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. 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.
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.
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 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.
2. Perfusate formulation
3. Ex Vivo lung perfusion circuit setup
4. Procurement of donor rat lung block
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 had stable LA pO2, while PolyhHb had a non-significant trend (p > 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 < 0.05), while it remained stable in the PolyhHb and Control perfusates with a non-significant trend (p > 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 < 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 < 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 < 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 < 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 < 0.05), while there was a non-significant decrease in the PolyhHb and Control perfusate (p > 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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 < 0.05. Please click here to view a larger version of this figure.
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 < 0.05. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µm 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.
The authors have nothing to disclose.
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.
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 |