D)+AND+%22Transfusion%22%5BJournal%5D)">Continued decline in blood collection and transfusion in the United States-2015.</a> Transfusion. 57, Suppl 2 1588-1598 (2017).</li>
<li>Cuddington, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pilot+scale+production+and+characterization+of+next+generation+high+molecular+weight+and+tense+quaternary+state+polymerized+human+hemoglobin%5BTitle%5D)+AND+%22Biotechnol+Bioeng%22%5BJournal%5D)">Pilot scale production and characterization of next generation high molecular weight and tense quaternary state polymerized human hemoglobin.</a> Biotechnol Bioeng. 119 (12), 3447-3461 (2022).</li>
<li>Moore, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+polymerized+hemoglobin+for+the+treatment+of+hemorrhagic+shock+when+blood+is+unavailable%3A+the+USA+multicenter+trial%5BTitle%5D)+AND+%22J+Am+Coll+Surg%22%5BJournal%5D)">Human polymerized hemoglobin for the treatment of hemorrhagic shock when blood is unavailable: the USA multicenter trial.</a> J Am Coll Surg. 208 (1), 1-13 (2009).</li>
<li>Shonaka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+human-derived+hemoglobin+based+oxygen+vesicles+as+a+machine+perfusion+solution+for+liver+donation+after+cardiac+death+in+a+pig+model%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Impact of human-derived hemoglobin based oxygen vesicles as a machine perfusion solution for liver donation after cardiac death in a pig model.</a> PLoS One. 14 (12), e0226183(2019).</li>
<li>Chen, G., Palmer, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hemoglobin-based+oxygen+carrier+and+convection+enhanced+oxygen+transport+in+a+hollow+fiber+bioreactor%5BTitle%5D)+AND+%22Biotechnol+Bioeng%22%5BJournal%5D)">Hemoglobin-based oxygen carrier and convection enhanced oxygen transport in a hollow fiber bioreactor.</a> Biotechnol Bioeng. 102 (6), 1603-1612 (2009).</li>
<li>Bucci, E., Kwansa, H., Koehler, R. C., Matheson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+zero-link+polymers+of+hemoglobin%2C+which+do+not+extravasate+and+do+not+induce+pressure+increases+upon+infusion%5BTitle%5D)+AND+%22Artif+Cells+Blood+Substit+Immobil+Biotechnol%22%5BJournal%5D)">Development of zero-link polymers of hemoglobin, which do not extravasate and do not induce pressure increases upon infusion.</a> Artif Cells Blood Substit Immobil Biotechnol. 35 (1), 11-18 (2007).</li>
<li>Schaer, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Haptoglobin+preserves+vascular+nitric+oxide+signaling+during+hemolysis%5BTitle%5D)+AND+%22Am+J+Respir+Crit+Care+Med%22%5BJournal%5D)">Haptoglobin preserves vascular nitric oxide signaling during hemolysis.</a> Am J Respir Crit Care Med. 193 (10), 1111-1122 (2016).</li>
<li>Muller, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safety+and+efficacy+of+human+polymerized+hemoglobin+on+guinea+pig+resuscitation+from+hemorrhagic+shock%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Safety and efficacy of human polymerized hemoglobin on guinea pig resuscitation from hemorrhagic shock.</a> Sci Rep. 12 (1), 20480(2022).</li>
<li>Greenfield, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biophysical+analysis+and+preclinical+pharmacokinetics-pharmacodynamics+of+tangential+flow+filtration+fractionated+polymerized+human+hemoglobin+as+a+red+blood+cell+substitute%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">Biophysical analysis and preclinical pharmacokinetics-pharmacodynamics of tangential flow filtration fractionated polymerized human hemoglobin as a red blood cell substitute.</a> Biomacromolecules. 24 (4), 1855-1870 (2023).</li>
<li>Cuddington, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polymerized+human+hemoglobin-based+oxygen+carrier+preserves+lung+allograft+function+during+normothermic+ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22Asaio+j%22%5BJournal%5D)">Polymerized human hemoglobin-based oxygen carrier preserves lung allograft function during normothermic ex vivo lung perfusion.</a> Asaio j. , 10.1097/mat.0000000000002118 (2024).</li>
<li>Cabrales, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+the+molecular+mass+of+tense-state+polymerized+bovine+hemoglobin+on+blood+pressure+and+vasoconstriction%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Effects of the molecular mass of tense-state polymerized bovine hemoglobin on blood pressure and vasoconstriction.</a> J Appl Physiol. 107 (5), 1548-1558 (2009).</li>
<li>Baek, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Down+selection+of+polymerized+bovine+hemoglobins+for+use+as+oxygen+releasing+therapeutics+in+a+guinea+pig+model%5BTitle%5D)+AND+%22Toxicol+Sci%22%5BJournal%5D)">Down selection of polymerized bovine hemoglobins for use as oxygen releasing therapeutics in a guinea pig model.</a> Toxicol Sci. 127 (2), 567-581 (2012).</li>
<li>Williams, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resuscitation+from+hemorrhagic+shock+with+fresh+and+stored+blood+and+polymerized+hemoglobin%5BTitle%5D)+AND+%22Shock%22%5BJournal%5D)">Resuscitation from hemorrhagic shock with fresh and stored blood and polymerized hemoglobin.</a> Shock. 54 (4), 464-473 (2020).</li>
<li>Muller, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resuscitation+from+hemorrhagic+shock+after+traumatic+brain+injury+with+polymerized+hemoglobin%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Resuscitation from hemorrhagic shock after traumatic brain injury with polymerized hemoglobin.</a> Sci Rep. 11 (1), 2509(2021).</li>
<li>Lamb, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+size+of+bioengineered+oxygen+carriers+determines+tissue+oxygenation+in+a+hypercholesterolemia+guinea+pig+model+of+hemorrhagic+shock+and+resuscitation%5BTitle%5D)+AND+%22Mol+Pharm%22%5BJournal%5D)">The molecular size of bioengineered oxygen carriers determines tissue oxygenation in a hypercholesterolemia guinea pig model of hemorrhagic shock and resuscitation.</a> Mol Pharm. 20 (11), 5739-5752 (2023).</li>
<li>Bobba, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+negative+pressure-flow+waveform+to+ventilate+lungs+for+normothermic+ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22Asaio+j%22%5BJournal%5D)">A novel negative pressure-flow waveform to ventilate lungs for normothermic ex vivo lung perfusion.</a> Asaio j. 67 (1), 96-103 (2021).</li>
<li>Nelson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+of+isolated+ex+vivo+lung+perfusion+in+a+rat+model%3A+lessons+learned+from+developing+a+rat+EVLP+program%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Method of isolated ex vivo lung perfusion in a rat model: lessons learned from developing a rat EVLP program.</a> J Vis Exp. (96), e52309(2015).</li>
<li>Nelson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+ex+vivo+lung+perfusion+as+a+platform+for+transplantation+research%5BTitle%5D)+AND+%22World+J+Exp+Med%22%5BJournal%5D)">Animal models of ex vivo lung perfusion as a platform for transplantation research.</a> World J Exp Med. 4 (2), 7-15 (2014).</li>
<li>Wong, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+therapeutic+targets+for+lung+repair+during+human+ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22Eur+Respir+J%22%5BJournal%5D)">Potential therapeutic targets for lung repair during human ex vivo lung perfusion.</a> Eur Respir J. 55 (4), 1902222(2020).</li>
<li>Machuca, T. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+the+endothelin-1+pathway+as+a+biomarker+for+donor+lung+assessment+in+clinical+ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22J+Heart+Lung+Transpl%22%5BJournal%5D)">The role of the endothelin-1 pathway as a biomarker for donor lung assessment in clinical ex vivo lung perfusion.</a> J Heart Lung Transpl. 34 (6), 849-857 (2015).</li>
<li>Gouchoe, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitsugumin+53+Mitigation+of+ischemia+reperfusion+injury+in+a+mouse+model%5BTitle%5D)+AND+%22J+Thorac+Cardiovasc+Surg%22%5BJournal%5D)">Mitsugumin 53 Mitigation of ischemia reperfusion injury in a mouse model.</a> J Thorac Cardiovasc Surg. 10, 10.1016/j.jtcvs.2023.08.005 (2023).</li>
<li>Gouchoe, D. A., Whitson, B. A., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+next+frontier+in+lung+transplantation%3A+Protecting+the+endothelium+and+repairing+organs+for+transplant+utilizing+MG53%5BTitle%5D)+AND+%22Clin+Transl+Dis%22%5BJournal%5D)">The next frontier in lung transplantation: Protecting the endothelium and repairing organs for transplant utilizing MG53.</a> Clin Transl Dis. 3 (6), e255(2023).</li>
<li>Gouchoe, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MG53+mitigates+warm+ischemic+lung+injury+in+a+murine+model+of+transplantation%5BTitle%5D)+AND+%22J+Thorac+Cardiovasc+Surg%22%5BJournal%5D)">MG53 mitigates warm ischemic lung injury in a murine model of transplantation.</a> J Thorac Cardiovasc Surg. , 10.1016/j.jtcvs.2023.10.056 (2023).</li>
</ol>

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 therapies39,40,41,42,43.
The PolyhHb described in this work is bracketed between 500 kDa and 0.2 &#181;m 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 new37,38, 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.

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><tbody><tr><td>10 cc insulin syringe 29 G x 1/2&quot; 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 (CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma Aldrich</td><td>10035-04-8</td><td>For modified Ringer&#039;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>&amp;nbsp;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&amp;deg;</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&lt;br/&gt; Canadian Blood Services&lt;br/&gt; 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 (C&lt;sub&gt;5&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; 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, C&lt;sub&gt;5&lt;/sub&gt;H&lt;sub&gt;9&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;S)</td><td>Sigma Aldrich</td><td>616-91-1 (A7250)</td><td>For modified Ringer&#039;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>&amp;nbsp;Ismatec</td><td>&amp;nbsp;ISM 827B</td><td></td></tr><tr><td>PES, 0.65 &amp;micro;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 &amp;micro;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>&amp;nbsp;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&amp;nbsp;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 (NaCNBH&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>25895-60-7</td><td></td></tr><tr><td>Sodium Dithionite (Na&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;)</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&#039;s lactate</td></tr><tr><td>Sodium Lactate (NaC&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>867-56-1</td><td>For modified Ringer&#039;s lactate</td></tr><tr><td>Sodium phosphate dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>7558-79-4</td><td>For PBS</td></tr><tr><td>Sodium phosphate monobasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</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>&amp;nbsp;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>&amp;nbsp;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>&amp;nbsp;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>&amp;nbsp;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&#039;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></tr></tbody></table>

exploring alternative perfusion solutions using next-generation polymerized hemoglobin-based oxygen carriers 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. 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 &lt; 0.05). When tested over the next several hours, both PolyhHb and Control perfusates had stable LA pO2, while PolyhHb had a non-significant trend (p &gt; 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 &lt; 0.05), while it remained stable in the PolyhHb and Control perfusates with a non-significant trend (p &gt; 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 &lt; 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 &lt; 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 &lt; 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 &lt; 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 &lt; 0.05), while there was a non-significant decrease in the PolyhHb and Control perfusate (p &gt; 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.
<img alt="Bioreactor setups for protein purification research, chromatography columns, laboratory equipment." class="xfigimg" src="/files/ftp_upload/66702/66702fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
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. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cardiac hemodynamics experiment; diagram and setup with mouse lung-heart system; blood flow analysis." class="xfigimg" src="/files/ftp_upload/66702/66702fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
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. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical instruments layout for procedures; includes forceps, scissors, retractors on sterile cloth." class="xfigimg" src="/files/ftp_upload/66702/66702fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
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. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Rat dissection process showing IVC exposure for surgical research; anatomical experiment sequence." class="xfigimg" src="/files/ftp_upload/66702/66702fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
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. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical procedure steps on animal tissue; includes suturing and dissection tools in sequence diagram." class="xfigimg" src="/files/ftp_upload/66702/66702fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
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. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical procedure diagram showing pulmonary artery isolation using clamps and sutures." class="xfigimg" src="/files/ftp_upload/66702/66702fig06.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
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. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical procedure images: suturing and connecting medical tubing in a heart transplant setup." class="xfigimg" src="/files/ftp_upload/66702/66702fig07.jpg" data-altupdatedat="1788304762000" data-alt="Figure 7"> 
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. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical anastomosis technique; sequential steps; blood vessel suturing; medical procedure education." class="xfigimg" src="/files/ftp_upload/66702/66702fig08.jpg" data-altupdatedat="1788304762000" data-alt="Figure 8"> 
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. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Dissection procedure with surgical tools; heart tissue isolation; biological experiment setup." class="xfigimg" src="/files/ftp_upload/66702/66702fig09.jpg" data-altupdatedat="1788304762000" data-alt="Figure 9"> 
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. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Post-perfusion blood gas changes; bar graphs showing PO₂, O₂, PCO₂, CO₂ pressure over time." class="xfigimg" src="/files/ftp_upload/66702/66702fig10.jpg" data-altupdatedat="1788304762000" data-alt="Figure 10"> 
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 &lt; 0.05. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Pulmonary vascular research charts: PVR, lung weight, compliance vs. time analyzed in perfusion study." class="xfigimg" src="/files/ftp_upload/66702/66702fig11.jpg" data-altupdatedat="1788304762000" data-alt="Figure 11"> 
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 &lt; 0.05. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Heart transplant procedure sequence diagram showing organ preparation and connection phases." class="xfigimg" src="/files/ftp_upload/66702/66702fig12.jpg" data-altupdatedat="1788304762000" data-alt="Figure 12"> 
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. <a href="https://www.jove.com/files/ftp_upload/66702/66702fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

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.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

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.

Isolated Lung Perfusion System in the Rabbit Model

Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs.

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Here, we describe the necessary steps for establishing a rat EVLP model and show the inflammatory profile associated with the perfused lungs. The aim is to propagate knowledge and experiences about the rat EVLP model, enabling the integral understanding of the biological responses associated with that revolutionary technique.

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

The objective is to develop a new ex vivo lung perfusion (EVLP) model in rats that can sustain consistent lung function for 4 h throughout EVLP and furnish the industry with implementation specifics that will offer a sturdy method for applying EVLP to rodent models.

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

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

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

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Explore polymerized hemoglobin-based oxygen carriers for ex vivo lung perfusion using a rat EVLP model. This method improves lung function metrics and extends preservation times for donor organ evaluation.

Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion (Video) | JoVE Journal

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

Explore More Videos