We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.
Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient’s circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.
Heart failure is a condition that affects an estimated 6 million adults in the United States and is projected to increase to 8 million adults by the year 20301. Cardiac transplantation is the gold standard treatment for end stage heart failure. However, it is not without its limitations and complications. It remains limited by the number of available donor hearts, primary graft dysfunction, rejection of the heart, and the side effects of long-term immunosuppression2. These limitations are particularly important in young recipients who may experience allograft failure and require subsequent re-transplantation to achieve normal life expectancy.
An ideal intervention to overcome these limitations would treat entire cardiac allografts with therapeutics prior to implantation into the recipient that can improve the viability of the allograft and confer "cardioprotection." Such interventions would be given prophylactically to minimize the incidence of ischemic insults, allograft rejection, cardiac allograft vasculopathy, and even repair marginal allografts. Translational studies for developing these types of interventions require a large-animal model of cardiac transplantation to allow for the long-term surveillance of the cardiac graft. The porcine, heterotopic heart transplantation model in the intraabdominal position has proven ideal for this purpose. Heart transplantation in this position allows for testing the effects of novel therapies and assessing the immunopathology of graft rejection. Additionally, the heterotopic model is advantageous over the orthotopic model due to better overall survival of the recipient, no requirement for cardiopulmonary bypass, and no requirement of the graft to maintain the recipient's circulation3.
Effective delivery of therapeutic interventions to the heart, such as gene, cell, or immuno- therapy, is a significant barrier to clinical application4,5. The technology introduced by ex vivo perfusion devices allows grafts to be continually perfused, maintaining them in a nonworking but metabolically active state6,7,8,9. This offers a unique opportunity to treat a whole heart with advanced therapeutics while minimizing the potential side effects of systemic delivery10,11,12,13. Another advantage of utilizing ex vivo perfusion devices for therapeutic delivery is that they allow the administration of medications to the coronary circulation over extended periods that are not feasible using traditional cold static storage methods. This allows for more global delivery of the therapeutics to the graft14. Using the protocol presented here, we successfully delivered the firefly luciferase gene to a whole porcine cardiac graft using adenoviral vectors15. The aim of this protocol is to provide a reproducible and robust approach for achieving delivery of a therapeutic to the entire cardiac allograft prior to transplantation.
NOTE: Two female Yucatan pigs are selected, with one designated to be the cardiac graft donor and the other the recipient. Pigs aged 6-8 months, weighing approximately 30 kg, and having compatible blood types are recommended. The overview of the protocol is demonstrated in Figure 1. Housing and the treatment procedures for the pigs are performed in accordance with the guidelines of the Animal Care and Use Committee of Duke University Medical Center.
1. Preparation of the ex vivo perfusion device
2. Initiation of anesthesia and IV access in the donor pig
3. Vital signs and central line settings
4. Median sternotomy of the donor pig
5. Cardiac arrest and cardiectomy of the donor pig
6. Washing the donor blood and priming the ex vivo perfusion device
NOTE: This step is necessary to remove any components from the donor serum that might neutralize the delivery of the therapeutic when it is introduced to the perfusate. Perform this step during the explantation of the donor heart to minimize the allograft ischemic time.
7. Backtable preparation of the donor heart and reanimating the heart
8. Administering the therapeutic
9. Preparation of the recipient and laparotomy with vascular exposure
10. Final arrest and removing the heart from the ex vivo perfusion device
11. Heterotopic implantation of the cardiac graft
12. Closure of the laparotomy
13. Postsurgical treatment and euthanasia
This group has successfully survived 9 pigs between 5 and 35 days following the protocol as presented here, depending on the study design. Out of 10 pigs that have undergone this protocol, only 1 died prematurely from surgical complications, yielding a 90% survival rate. Demonstrated in Figure 2 is a diagram of the configuration of a heterotopic heart transplanted in the intraabdominal position in a pig. When determining the site for anastomosis of the allograft, select a site that minimizes any tension or kinking on the anastomosis. This ensures that the anastomoses heal properly and that the allograft receives optimal perfusion and drainage of blood.
A representative image of a cardiac allograft being perfused on a normothermic ex vivo perfusion device is shown in Figure 3. Figure 4 outlines representative perfusion parameters acquired during a successful experiment (circulatory flow rate, aortic pressure, heart rate, temperature, mixed venous oxygen saturation, and hematocrit). Inability to achieve the parameter values demonstrated here may lead to compromised allograft function after transplantation. Figure 5 demonstrates an image of an intraabdominal heterotopic heart in situ 35 days after successful transplantation. Representative results of the effectiveness of using the protocol presented here for therapeutic delivery were previously demonstrated by this group15. The cardiac allografts (n = 3) were perfused with perfusate treated with an adenoviral vector carrying the transgene for luciferase. Gene expression proved to be global and robust within the allografts 5 days after the treatment and transplantation. Figure 6 shows an atlas of luciferase protein activity measured and presented as average fold-change in activity from each region of the explanted cardiac allograft in comparison to the thoracic heart of the recipients.
Figure 1: Protocol schematic for therapeutic delivery to an entire cardiac allograft using normothermic ex vivo sanguinous perfusion. (A) The heart and blood are procured from the donor pig. (B) The blood is washed using a cell saver device to remove any therapeutic neutralizing components from the donor serum. (C) The cardiac allograft is mounted onto the normothermic ex vivo perfusion device and perfused for 2 h. (D) Soon after the allograft is mounted, the therapeutic of interest is added to the perfusate. (E) After the allotted ex vivo perfusion period, the allograft is transplanted into the recipient pig in the intraabdominal, heterotopic position. This figure has been modified from15. Please click here to view a larger version of this figure.
Figure 2: Porcine heterotopic heart model in the intraabdominal position. Diagram of the heterotopic heart model where the allograft is transplanted in the intraabdominal position while the recipient's native heart remains in its natural location. The pulmonary artery of the allograft is anastomosed to the infra-renal inferior vena cava, while the aorta of the allograft is anastomosed to the infra-renal aorta of the recipient. Please click here to view a larger version of this figure.
Figure 3: Cardiac allograft on ex vivo perfusion device. The cardiac allograft mounted on a normothermic, ex vivo perfusion device where it is perfused with therapeutic-infused perfusate for 2 h prior to implantation into the recipient. Please click here to view a larger version of this figure.
Figure 4: Representative ex vivo perfusion parameters. (A) Circulatory flow rates measured from the pulmonary artery (blue), the aorta (green), and the coronary arteries (red). (B) Representative aortic pressure measurements: mean pressure (blue), systolic pressure (red), diastolic pressure (green). (C) Heart rate of a cardiac allograft during ex vivo perfusion. (D) Recorded temperature of the cardiac allograft during ex vivo perfusion. (E) demonstrates the values of SvO2 measured from the perfusate during the perfusion period. (F) Hematocrit values measured from the perfusate during the perfusion period. Abbreviations: hct = hematocrit; SvO2 = mixed venous oxygen saturation. Please click here to view a larger version of this figure.
Figure 5: Cardiac allograft transplanted in the recipient. A cardiac allograft on postoperative day 35 treated with therapeutic at the time of implantation. The donor was selected to be a perfect SLA match with the recipient. Abbreviation: SLA = Swine Leukocyte Antigen. Please click here to view a larger version of this figure.
Figure 6: Luciferase activity after transduction of cardiac allografts. Presented are the results of three cardiac allografts that were transduced with adenoviral vectors carrying a luciferase transgene. Demonstrated is the average fold-change in luciferase protein activity in each area of the cardiac allograft. This figure has been modified from Bishawi et al.15. Please click here to view a larger version of this figure.
Delivery of therapeutics during ex vivo perfusion in cardiac transplantation offers a strategy to modify the allograft and potentially improve transplant outcomes. The protocol presented here incorporates the state-of-the-art normothermic ex vivo sanguinous perfusion storage and offers promising potential to test isolated delivery of cell, gene, or immunotherapies to the allograft11,12,13. To date, cardiac delivery techniques for these putative therapies for cardiovascular disease and end-stage heart failure have relied on systemic administration, intracoronary perfusion via catheterization, and direct intramyocardial injections, all of which have achieved poor results in terms of myocardial delivery5,16. We had previously demonstrated robust and global expression of a reporter gene to entire cardiac allografts when a viral vector was administered into the perfusate during ex vivo perfusion prior to transplantation15. This is particularly important in the context of cardiac transplantation, where global expression and effect of the therapeutic should reach all areas of the allograft to achieve the desired "cardioprotection" of the whole allograft. This protocol achieves this in a manner that has not been previously achieved using traditionally described routes of administration for therapeutics.
There are several critical steps presented in this protocol to highlight. (1) Every precaution must be taken to minimize blood loss during the procurement of the heart from the donor. At least 1 L of blood needs to be attained from the donor for the perfusion device to achieve adequate flow rates. (2) For therapeutic delivery using normothermic ex vivo sanguinous perfusion, it is necessary to wash the donor blood before adding it to the perfusate to remove any neutralizing components in the donor serum that may negatively affect the delivery of the therapeutic to the heart. (3) Minimize dissection of the heart in the donor until after cardioplegic arrest to avoid fatal arrhythmias. (4) When introducing the therapeutic to the perfusion device, it is important to introduce it through the port closest to the aortic root and always flush the port to ensure complete delivery of the suspension. This is to minimize any potential loss of the therapeutic to the oxygenator or tubing within the circuit and ensure that the graft is receiving as high of a therapeutic concentration as possible. (5) Finally, when selecting the site for graft implantation, it is critical that the location minimizes the potential for tension on the anastomosis and that there be no kinking of the blood vessels/anastomoses.
It is also recommended that the pigs be Swine Leukocyte Antigen (SLA)-typed (i.e., porcine major histocompatibility complex, MHC) beforehand to select for the appropriate degree of matching/mismatching across SLA haplotypes comprising the cell-surface class I (SLA-1, SLA-2, and SLA-3) and/or class II (DR and DQ) antigens based on the investigator's needs (SLA-typing performed by SH as previously described with slight modifications made to the typing primer panels)17,18. For example, ensuring that pigs match across all SLA antigens minimizes the risk of allograft rejection, whereas using pigs with mismatch across all SLA antigens maximizes the incidence of allograft rejection.
A limitation of this model is that while it allows for the study of the immunologic effects on the cardiac graft, it does not allow for a full assessment of the graft's ability to support the cardiovascular system following an intervention. To achieve that, the graft would need to be implanted orthotopically. However, orthotopic transplantation in large-animal models has higher associated mortality and requires cardiopulmonary bypass3. Another limitation of this model is limited access to an ex vivo perfusion device to conduct effective gene delivery to the graft. As these devices become more available in the field of organ transplantation, access is expected to improve. Furthermore, a non-commercial device may be an option for experimental purposes.
Cardiac transplantation offers a unique setting where therapeutics can be introduced to the allograft via ex vivo perfusion prior to implantation into the recipient. The use of an ex vivo perfusion device allows for grafts to be in transit from the donor to the recipient for periods that are much longer than what is safe using traditional cold static storage6. This extended perfusion period enables effective isolated delivery of therapeutics. This model serves as a translational step between preclinical animal testing of therapeutics and transformative clinical therapies.
The authors have nothing to disclose.
We would like to thank Duke Large Animal Surgical Core and Duke Perfusion Services for their assistance during these procedures. We would also like to thank Paul Lezberg and TransMedics, Inc. for support.
0 Looped Maxon suture | Covidien | GMM-341L | Used to close fascia of the laparotomy incision |
0 Silk ties | Medtronic, Inc | S346 | |
18 G Angiocath | BD | 381144 | Used to de-air the left ventricle of the donor heart after implantation |
20 Fr LV vent | Medtronic, Inc | 12002 | |
2-0 Silk sutures | Ethicon, Inc. | SA11G | |
2-0 Silk ties | Ethicon, Inc. | SA65H | |
2-0 Vicryl suture | Ethicon, Inc. | J259H | |
24 Fr venous cannula | Medtronic, Inc | 68124 | |
3-0 Prolene sutures | Ethicon, Inc. | 8522 | |
4-0 Monocryl suture | Ethicon, Inc. | Y469G | |
4-0 Prolene sutures | Ethicon, Inc. | 8521 | |
Animal hair cutting clipper | Wahl | 8786-452 | |
Aortic clamp | V. Mueller | CH6201 | |
Army Navy retractor | V. Mueller | SU3660 | |
ATF 40, Cell saver disposable set | Fresenius Kabi | 9108494 | Cell saver device insert |
Balfour retractor | V. Mueller | SU3042 | Used as an abdominal wall retractor |
C.A.T.S cell saver | Fresenius Kabi | ES0019 | Cell saver device used to wash donor blood |
Cardiac defibrillator | Zoll | M Series | Cardiac defibrillator |
Castro needle holder | V. Mueller | CH8589 | |
CG4 iStat cartridges | Abbott | 03P85-25 | POC testing |
CG8 iStat cartridges | Abbott | 03P88-25 | POC testing |
DeBakey forceps | V. Mueller | CH5902 | |
Electrocautery disposable pencil | Covidien | E2450H | |
Gerald forceps | V. Mueller | NL1451 | |
Hemotherm 400CE Dual Reservoir Cooler/Heater | Cincinnati Sub-Zero | 86022 | Heater cooler used to regulate perfusion temperature on the ex vivo perfusion device |
iSTAT 1 | Abbott | 04P75-03 | POC testing device |
Kocher clamp | V. Mueller | SU2790 | |
Large clip applier | Sklar | 50-4300 | |
Large clips | Teleflex | 4200 | |
Large soft pledgets | Covidien | 8886867901 | |
Medium clip applier | Sklar | 50-4335 | |
Medium clips | Teleflex | 2200 | |
Metzenbaum scissor | V. Mueller | CH2006-001 | |
No. 10 scalpel blade | Swann-Mortan | 301 | Used for skin incision |
No. 11 scalpel blade | Kiato Plus | 18111 | Used for vascular incision |
OCS device with base | TransMedics, Inc. | Ex vivo perfusion device | |
OCS disposable | TransMedics, Inc. | Ex vivo perfusion device insert with perfusion kits | |
Pacing cable | Remington Medical | FL-601-97 | |
Pediatric cardioplegia catheter (4Fr) | Medtronic, Inc | 10218 | Used to deliver cardioplegia to the donor aortic root |
Pediatric Foley catheter | Teleflex | RSH170003080 | Placed pre-op to decompress the recipient's bladder |
Potts scissors | V. Mueller | CH13038 | |
Pressure bag x2 (1,000 mL) | Novaplus | V4010H | Used to deliver cardioplegia at a set pressure |
Satinsky clamp | V. Mueller | CH7305 | Vascular clamp used for creating anastomoses between donor heart and recipient vessels |
Scissors | Felco | FELCO 200A-50 | Used to perform sternotomy |
Small hard pledgets | Covidien | 8886867701 | |
Sternal retractor | V. Mueller | CH6950-007 | |
Temporary cardiac pacing wires | Ethicon, Inc. | TPW32 | |
Temporary dual chamber pacemaker | Medtronic, Inc | 5388 | Cardiac pacing device |
Tourniquet kit | Medtronic, Inc | 79005 | Rummel tourniquets |
Umbilical tape | Covidien | 8886861903 | |
Vessel loops | Covidien | 31145686 |