Name: A High-Fidelity Porcine Model of Orthotopic Heart Transplantation Following Donation after Circulatory Death (Video) | JoVE Journal
Uploaded: 2025-06-06T14:45:00.000+00:00
Duration: 7 min 8 s
Description: Learn a high-fidelity porcine model for orthotopic heart transplantation following donation after circulatory death. This method enables evaluation of DCD pathophysiology and supports translational research to improve allograft recovery.

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5 minutes

We thank the veterinary technical support from the Duke Laboratory Animal Resources, perfusion support from Centrifugal Solutions, and the Duke cardiovascular anesthesia teams for their invaluable support of these surgical experiments. We also sincerely acknowledge Paul Lezberg and TransMedics, Inc. for their support.

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<li>Messer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+5-year+single-center+early+experience+of+heart+transplantation+from+donation+after+circulatory-determined+death+donors%5BTitle%5D)+AND+%22J+Heart+Lung+Transplant%22%5BJournal%5D)">A 5-year single-center early experience of heart transplantation from donation after circulatory-determined death donors.</a> J Heart Lung Transplant. 39 (12), 1463-1475 (2020).</li>
<li>Messer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Outcome+after+heart+transplantation+from+donation+after+circulatory-determined+death+donors%5BTitle%5D)+AND+%22J+Heart+Lung+Transplant%22%5BJournal%5D)">Outcome after heart transplantation from donation after circulatory-determined death donors.</a> J Heart Lung Transplant. 36 (12), 1311-1318 (2017).</li>
<li>Jawitz, O. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+the+United+States+heart+transplant+donor+pool+with+donation+after+circulatory+death%5BTitle%5D)+AND+%22J+Thorac+Cardiovasc+Surg%22%5BJournal%5D)">Increasing the United States heart transplant donor pool with donation after circulatory death.</a> J Thorac Cardiovasc Surg. 159 (5), e307-e309 (2020).</li>
<li>White, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+hearts+donated+after+circulatory+death%5BTitle%5D)+AND+%22Front+Cardiovasc+Med%22%5BJournal%5D)">Transplantation of hearts donated after circulatory death.</a> Front Cardiovasc Med. 5, 8(2018).</li>
<li>Lee, C., Tsai, C., Adler, E., Pretorius, V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLee%2C+C+%22Emerging+frontier+in+heart+transplantation%3A+donation+after+circulatory+death%22">Emerging frontier in heart transplantation: donation after circulatory death</a>. , American College of Cardiology. https://www.acc.org/Latest-in-Cardiology/Articles/2022/11/21/13/31/Emerging-Frontier-in-Heart-Transplantation (2022).</li>
<li>Messer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+national+pilot+of+donation+after+circulatory+death+%28DCD%29+heart+transplantation+within+the+United+Kingdom%5BTitle%5D)+AND+%22J+Heart+Lung+Transplant%22%5BJournal%5D)">A national pilot of donation after circulatory death (DCD) heart transplantation within the United Kingdom.</a> J Heart Lung Transplant. 42 (8), 1120-1130 (2023).</li>
<li>Kwon, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+outcomes+of+heart+transplantation+using+donation+after+circulatory+death+donors+in+the+United+States%5BTitle%5D)+AND+%22Circ+Heart+Fail%22%5BJournal%5D)">Early outcomes of heart transplantation using donation after circulatory death donors in the United States.</a> Circ Heart Fail. 15 (12), e009844(2022).</li>
<li>Pasrija, C., Tipograf, Y., Shah, A. S., Trahanas, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normothermic+regional+perfusion+for+donation+after+circulatory+death+donors%5BTitle%5D)+AND+%22Curr+Opin+Organ+Transplant%22%5BJournal%5D)">Normothermic regional perfusion for donation after circulatory death donors.</a> Curr Opin Organ Transplant. 28 (2), 71-75 (2023).</li>
<li>Benkert, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((heart+transplant+experience+with+normothermic+regional+perfusion+following+donation+after+circulatory+death%5BTitle%5D)+AND+%22JACC+Heart+Fail%22%5BJournal%5D)">heart transplant experience with normothermic regional perfusion following donation after circulatory death.</a> JACC Heart Fail. 12 (12), 2073-2083 (2024).</li>
<li>Kobashigawa, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Report+from+a+consensus+conference+on+primary+graft+dysfunction+after+cardiac+transplantation%5BTitle%5D)+AND+%22J+Heart+Lung+Transplant%22%5BJournal%5D)">Report from a consensus conference on primary graft dysfunction after cardiac transplantation.</a> J Heart Lung Transplant. 33 (4), 327-340 (2014).</li>
<li>Iyer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathophysiological+trends+during+withdrawal+of+life+support%3A+implications+for+organ+donation+after+circulatory+death%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Pathophysiological trends during withdrawal of life support: implications for organ donation after circulatory death.</a> Transplantation. 100 (12), 2621-2629 (2016).</li>
<li>White, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiologic+changes+in+the+heart+following+cessation+of+mechanical+ventilation+in+a+porcine+model+of+donation+after+circulatory+death%3A+implications+for+cardiac+transplantation%5BTitle%5D)+AND+%22Am+J+Transplant%22%5BJournal%5D)">Physiologic changes in the heart following cessation of mechanical ventilation in a porcine model of donation after circulatory death: implications for cardiac transplantation.</a> Am J Transplant. 16 (3), 783-793 (2016).</li>
<li>Arnold, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+considerations+in+direct+procurement+and+perfusion+protocols+with+DCD+heart+transplantation%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Metabolic considerations in direct procurement and perfusion protocols with DCD heart transplantation.</a> Int J Mol Sci. 25 (8), 4153(2024).</li>
<li>Dann, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Donor+heart+refusal+after+circulatory+death%3A+an+analysis+of+United+Network+for+Organ+Sharing+refusal+codes%5BTitle%5D)+AND+%22J+Thorac+Cardiovasc+Surg+Open%22%5BJournal%5D)">Donor heart refusal after circulatory death: an analysis of United Network for Organ Sharing refusal codes.</a> J Thorac Cardiovasc Surg Open. 27 (18), 91-103 (2024).</li>
<li>Pla, M. M., Bowles, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+gene+therapy+in+organ+transplantation%3A+considerations+and+clinical+translation%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">Ex vivo gene therapy in organ transplantation: considerations and clinical translation.</a> Hum Gene Ther. 35 (7-8), 284-297 (2024).</li>
<li>Pla, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+gene+delivery+to+porcine+cardiac+allografts+using+a+myocardial-enhanced+adeno-associated+viral+vector%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">Ex vivo gene delivery to porcine cardiac allografts using a myocardial-enhanced adeno-associated viral vector.</a> Hum Gene Ther. 34 (7-8), 303-313 (2023).</li>
<li>Bishawi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+normothermic+ex+vivo+organ+perfusion+delivery+method+for+cardiac+transplantation+gene+therapy%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy.</a> Sci Rep. 9 (1), 8029(2019).</li>
<li>Silvis, M. J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Damage-associated+molecular+patterns+in+myocardial+infarction+and+heart+transplantation%3A+the+road+to+translational+success%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Damage-associated molecular patterns in myocardial infarction and heart transplantation: the road to translational success.</a> Front Immunol. 11, 599511(2020).</li>
<li>Zuurbier, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+metabolism+as+a+driver+and+therapeutic+target+of+myocardial+infarction%5BTitle%5D)+AND+%22J+Cell+Mol+Med%22%5BJournal%5D)">Cardiac metabolism as a driver and therapeutic target of myocardial infarction.</a> J Cell Mol Med. 24 (11), 5937-5954 (2020).</li>
<li>Heinis, F. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Considerations+for+the+use+of+porcine+organ+donation+models+in+preclinical+organ+donor+intervention+research%5BTitle%5D)+AND+%22Anim+Models+Exp+Med%22%5BJournal%5D)">Considerations for the use of porcine organ donation models in preclinical organ donor intervention research.</a> Anim Models Exp Med. 7, 283-296 (2024).</li>
<li>Hubacher, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Open-+vs.+closed-chest+pig+models+of+donation+after+circulatory+death%5BTitle%5D)+AND+%22Front+Cardiovasc+Med%22%5BJournal%5D)">Open- vs. closed-chest pig models of donation after circulatory death.</a> Front Cardiovasc Med. 11, 1325160(2024).</li>
</ol>

CAM has received stock compensation for serving as a consultant for TransMedics Inc.

Despite the efficacy of heart transplantation for the treatment of end-stage heart failure, significant challenges persist in this field. To combat the limited availability of donor organs, advancements in heart allograft preservation methods (e.g., normothermic machine perfusion) have risen to the forefront in recent years. These advancements led to the adoption of the transplantation of cardiac allografts following donation after circulatory death (DCD HT). While utilization of DCD HT allografts expanded the donor pool and has short-term outcomes that are non-inferior to DBD HT allografts, there remains an approximately 5% risk of early peri-transplant mortality associated with both donor types3. The early mortality risk in heart transplantation is driven predominantly by PGD. While PGD is multifactorial in etiology with some contribution of defined donor, recipient, and preservation variables, the molecular mechanisms underlying PGD remain generally poorly understood. Furthermore, given the added warm ischemic injury incurred by the DCD HT process, it is not surprising that these hearts, compared to DBD allografts, may be at higher risk of PGD. Therefore, a better understanding of PGD is important to help reduce short-term mortality risk after orthotopic heart transplant, and this may be particularly true for DCD HT.
Here, we describe a high-fidelity porcine surgical model of DCD HT. The benefits of modeling DCD HT in vivo in a large animal model include not only the ability to further understand the pathophysiologic changes that occur in an allograft procured during DCD HT but also the ability to test targeted interventions aimed at ensuring optimal allograft quality. Evaluation of post-implantation left ventricular function, and pressure-volume loop analysis suggests that this porcine surgical model is able to recapitulate early cardiac allograft dysfunction following DCD HT. Thus, the technique detailed here creates a reproducible large animal model of PGD following DCD HT and is amenable to evaluating therapeutic strategies at many points along the DCD HT process. In fact, the use of ex vivo perfusion as a means for delivering novel therapeutics, such as viral vector-mediated gene therapy, is an active area of focus in our lab and others18,19,20. We previously demonstrated the ability to robustly and homogenously deliver a transgene to a cardiac allograft using a porcine non-DCD HT model; the same techniques can be applied to a DCD HT model19,20. Other potential therapeutic strategies include chemical modification of the perfusate, small molecule delivery aimed at decreasing oxidative stress, providing metabolic substrates to support metabolism during the ischemic period, etc16,21,22. Furthermore, the model presented herein can easily be adapted to evaluate different variables associated with DCD HT, including the different procurement techniques used clinically. For example, we describe a direct procurement and ex vivo perfusion strategy; however, the model can easily be adapted to incorporate alternate procurement perfusion methods, including normothermic regional perfusion.
The surgical technique employed in this model closely mirrors that used in the clinical setting; however, there are some key differences. First, the sternotomy and cardiac exposure occur prior to the cessation of cardiopulmonary support, agonal phase, determination of death, and ethically mandated standoff period (open-chest model)23. The sternotomy is done in this order so that baseline donor heart evaluation, including myocardial biopsies, can occur. Prior evaluation of the timing of sternotomy in a DCD HT porcine model demonstrated that the progression from withdrawal of life-sustaining measures to death (warm ischemic time) is more rapid with accompanying less pronounced hemodynamic changes in the open-chest model; these allografts may experience less-damaging conditions during procurement. However, there were no significant differences in biochemical (lactate, glucose, catecholamine, etc.) lab values or markers of cell death between the closed and open-chest groups24. Furthermore, due to the inclusion of the 10-min standoff period in the model, the time from withdrawal of ventilation to functional warm ischemia in this model more closely resembles the timing of the cited study&#39;s closed-chest group. Additionally, within this model, the time of functional warm ischemia is a variable that can be modified to fit experimental goals for a particular study. Another potential difference between the reported model here and the clinical setting is that pulseless electrical activity (PEA; mechanical asystole) is used to define death in this model. In the clinical context, a declaration of death is made by a physician who is not involved in the transplant process; either PEA, along with the absence of other signs of life or electrical asystole, would be acceptable for meeting the criteria for death declaration. The porcine hearts sustained electrical activity for a prolonged period following cessation of contractility. In order to avoid warm ischemic damage that is out of proportion to that seen in the clinical context, mechanical asystole (PEA) was used to define death. For investigators using a closed-chest model of DCD, pulseless electrical activity (lack of pulsatility on the arterial line) can still be used as the death-defining criteria. We did not find that peripheral oxygen saturation was a relevant marker to define the onset of the agonal period or to define death.
Lastly, what we have described herein is a time and resource-intensive model. From preparation of the donor pig to the death of the recipient animal takes approximately 10 h and requires a large collaborative effort. The procedural team includes experienced cardiac surgeons, cardiac anesthesiologists, perfusionists, and veterinary and laboratory staff who help with sample collection and processing. A large team such as this is integral during the complex procedure of DCD HT. However, with an experienced team, the creation of a high-fidelity porcine model of DCD HT is possible, as described within this manuscript.

For patients with end-stage heart failure refractory to medical management, heart transplantation remains the therapy associated with the best long-term survival and quality of life. Historically, heart transplantation required the use of a heart allograft procured from a brain-dead donor (DBD HT) and transported while preserved with hypothermic static storage. However, the number of patients who require a heart transplant exceeds the number of available donors. While more than 5,000 heart transplants are performed annually worldwide, it is estimated that 50,000 candidates await a heart transplant1. In addition, the utilization of organs from identified donors remains as low as 30%2. In order to improve donor organ utilization, alternative donation and procurement methods have been developed in recent years, including heart transplantation following donation after circulatory death (DCD HT)3,4,5,6.
DCD HT donors do not meet formal brain-death criteria but have a non-recoverable neurologic injury for which ongoing medical care is deemed futile. During a DCD HT procurement, life-sustaining measures are withdrawn, and the patient is monitored for progression to apnea and circulatory arrest. Death in these situations is declared by a physician not participating in the transplant or organ procurement process. Once death has been declared, there is an additional standoff period (usually 5 min) where the potential donor is observed to ensure there is no recovery or signs of life, after which declaration of death is reconfirmed prior to proceeding with organ procurement7,8. DCD allografts are, therefore, exposed to a variable period of warm ischemia (at least 10 min) to which DBD donor allografts are not exposed. This period of warm ischemia previously deterred the use of heart transplantation with DCD donors. However, within the last five years, two procurement reperfusion methods were developed for allograft recovery following the warm ischemic time associated with DCD. First, direct procurement and perfusion (DPP) involves donor cardiectomy upon the confirmation of circulatory death, followed by allograft resuscitation and preservation by ex vivo perfusion. Alternatively, normothermic regional perfusion (NRP) uses extracorporeal circulation with the exclusion of the cerebral circulation to reperfuse and reanimate the donor heart in situ prior to cardiectomy9,10,11,12.
Thus far, short-term survival associated with DCD HT has been similar to that observed with DBD HT. However, early studies also suggest there is a higher risk of severe primary graft dysfunction (PGD) with DCD HT compared to DBD HT3. PGD is a term used to describe the clinical circumstance where, following heart transplantation, early allograft function is insufficient to meet the recipient&#39;s circulatory needs. A system for grading the severity of PGD was described by the International Society of Heart and Lung Transplantation. In severe PGD, mechanical circulatory support is required to support adequate circulation in the post-transplant period13. This condition is the main driver of early postoperative mortality after heart transplantation. The etiology of PGD for both DBD HT and DCD HT is poorly understood but is likely multifactorial, with donor, preservation, and recipient variables all contributing. For DCD allografts, the warm ischemic injury during DCD procurement, as well as deleterious donor-specific responses, including hemodynamic instability, metabolic derangements, and surges in catecholamines, cytokines, lactate, and potassium, are proposed factors that may contribute to an increased risk of PGD compared to DBD allografts.&#160;14,15,16. However, the mechanisms underlying PGD in this clinical scenario are poorly understood at the molecular level. In addition, perhaps owing to these concerns, DCD hearts are 3.37 times more likely to be declined than DBD organs17. As a result, there is still an unmet need to enhance organ utilization and improve transplant outcomes by optimizing the organ preservation process.
In this study, we describe a porcine DCD HT using DPP that mirrors current clinical DPP DCD HT with high fidelity. This model includes elements throughout the DCD transplant process: 1) DCD procurement, 2) ex vivo perfusion preservation with an Organ Care System (OCS), and 3) recipient implantation. This comprehensive model provides an opportunity to better characterize mechanisms underlying PGD in DCD HT. This then allows for the development of targeted and rational therapeutic strategies aimed at improving allograft preservation and performance. Furthermore, this model provides a platform for the preclinical evaluation of such novel therapeutics, which will be important to help advance the field of heart transplant in a safe and expeditious manner.

<table><tbody><tr><td>0-0 silk suture with needle</td><td>DemeTECH</td><td>SK260026B0P</td><td></td></tr><tr><td>0-0 silk ties</td><td>DemeTECH</td><td>SK6X2600</td><td></td></tr><tr><td>1/4&quot; x 1/4&quot; straight connector</td><td>Liva Nova</td><td>5050400</td><td></td></tr><tr><td>10% Formalin</td><td>VWR</td><td>16004-126</td><td></td></tr><tr><td>2-0 Ethibond SH</td><td>Covidien</td><td>3369-51</td><td></td></tr><tr><td>2-0 silk pops</td><td>Covidien</td><td>GS62M</td><td></td></tr><tr><td>2-0 silk suture with needle</td><td>DemeTECH</td><td>SK262026B0P</td><td></td></tr><tr><td>2-0 silk ties</td><td>DemeTECH</td><td>SK13X6620W</td><td></td></tr><tr><td>5 Fr micropuncture</td><td>Cook Medical</td><td>G48007</td><td></td></tr><tr><td>6 Fr introducer sheath</td><td>Terumo</td><td>RSS605</td><td></td></tr><tr><td>7.0 Fr Triple Lumen central venous line</td><td>Cook Medical</td><td>G47833</td><td></td></tr><tr><td>Aggrastat (tirofiban HCl) 2 mg</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Albumin 25% 12.5 g/50 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Blood access sample for autologous blood recovery (spike with one-way stopcock)</td><td>Liva Nova</td><td>7016000</td><td></td></tr><tr><td>Blood typing kit</td><td>Eldon Biologicals</td><td>892165002056</td><td></td></tr><tr><td>Calcium gluconate 1 mg/10 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Calcium gluconate 1 mg/10 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Cefazolin 1 g</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Cefazolin 1 g</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Ciprofloxacin in D5W 200 mg/100 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Core needle biopsies 18 G TEMNO Care Fusion</td><td>Merit Medical</td><td>CA1820</td><td></td></tr><tr><td>CPT 8ml tubes for PBMCs</td><td>BD Bioscience</td><td>362761</td><td></td></tr><tr><td>Cryogenic laster labels for frozen vials and containers - 1.28&quot; x 0.5&quot;</td><td>LabTAG</td><td>LCS-23</td><td></td></tr><tr><td>D5W 500 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Del Nido cardioplegia 1 L</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>DLP 0.64cm (1/4 in) perfusion adapter</td><td>Medtronic</td><td>10007</td><td></td></tr><tr><td>Dopamine 200 mg/5 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Double-armed 4-0 prolene on BB needle</td><td>DemeTECH</td><td>PM1094017G0P</td><td></td></tr><tr><td>Double-armed 4-0 prolene on RB-1 needle</td><td>DemeTECH</td><td>PM1094017C0P</td><td></td></tr><tr><td>Echo probe covers</td><td>Microtek Medical</td><td>PC1292</td><td></td></tr><tr><td>EDTA 10 mL blood tubes: BD Vacutainer venous blood collection tubes BD Medical&amp;nbsp;</td><td>VWR</td><td>BD-366643</td><td></td></tr><tr><td>Epicardial pacing wires</td><td>A&amp;amp;E Medical</td><td>024-200</td><td></td></tr><tr><td>Epinephrine 1 mg/mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Epinephrine 1 mg/mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Esmolol 100 mg/10 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Heparin 10,000 unit/10 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Insulin regular (humulin R) 100 unit/1 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>ISTAT Activated Clotting Time (ACT) Kaolin cartridges</td><td>Abbott</td><td>03P87-25</td><td></td></tr><tr><td>ISTAT CG8+ cartridges</td><td>Abbott</td><td>03P88-25</td><td></td></tr><tr><td>IV Amiodarone 150 mg/3 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>IV Heparin 10,000 U/10 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>IV Lidocaine</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>IV Methylprednisolone 125 mg/2 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Lidocaine 2% hydrochloride injection USP 100 mg/5 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Long 3-0 prolene on SH needle</td><td>DemeTECH</td><td>PM1093026C0P</td><td></td></tr><tr><td>Methylprednisolone 125 mg/2 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Microcentrifuge tube with flat screw-cap</td><td>VWR</td><td>16466-060</td><td></td></tr><tr><td>Multivitamin (infuvite adult)</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Nalgene sterile specimen cryogenic vial with screw closure</td><td>VWR</td><td>66008-740</td><td></td></tr><tr><td>Norepinephrine 4 mg/4 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>OCS disposable Heart kit</td><td>TransMedics</td><td>N/A</td><td></td></tr><tr><td>Organ Care System (OCS) Heart Module</td><td>TransMedics</td><td>N/A</td><td></td></tr><tr><td>Oxygen tank</td><td>TransMedics</td><td>N/A</td><td></td></tr><tr><td>Pacing cables</td><td>Remington Medical</td><td>ADAP-2000</td><td></td></tr><tr><td>Phenylephrine hydrochloride 100 mg/10 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Pledgets</td><td>Covidien</td><td>8677-01</td><td></td></tr><tr><td>Pressure-volume loop catheter (Ventricath 512, 5Fr, 122 cm)</td><td>AD Instruments</td><td>Ventricath-512</td><td></td></tr><tr><td>Protamine 50 mg/5 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>RNAlater</td><td>Thermo Fisher</td><td>&amp;nbsp;AM7024</td><td></td></tr><tr><td>Scigen Tissue-Plus O.C.T compound</td><td>FisherSci</td><td>23-730-571</td><td></td></tr><tr><td>Smart Perfusion Pack: double rapid prime line stock</td><td>Liva Nova</td><td>26020000</td><td></td></tr><tr><td>Sodium bicarbonate 50 mEq/50 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Sodium bicarbonate 50 mEq/50 mL</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Sterile water vial (10 mL)</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Tissue-Tek Cryomold molds/adapters, Sakura Finetek</td><td>VWR</td><td>25608-924 &amp;amp; 25608-916</td><td></td></tr><tr><td>Tissue-Tek Mega-Cassette System, Sakura Finetek</td><td>VWR</td><td>25608-844</td><td></td></tr><tr><td>Umbilical tape</td><td>CP Medical</td><td>CP12A</td><td></td></tr><tr><td>Vasopressin</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Vecuronium 10 mg vial</td><td>obtained from institutional pharmacy</td><td>N/A</td><td></td></tr><tr><td>Vessel loops</td><td>Medline</td><td>DYNJVL03</td><td></td></tr><tr><td>Weck Horizon Titanium Ligating Clips, Large</td><td>Teleflex</td><td>4200</td><td></td></tr><tr><td>Weck Horizon Titanium Ligating Clips, Medium</td><td>Teleflex</td><td>2200</td><td></td></tr><tr><td>Weck Horizon Titanium Ligating Clips, Small</td><td>Teleflex</td><td>1201</td><td></td></tr></tbody></table>

a high-fidelity porcine model of orthotopic heart transplantation following donation after circulatory death

A High-Fidelity Porcine Model of Orthotopic Heart Transplantation Following Donation after Circulatory Death

The number of advanced heart failure patients who can receive a heart transplant is limited by a shortage of suitable organ donors. In efforts to expand the donor pool, alternative donation&#160;and procurement methods have been developed,&#160;including heart transplantation following donation after circulatory death (DCD HT). While short-term survival following DCD HT is non-inferior to heart transplantation with brain-dead&#160;donors, there may be an increased rate of primary graft dysfunction (PGD) associated with DCD HT allografts. The underlying etiology of PGD is multifactorial and incompletely understood. For DCD HT allografts, the period of warm ischemic injury during DCD procurement is a potential risk factor for PGD to which brain death allografts are not exposed. The functional warm ischemic time thus may be an important driver of PGD in DCD HT. However, the mechanisms underlying PGD in this clinical scenario are poorly understood at the molecular level. The work presented herein aims to describe the development and validation of a high-fidelity non-survival porcine model of DCD orthotopic heart transplantation. We hypothesize that the use of this translational large animal model is critical to elucidate molecular mechanisms contributing to PGD, as well as to investigate interventions designed to optimize allograft preservation and early performance. This model replicates the perioperative and surgical approach used in DCD HT clinically, with modifications to account for porcine anatomy and physiology. The development of this large animal surgical model will not only provide mechanistic insights into the development of PGD but also can be modified to enhance translational research efforts aimed at improving organ recovery following DCD HT.

A total of 6 porcine DCD transplants were performed for a pilot analysis using the protocol described here. Over the course of these six pilot experiments, components of the protocol were refined to better suit the needs of the model, enhance reproducibility, and account for logistical constraints. The final resulting protocol, as written here, is summarized in Figure 1.
Unlike in the human-controlled circulatory death process, porcine hearts subjected to ischemia sustained electrical activity for much longer than expected, even after contractility ceased. Upon recognition of this difference in the first experiment, electrical asystole was deemed to be unreliable as a measure for declaration of death. In order to avoid warm-ischemic damage to the allograft out of proportion to that seen in the clinical context, mechanical asystole (pulseless electrical activity) was utilized to determine the time of death for the remaining transplant procedures.
Additionally, the standoff period was initially 15 min prior to proceeding with donor cardiectomy. This was utilized for the first three experiments of the pilot set. Part of the rationale for this 15 min warm ischemia period was that we were aiming to have severe enough injury to have a degree of post-transplantation graft dysfunction that would be suitable for study. We found that graft dysfunction in these early experiments was so severe that we had difficulty sustaining the recipient animal for an hour following separation from CPB, such that the recipient animal required high doses of inotropic and pressor support to meet the 1-h timepoint. Given these difficulties, we reduced the period of warm-ischemic injury to 10 min, which more closely approximates the clinical DCD HT with ex vivo perfusion. Figure 2 depicts representative images of the allograft at this stage as well as during ex vivo perfusion and after implantation. Lastly, adjustments were made to the titration of vasopressor and inotrope infusions in the post-CPB wean phase to support the newly transplanted heart. The need for an experienced team with specific expertise in cardiovascular anesthesiology to enhance success in this post-CPB phase cannot be emphasized enough.
Operative times were tracked across 4 of the 6 experiments and are summarized in Table 1. In the DCD procurement phase of the protocol, the elapsed time from cessation of life-sustaining measures in the donor pig to the declaration of death was 14.25 (+/- 2.6) min. Operative cardiopulmonary bypass time and cross-clamp time remained consistent throughout the pilot experiments at about 3 h and 1.5 h, respectively. Experiments were ended with euthanasia approximately 1 h after separation from CPB. There was variability in the degree of stability of the animals at the time of euthanasia. Some animals demonstrated relatively good stability and only mild graft dysfunction, while others demonstrated significant hemodynamic instability and severe graft dysfunction. Representative still echocardiography displays are demonstrated in Figure 3. This and other functional assessments, such as PV loops, can be utilized to determine differences in allograft function from baseline as well as before and after the introduction of therapeutic interventions.
A summary of samples collected, processed, and stored through the experiment is displayed in Figure 4. Explanted hearts were immediately placed in ice-cold PBS and stored in the lab for tissue and molecular analysis. The recipient's native heart was utilized as a control, while the transplanted allograft was stored as the experimental tissue. The hearts were cut into 4 cross-sections from apex to base. From each of these, representative tissue samples of each chamber (left ventricle, right ventricle, septum, and both atria) were flash-frozen in liquid nitrogen and stored at -80 °C for future analysis. Similarly, representative tissue samples from each of these levels and chambers were incubated in RNAlater and flash-frozen. The remaining sample of tissue was preserved in formalin for histopathologic analysis. Blood samples from any time point were obtained in duplicate and stored in either EDTA or CPT tubes. Blood stored in EDTA tubes was spun down to isolate plasma, which was flash-frozen. Blood in the CPT tubes was processed for PBMCs using a modified protocol provided by the CPT tube vendor.
<img alt="Heart transplantation process: diagram of circulatory death, DCD procurement, ex-vivo perfusion study." class="xfigimg" src="/files/ftp_upload/68090/68090fig1.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Porcine DCD HT protocol schematic. The timeline of events that occur in the porcine DCD HT procedure is depicted here. In this pilot study, 6 DCD HTs were conducted. Baseline myocardial assessments are performed on the donor pig heart allograft, after which controlled circulatory death commences. After the declaration of death, there is a 10-min standoff period. The allograft is then explanted and transported to the ex vivo perfusion device, where it is reperfused for 2-3 h. After the preparation of the recipient animal, the donor allograft is implanted with a biatrial technique. Following 1 h of reperfusion on CPB, the recipient is weaned from bypass support. Euthanasia occurs 1 h after separation from CPB. The implanted allograft is then processed for tissue analysis. DCD HT = heart transplantation following donation after circulatory death; CPB = cardiopulmonary bypass. <a href="https://www.jove.com/files/ftp_upload/68090/68090fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Heart transplantation process; diagram; surgical procedure; organ preservation; medical technique." class="xfigimg" src="/files/ftp_upload/68090/68090fig2.jpg" data-altupdatedat="1783396841000" data-alt="Figure 2"> 
Figure 2: Porcine cardiac allograft at different stages along the DCD OHT process. (A) Following controlled circulatory death, the donor allograft is distended, ischemic, and edematous. The white arrowhead shows the aortic root cannula. (B) Once the allograft is explanted from the donor, it is placed on the EVP device for ex vivo perfusion. Note the orientation of the heart, with the posterior aspect facing outwards. The black arrowhead points to the aortic adaptor used to connect the allograft to the device. (C) After implantation in the recipient, the allograft is reperfused on cardiopulmonary bypass for one hour prior to weaning from CPB. The white arrowhead shows the aortic cannula; white arrows point to the bicaval venous cannulas. EVP = ex vivo perfusion; CPB = cardiopulmonary bypass. <a href="https://www.jove.com/files/ftp_upload/68090/68090fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cardiac ultrasound images showing heart chambers; labeled LV, RV, RA, LA; diagnostic analysis." class="xfigimg" src="/files/ftp_upload/68090/68090fig3.jpg" data-altupdatedat="1783396841000" data-alt="Figure 3"> 
Figure 3: Cardiac epicardial echocardiography. Throughout the DCD OHT procedure, multiple epicardial echocardiographic images were acquired to evaluate ventricular function. (A) Standard short-axis, (B) 4-chamber, and (C) 2-chamber views are shown here. LV = left ventricle; LA = left atrium; RV = right ventricle; RA = right atrium. <a href="https://www.jove.com/files/ftp_upload/68090/68090fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Pig heart transplantation diagram showing ex-vivo perfusion, biopsy, and blood collection process." class="xfigimg" src="/files/ftp_upload/68090/68090fig4.jpg" data-altupdatedat="1783396841000" data-alt="Figure 4"> 
Figure 4: Specimen collection and processing workflow. Schematic of sample collection and processing at each step of the procedure. CBC = complete blood count; CMP = comprehensive metabolic panel; PBMCs = peripheral blood mononuclear cells. Figure created in BioRender. <a href="https://www.jove.com/files/ftp_upload/68090/68090fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2"></td>
			<td>Length of Time (min)</td>
		</tr>
		<tr>
			<td>(mean ±  SD; n = 4)</td>
		</tr>
		<tr>
			<td>Time from cessation of ventilation to declaration of death</td>
			<td>14.25 (2.6)</td>
		</tr>
		<tr>
			<td>Ex vivo perfusion duration</td>
			<td>147 (18)</td>
		</tr>
		<tr>
			<td>CPB duration </td>
			<td>174 (4)</td>
		</tr>
		<tr>
			<td>Cross-clamp time</td>
			<td>90 (12.5)</td>
		</tr>
		<tr>
			<td>Time from CPB wean to death</td>
			<td>50 (37)</td>
		</tr>
	</tbody>
</table>
Table 1. Procedural information. The average duration of each critical step in the DCD HT procedure. CPB = cardiopulmonary bypass.

The protocol here describes a high-fidelity porcine model of heart transplantation following donation after circulatory death utilizing ex vivo perfusion of the allograft.

The number of advanced heart failure patients who can receive a heart transplant is limited by a shortage of suitable organ donors. In efforts to expand ...

We present a high fidelity porcine model of heart transplantation after circulatory death, enabling evaluation of DCD related pathophysiology and supporting translational research to improve allograft recovery. Since 2019, clinical adoption DCD heart transplantation has grown in the United States with recent trials showing non-inferior short-term survival when compared to concessional brain death donation methods. Outcomes after DCD heart transplantation have further been improved by advances in RAF free profusion methods, including direct procurement and normothermic ex vivo perfusion, as well as normothermic regional perfusion.Primary graft dysfunction remains a key driver of short-term mortality after DCD heart transplantation. While warm ischemic injury and metabolic derangement are proposed contributing factors, the underlying mechanism behind PGD still remains unknown. This porcine model replicates clinical DCD procedures with adjustments for species-specific anatomy.In addition, ex vivo perfusion supports the development of targeted therapies to enhance allograft preservation and function. After performing a carotid cutdown and exposing the donor heart, perform baseline analysis of the allograft, including pressure volume loop recordings. To begin, measure the length of the solid state pressure volume catheter from the cannulation site to the apex of the swine heart.Introduce the catheter sequentially into the carotid artery and internal jugular vein. Guide the catheter to the left and right ventricles under epicardial ultrasound guidance. Prepare the donor heart for explant by cannulating the aortic root with a seven French vent, securing it with a room tourniquet and placing a purse string stitch in the right atrial appendage.Cease mechanical ventilation to initiate the controlled circulatory death process. The heart may distend. Wait for 10 minutes after confirmed death defined by pulseless electrical activity and no signs of life before proceeding.Slow pulseless electrical activity persists in swine longer than in humans, resulting in increased allograft ischemic insult. For ex vivo profusion device priming, use a number 11 blade to make a stab incision into the right atrial appendage and cannulate with the 34 French venous cannula. Secure the cannula with a rummel tourniquet and connect it to the ex vivo perfusion devices collection bag.Collect approximately 1, 200 to 1, 500 milliliters of donor blood into the manufacturer provided collection bag. Following collection of the donor blood, apply the aortic cross clamp. Administer one liter of del Nido cardioplegia into the aortic root, targeting a pressure of 60 to 100 millimeters of mercury.Transect the inferior vena cava and left atrial appendage to vent both ventricles during cardioplegia delivery. Divide the inferior vena cava, superior vena cava, aorta just distal to the innominate artery and pulmonary artery at the bifurcation. Then transect the donor left atrium, ensuring a sufficient cuff remains.Remove the heart and place it in a basin filled with cold, sterile slush for back table preparation. Place four equidistant pledged horizontal mattress sutures using 4-0 Prolene suture inside the distal end of the aorta. Then insert the ex vivo profusion aortic adapter and secure with 0-0 silk suture or umbilical tape.Transport the allograft to the ex vivo perfusion device and connect the adapter. Position the heart with the posterior side facing up and obtain perfuse 8 samples, blood work and biopsy samples during this stage. In this model, the allograft remained on the ex vivo perfusion device at 34 degrees Celsius for two to three hours before implantation into the recipient.After exposing the recipient's native heart, prepare for cardiectomy. Cannulate the distal ascending aorta, and then perform by caval venous cannulation. Apply the aortic cross clamp proximal to the cannula and explant the recipient heart.Leave intact atrial cuffs for biatrial implantation. Transect the aorta and pulmonary artery close to the root. Once the recipient's native heart has been explanted, begin by atrial implantation of the donor allograft and start the left atrial anastomosis using 4-0 Proline suture.Then complete the pulmonary artery, aortic and right atrial anastomosis with continuous 4-0 Proline suture. After completing all anastomosis, release the aortic cross clamp to reperfuse the allograft. Ensure hemostasis of all anastomosis.After 60 minutes of reperfusion, attempt to wean the recipient from cardiopulmonary bypass. The recipient animal was supported for one hour post-separation from cardiopulmonary bypass. In the DCD procurement phase, the elapsed time from cessation of life-sustaining measures in the donor pig to the declaration of death was approximately 14.25 minutes.Operative cardiopulmonary bypass time in the recipient animals was around three hours with a cross clamp time of on average 1.5 hours. Post-transplant epicardial echocardiographic assessment was performed to evaluate graft function following implantation. Standard short axis, fourth chamber, and two chamber views were obtained.Post-transplant graft function varied from mild graft dysfunction with stable hemodynamics to severe dysfunction with hemodynamic instability.

a-high-fidelity-porcine-model-orthotopic-heart-transplantation

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JoVE Journal

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Learn a high-fidelity porcine model for orthotopic heart transplantation following donation after circulatory death. This method enables evaluation of DCD pathophysiology and supports translational research to improve allograft recovery.

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