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 purific...

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

The development and testing of perfusion solutions is a novel endeavor that many throughout the globe are embarking on. Traditionally, standard perfusates offer the ability to suspend ischemic time and mitigate the associated injuries with ischemia, as well as reperfusion18. However, the next evolution of EVLP is to improve current perfusate technology as well as incorporate repair and reconditioning therapies39,40,<sup class="x...

Like any field in solid organ transplantation, lung transplantation suffers from a shortage of donor organs. In order to increase the donor pool, significant research has been dedicated to investigating the potential of allografts that were once thought to be unsuitable for transplantation, i.e., extended criteria donors (ECD). These allografts can be considered ECD for a milieu of reasons, including questionable quality, poor function, infection, trauma, prolonged warm or cold ischemic times, and advanced age1,2. In certain cases, where these lungs are suitable for immediate transplant3, it is often advantageous to providers and recipients alike to evaluate these lungs for an additional time to determine their suitability for transplantation. Ex vivo lung perfusion (EVLP) is such a technology that allows for extended assessment of potential lung allografts in a closed circuit outside the donor2,4,5,6,7, affording the transplant provider the ability to determine transplantation suitability. EVLP has shown the ability to adequately assess donor organs8,9,10,11, decrease the effects of ischemic reperfusion injury (IRI)12,13 and increase the donor pool14,15 thus making lung transplantation a more accessible treatment for all.
In general, an EVLP system is a closed system with a ventilatory circuit (achieved by connecting a ventilator to the trachea to introduce air into the system) and a vascular circuit (achieved by connecting the left atrium (LA) to the pulmonary artery (PA) with tubing)7. The vascular circuit has perfusate running through the tubing to give the lung vital nutrients and oxygen while limiting the cold ischemic time (CIT)5,8,16,17. This solution is either blood-based (i.e., via the addition of packed red blood cells (PRBCs))16,17 or acellular-based (i.e., no PRBCs)4,5. However, there are several notable disadvantages to using PRBCs. If using PRBCs from donors who died from trauma or brain-dead donors (BDD), these fluids often contain large amounts of inflammatory cytokines, which may increase cellular damage during EVLP as well as increase levels of cell-free hemoglobin (Hb), heme, iron, and cell fragments which deliver additional damage to cells18,19. Furthermore, as these donors are often multi-organ, the collection of PRBCs prior to procurement could lead to decreasing blood volume in the donor and subsequently increasing ischemia to all organs. If using PRBCs from another source, providers could face blood shortages as this is a scarce material in and of itself20,21. Finally, PRBCs are prone to mechanical lysis on the EVLP circuit regardless of their source, releasing Hb and other components that contribute to cellular damage.
Thus, for many reasons, it could be advantageous to use an artificial red blood cell substitute, i.e., hemoglobin-based oxygen carriers (HBOCs), as a perfusate supplement. One particularly promising HBOC is polymerized human hemoglobin (PolyhHb). PolyhHb is synthesized from Hb purified from expired PRBCs that were deemed unsuitable for immediate transfusion22. They have been shown to be viable blood substitutes in hemorrhagic shock23 and transplantation24 and can be produced in large quantities22. However, large-scale adoption of PolyhHb has been unsuccessful due to unforeseen complications such as vasoconstriction, increasing blood pressure, and cardiac arrest23,25. The reasons behind these findings were likely due to the presence of cell-free Hb or low molecular weight Hb polymers (&#60; 500 kDa) in the PolyhHb solution, as they have a propensity to extravasate into the tissue space, which resulted in decreased nitric oxide availability, subsequent vasoconstriction, systemic hypertension, and ultimately oxidative tissue injury26,27. To improve upon these issues, the Palmer Laboratory has worked to develop a next-generation PolyhHb that contains minimal low MW species and cell-free Hb, which has demonstrated improved biophysical characteristics and in vivo responses22,28,29,30. Several transfusion studies in animals have shown that if low molecular weight Hb polymers are eliminated from the HBOC, vasoconstriction, systemic hypertension, and oxidative damage can be mitigated28,29,31,32,33,34,35. Therefore, making this next-generation PolyhHb a promising perfusate candidate.
Here, we describe the application of a next-generation PolyhHb to be used in a perfusate and the protocol by which this perfusion solution can be tested in a model of rat EVLP. The goal of this study is to provide the lung transplant community with key information in designing and developing novel perfusion solutions, as well as provide protocols to test them in clinically relevant translational transplant models.

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

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