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

Published: June 14, 2024

doi:

Doug A. Gouchoe*^1,2, Yong Gyu Lee*^1,2, Alisyn Greenfield, Clayton Cuddington, Jung-Lye Kim², Sylvester M. Black, Andre F. Palmer, Bryan A. Whitson²

¹COPPER Laboratory, Department of Surgery,The Ohio State University Wexner Medical Center, ²Division of Cardiac Surgery, Department of Surgery,The Ohio State University Wexner Medical Center, ³William G. Lowrie Department of Chemical and Biomolecular Engineering, College of Engineering,The Ohio State University

Summary

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

Abstract

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

Introduction

Like any field in solid organ transplantation, lung transplantation suffers from a shortage of donor organs. In order to increase the donor pool, significant research has been dedicated to investigating the potential of allografts that were once thought to be unsuitable for transplantation, i.e., extended criteria donors (ECD). These allografts can be considered ECD for a milieu of reasons, including questionable quality, poor function, infection, trauma, prolonged warm or cold ischemic times, and advanced 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 (< 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.

Protocol

Sprague-Dawley rats (300 g body weight) were commercially obtained and housed under pathogen-free conditions at The Ohio State University Wexner Medical Center Animal Facility. All procedures were humanely performed according to the NIH and the National Research Council's Guide for the Humane Care and Use of Laboratory Animals and with the approval of The Ohio State University Institutional Animal Care and Use Committee (IACUC Protocol 2023A00000071). 1. PolyhHb synthesis and purific…

Representative Results

The validation of our PolyhHb-based perfusate, and furthermore, the stability of this perfusate over several hours, is demonstrated in Figure 10. Over the first 1 h, all perfusates tested (PolyhHb, Control (Williams Media + 5% HSA), RBC based) showed a slight decrease in LA pO2 (Post pO2). However, the RBC-based perfusate showed a significant decrease at 1 h compared to PolyhHb (p < 0.05). When tested over the next several hours, both PolyhHb and Control perfusates …

Discussion

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

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

Riferimenti

Valapour, M., et al. OPTN/SRTR 2021 annual data report: Lung. Am J Transplant. 23 (2 Suppl 1), 379-442 (2023).
Gouchoe, D. A., et al. 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. J Thorac Cardiovasc Surg. , (2024).
Bobba, C. M., et al. Trends in donation after circulatory death in lung transplantation in the United States: Impact of era. Transpl Int. 35, 10172 (2022).
Steen, S., et al. Transplantation of lungs from a non-heart-beating donor. Lancet. 357 (9259), 825-829 (2001).
Cypel, M., et al. Normothermic ex vivo lung perfusion in clinical lung transplantation. N Engl J Med. 364 (15), 1431-1440 (2011).
Cypel, M., Neyrinck, A., Machuca, T. N. Ex vivo perfusion techniques: state of the art and potential applications. Intens Care Med. 45 (3), 354-356 (2019).
Gouchoe, D. A., et al. XPS™ Jensen lung as a low-cost, high-fidelity training adjunct to ex-vivo lung perfusion. Artif Organs. , (2023).
Van Raemdonck, D., Rega, F., Rex, S., Neyrinck, A. Machine perfusion of thoracic organs. J Thorac Dis. 10 (Suppl 8), 910-923 (2018).
Andreasson, A. S., Dark, J. H., Fisher, A. J. Ex vivo lung perfusion in clinical lung transplantation–state of the art. Eur J Cardiothorac Surg. 46 (5), 779-788 (2014).
Ahmad, K., Pluhacek, J. L., Brown, A. W. Ex vivo lung perfusion: A review of current and future application in lung transplantation. Pulm Ther. 8 (2), 149-165 (2022).
Kim, J. L., et al. Biometric profiling to quantify lung injury through ex vivo lung perfusion following warm ischemia. Asaio j. 69 (8), 368-375 (2023).
Jeon, J. E., et al. Acellular ex vivo lung perfusate silences pro-inflammatory signaling in human lung endothelial and epithelial cells. J Transl Med. 21 (1), 729 (2023).
Baciu, C., et al. Altered purine metabolism at reperfusion affects clinical outcome in lung transplantation. Thorax. 78 (3), 249-257 (2023).
Peel, J. K., et al. Evaluating the impact of ex vivo lung perfusion on organ transplantation: A retrospective cohort study. Ann Surg. 278 (2), 288-296 (2023).
Peel, J. K., et al. Determining the impact of ex vivo lung perfusion on hospital costs for lung transplantation: A retrospective cohort study. J Heart Lung Transpl. 42 (3), 356-367 (2023).
Warnecke, G., et al. 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. Lancet Respir Med. 6 (5), 357-367 (2018).
Loor, G., et al. 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. Lancet Resp Med. 7 (11), 975-984 (2019).
Loor, G., et al. Prolonged EVLP using OCS lung: Cellular and acellular perfusates. Transplantation. 101 (10), 2303-2311 (2017).
Bansal, S., Biswas, G., Avadhani, N. G. Mitochondria-targeted heme oxygenase-1 induces oxidative stress and mitochondrial dysfunction in macrophages, kidney fibroblasts and in chronic alcohol hepatotoxicity. Redox Biol. 2, 273-283 (2014).
Park, S., et al. Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as an alternative to human red blood cells for transfusion. Front Immunol. 14, 1298035 (2023).
Ellingson, K. D., et al. Continued decline in blood collection and transfusion in the United States-2015. Transfusion. 57 Suppl 2 (Suppl 2), 1588-1598 (2017).
Cuddington, C. T., et al. Pilot scale production and characterization of next generation high molecular weight and tense quaternary state polymerized human hemoglobin. Biotechnol Bioeng. 119 (12), 3447-3461 (2022).
Moore, E. E., et al. Human polymerized hemoglobin for the treatment of hemorrhagic shock when blood is unavailable: the USA multicenter trial. J Am Coll Surg. 208 (1), 1-13 (2009).
Shonaka, T., et al. Impact of human-derived hemoglobin based oxygen vesicles as a machine perfusion solution for liver donation after cardiac death in a pig model. PLoS One. 14 (12), 0226183 (2019).
Chen, G., Palmer, A. F. Hemoglobin-based oxygen carrier and convection enhanced oxygen transport in a hollow fiber bioreactor. Biotechnol Bioeng. 102 (6), 1603-1612 (2009).
Bucci, E., Kwansa, H., Koehler, R. C., Matheson, B. Development of zero-link polymers of hemoglobin, which do not extravasate and do not induce pressure increases upon infusion. Artif Cells Blood Substit Immobil Biotechnol. 35 (1), 11-18 (2007).
Schaer, C. A., et al. Haptoglobin preserves vascular nitric oxide signaling during hemolysis. Am J Respir Crit Care Med. 193 (10), 1111-1122 (2016).
Muller, C. R., et al. Safety and efficacy of human polymerized hemoglobin on guinea pig resuscitation from hemorrhagic shock. Sci Rep. 12 (1), 20480 (2022).
Greenfield, A., et al. Biophysical analysis and preclinical pharmacokinetics-pharmacodynamics of tangential flow filtration fractionated polymerized human hemoglobin as a red blood cell substitute. Biomacromolecules. 24 (4), 1855-1870 (2023).
Cuddington, C., et al. Polymerized human hemoglobin-based oxygen carrier preserves lung allograft function during normothermic ex vivo lung perfusion. Asaio j. , (2024).
Cabrales, P., et al. Effects of the molecular mass of tense-state polymerized bovine hemoglobin on blood pressure and vasoconstriction. J Appl Physiol (1985). 107 (5), 1548-1558 (2009).
Baek, J. H., et al. Down selection of polymerized bovine hemoglobins for use as oxygen releasing therapeutics in a guinea pig model. Toxicol Sci. 127 (2), 567-581 (2012).
Williams, A. T., et al. Resuscitation from hemorrhagic shock with fresh and stored blood and polymerized hemoglobin. Shock. 54 (4), 464-473 (2020).
Muller, C. R., et al. Resuscitation from hemorrhagic shock after traumatic brain injury with polymerized hemoglobin. Sci Rep. 11 (1), 2509 (2021).
Lamb, D. R., et al. The molecular size of bioengineered oxygen carriers determines tissue oxygenation in a hypercholesterolemia guinea pig model of hemorrhagic shock and resuscitation. Mol Pharm. 20 (11), 5739-5752 (2023).
Bobba, C. M., et al. A novel negative pressure-flow waveform to ventilate lungs for normothermic ex vivo lung perfusion. Asaio j. 67 (1), 96-103 (2021).
Nelson, K., et al. Method of isolated ex vivo lung perfusion in a rat model: lessons learned from developing a rat EVLP program. J Vis Exp. (96), 52309 (2015).
Nelson, K., et al. Animal models of ex vivo lung perfusion as a platform for transplantation research. World J Exp Med. 4 (2), 7-15 (2014).
Wong, A., et al. Potential therapeutic targets for lung repair during human ex vivo lung perfusion. Eur Respir J. 55 (4), 1902222 (2020).
Machuca, T. N., et al. The role of the endothelin-1 pathway as a biomarker for donor lung assessment in clinical ex vivo lung perfusion. J Heart Lung Transpl. 34 (6), 849-857 (2015).
Gouchoe, D. A., et al. Mitsugumin 53 Mitigation of ischemia reperfusion injury in a mouse model. J Thorac Cardiovasc Surg. , (2023).
Gouchoe, D. A., Whitson, B. A., Zhu, H. The next frontier in lung transplantation: Protecting the endothelium and repairing organs for transplant utilizing MG53. Clin Transl Dis. 3 (6), 255 (2023).
Gouchoe, D. A., et al. MG53 mitigates warm ischemic lung injury in a murine model of transplantation. J Thorac Cardiovasc Surg. , (2023).