We gratefully acknowledge funding to SNT from the US National Institute of Health (K99/R00 HL1431149; R01HL157803; R01DK134590; R24OD034189), the National Science Foundation under Grant No. EEC 1941543, the Claflin Distinguished Scholar Award on behalf of the MGH Executive Committee on Research, and the Polsky Family Award for Leaders in Surgery.&#160;We acknowledge research funding to AAO from the Hassenfeld Family Foundation, the MGH Executive Committee&#160;on Research, and the MGH Center for Diversity and Inclusion.&#160;We acknowledge research funding to GO from the Sarnoff Cardiovascular Research Foundation.

This study was conducted in accordance with the Institutional Animal Care and Use Committee (IACUC), Massachusetts General Hospital, and Jove&#39;s animal guidelines. Hearts (170 - 250 g) were harvested from Yorkshire pigs (30 - 35 kg, age 3-4 months, mixed sex) using a model of donation after brain death and perfused retrogradely (Langendorff) for 6 h prior to loading. All grafts were exposed to a cold ischemia time of approximately 1h during instrumentation.
1. System design</...

Enhanced Cardiac Assessment in Ex Vivo Perfusion with Left Ventricular Loading

DV is an employee and Founder of VentriFlo, Inc., Pelham, NH, and has patent applications relevant to this study. For more information see https://ventriflo.com/patents/.&#160;SNT has patent applications relevant to this study and serves on the Scientific Advisory Board for Sylvatica Biotech Inc., a company focused on developing organ preservation technology. All competing interests are managed by the MGH and Partners HealthCare in accordance with their conflict-of-interest policies.&#160;AR and AAO receive research funding from Paragonix Technologies Inc.

Normothermic machine perfusion is a powerful modality for organ preservation and assessment that has greatly impacted the field of cardiac transplantation by expanding the donor pool of adult hearts36. This expansion is the result of the ability to currently utilize a small pool of hearts previously considered unsuitable for transplantation. Normothermic machine perfusion preserves cardiac grafts in a beating state, offering the opportunity for both functional and metabolic assessment. However, de...

Orthotopic heart transplantation is the current gold standard of care for end-stage heart failure1. Unfortunately, the field is significantly limited by a severe donor shortage crisis, resulting in only 2,000 heart transplants being performed each year when over 20,000 people would benefit from the lifesaving procedure2. This organ shortage is expected to worsen as the prevalence of heart failure in the United States alone is projected to surpass 8 million individuals by 20303. Steady increases in waitlist survival times - as a result of improved medical management, advances in mechanical circulatory support, and amendments to the UNOS allocation policy - have resulted in a further increase in the number of patients in need of transplantation at any given moment4,5.
Ex vivo machine perfusion or normothermic machine perfusion (NMP) is a preservation modality that has facilitated the expansion of the supply pool by allowing the use of organs donated after circulatory death (DCD) while achieving some extension of preservation times5,6,7,8. Unlike static cold storage, the current gold standard for preservation, NMP maintains organs in a metabolically active state, which creates the opportunity for real-time monitoring and graft assessment, becoming the standard preservation method for DCD grafts8,9. However, NMP devices currently used clinically are restricted to the Langendorff perfusion mode, which lacks quantitative metrics to predict transplantation outcomes and is unable to capture functional parameters6. For instance, lactate accumulation during Langendorff perfusion has been denoted as the best metabolic predictor of post-transplantation outcomes and is currently used in the clinical setting as a proxy for cardiac graft health10. However, even as the best assessment biomarker, it fails to reliably anticipate the need for mechanical circulatory support post-transplantation11,12. Similarly, the predictive capabilities of commonly utilized hemodynamic parameters (i.e., aortic pressure and coronary blood flow) are largely limited by the retrograde nature of current clinically used configurations for heart machine perfusion9.
The development of assessment protocols for accurate and precise determination of cardiac graft health during NMP would have an immense impact in the field beyond improving post-transplantation outcomes. Objective predictive tools would enable the reliable evaluation and likely utilization of marginal or extended criteria organs (i.e., prolonged warm (&#62; 30 min) and cold ischemia times (&#62; 6 h), increased donor age (&#62; 55), other comorbidities, etc.) from both DCD and brain death donors (DBD) that are currently rejected for transplantation due to the stringent selection criteria13. By enabling the use of marginal hearts, NMP could facilitate an increase in the organ supply as it is estimated that successful transplantation of half the hearts that currently go unused would be sufficient to eliminate the heart waitlist within 2-3 years14. Hemodynamic measurements obtained from left ventricular loading during NMP have garnered significant attention within the field due to their potential as objective assessment parameters. Previous studies have demonstrated that these parameters, such as left ventricular pulse pressure, contractility, and relaxation, are more indicative of cardiac graft function than metabolic trends15,16,17.
In effect, efforts have been dedicated to the development and identification of optimal loading methods to maximize assessment accuracy. Through these efforts, other groups have identified the most relevant mode of aortic perfusion during loading, whereby a stronger correlation between hemodynamic parameters and post-transplantation function was seen when implementing a passive afterload (i.e., no retrograde perfusion to the aorta during loading) when compared with pump-supported afterload (i.e., retrograde perfusion to the aorta during loading)18. This indicates that assisted coronary perfusion likely masks functional deficiencies. Previous studies have successfully incorporated passive afterloads into perfusion setups by implementing systems that mimic the Windkessel effect18,19,20. The Windkessel effect aids in dampening the fluctuation in blood pressure, maintaining continuous blood flow to the tissues and improving coronary perfusion. This protocol achieves the Windkessel-based passive afterload using a modified intravenous (IV) bag enclosed in two spring-loaded plates, where coronary perfusion is exclusively dependent on heart ejection (patent pending).
The use of passive left atrium (LA) pressurization (i.e., gravity-dependent pressurization) during loading, although common practice in small animal heart perfusions, is rarely utilized in the loading of large hearts21,22,23. Instead, the large majority of methods reported in the literature rely on secondary pumps for LA pressurization18,24,25,26,27,28. The pressurization of the LA through a gravity-dependent reservoir, rather than by pump, significantly simplifies the implementation of loading protocols. The use of gravity provides a fixed and constant pressure source, which greatly decreases the need for complicated control systems to achieve and maintain adequate LA pressurization. Moreover, through this pressurization approach, the requirement for a secondary pump is eliminated, facilitating the incorporation of loading capabilities into current Langerdoff setups, as only an extra reservoir is needed. The integration of loading capabilities into clinically utilized machine perfusion systems would amplify the application of cardiac NMP devices by facilitating detailed assessment of cardiac grafts during the preservation period. In effect, maximizing the utility of a system that poses significant financial commitment for patient care due to transportation and device utilization29.
This protocol demonstrates the feasibility of employing both passive afterload and passive LA pressurization during left ventricular loading. Through the validation of passive afterload/LA pressurization as a loading method, this protocol also provides an easy and effective manner of incorporating loading capabilities into established Langendorff perfusion systems. Importantly, it highlights the capability of functional assessment to uncover differences in viable versus failing hearts after extended periods of preservation (&#707;6 h).

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cardiac loading using passive left atrial pressurization and passive afterload for graft assessment

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. Despite the immense opportunity for assessment due to the beating state of the heart, current clinical practice depends on limited metabolic trends for graft evaluation. Hemodynamic measurements obtained from left ventricular loading have garnered significant attention within the field due to their potential as objective assessment parameters. In effect, this protocol provides an easy and effective manner of incorporating loading capabilities to established Langendorff perfusion systems through the simple addition of an extra reservoir. Furthermore, it demonstrates the feasibility of employing passive left atrial pressurization for loading, an approach that, to our knowledge, has not been previously demonstrated. This approach is complemented by a passive Windkessel base afterload, which acts as a compliance chamber to maximize myocardial perfusion during diastole. Lastly, it highlights the capability of capturing functional metrics during cardiac loading, including left ventricular pulse pressure, contractility, and relaxation, to uncover deficiencies in cardiac graft function after extended periods of preservation times (&#707;6 h).

Author Spotlight: Enhancing Graft Viability Assessment Through Quantitative Metrics and Innovative Reservoir Systems

Cardiac Loading using Passive Left Atrial Pressurization and Passive Afterload for Graft Assessment

The scope of this research covers two important areas of cardiac ex vivo machine perfusion. First, it highlights the shortcomings of current clinically-used assessment metrics in determining graft viability and transplantability. Second, it provides an alternative assessment technique capable of discerning between viable and unviable grafts, even when conventional metrics fail to do so.Through this work, we developed a gravity-dependent loading reservoir that significantly simplifies and standardize the incorporation of loading capabilities into existing ex vivo machine perfusion systems. This, in combination with passive afterload, seems to significantly increase the predictive capabilities of the technique. The field has been in dire need of quantitative assessment metrics that eliminate the subjectivity of current practices.This work offers a method to obtain quantitative objective metrics for assessing graft variability. This assessment method is expected to significantly improve the ability to draw conclusions from both experimental and clinical setups.

Hearts from 4 Yorkshire pigs (30 - 35 kg) were harvested and preserved via Langendorff NMP for 6 h prior to 4 h of continuous loading. This experimental condition was chosen since 6 h is the average clinical preservation duration (5.1 &#177; 0.7 h)34. Through the addition of 4 extra hours of continuous loading (total of 10 h ex vivo time), some degree of heart failure was expected as a clear correlation between perfusion time and myocardial function decline has been previously reported<su...

The protocol describes a porcine ex vivo heart perfusion system in which direct loading of the left ventricle may serve as an assessment technique for graft health while simultaneously providing a holistic evaluation of graft function. A discussion of the system design and possible assessment metrics is also provided.

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. ...

cardiac-loading-using-passive-left-atrial-pressurization-passive

Research

JoVE Journal

Medicine

Designing a Porcine Ex Vivo Heart Perfusion System for Graft Health Assessment

To begin, start mounting the organ chamber on its stand, followed by the windkessel bag and the smaller reservoir. Verify that all components are securely connected. Then attach one end of a quarter inch silicone tubing to the outflow port of the oxygenator, and a Y connection at the distal end.To one side of the Y connector, attach another quarter inch silicone tubing with a lure connection and connect the other end to the bottom port on the windkessel bag. After that, fix a three-way lure valve to the expander's lure connection for adenosine drip delivery. Place a Hoffman clamp on an additional piece of quarter inch silicone tubing.For loaded profusion, attach one end of this line to the second end of the Y connector, and the other end to the top of the smaller reservoir tubing to control reservoir filling. Ensure that the clamp is fully engaged at this time. Then connect an overflow line from the top of the loading reservoir to the larger reservoir.Fit a quarter inch tube to the bottom of the loading reservoir and connect it to a port at the base of the organ chamber. Ensure that the line has a one-way valve. Next, insert two three-way lure valves midway through the tubing for adenosine delivery and a one-way valve before reaching the organ chamber.Fit the second overflow port at the bottom of the windkessel bag with three by eight inch tubing, connecting it to any inflow port of the venous reservoir. Afterward, close the Hoffman clamp completely during Langendorff mode and adjust it during loaded mode to modulate aortic pressures. Then connect a three-way lure valve to the overflow port at the top of the windkessel bag.Use a quarter inch tubing to connect the overflow port at the top windkessel bag to any inflow port in the venous reservoir. Connect the third outflow port on the windkessel bag to the aortic port on the organ chamber, inserting a temperature probe in the lower three-fourths of the tubing length.

Graft Preparation for Loading in Ex Vivo Normothermic Machine Perfusion Systems

To begin, take the isolated pig's heart and cut the branches of the aortic arch to form a single outflow tract. Insert the aortic cannula through this tract and secure it using a zip tie and 4-0 silk suture. Then place a bipolar pacing wire on the posterior wall of the right ventricle.Create two per string 4-0 prolene sutures around the perimeter of the left atrium tract. Secure the sutures with tourniquet snares, but do not tie them until loading. Lastly, close the left atrium appendage with a simple continuous 4-0 prolene suture.Then record the initial heart weight.

Cardiac Graft Revival and Left Ventricular Loading in the Langendorff Perfusion System

To begin clamp the 3 by 8 inch tubing right before the aortic port to stop perfusate flow. Position the prepared pig's heart with its posterior wall facing the operator and angle the organ chamber at roughly 20 degrees. Then, place the aortic cannula at a 90 degree angle from the aortic port, and unclamp the aortic line slowly.De-air the aortic cannula by allowing perfusate to flow into it gradually. Now, slowly decrease the angle of the aortic cannula until it aligns with the aortic port, then fully connect it to the aortic line. Once fully connected, gently massage the heart intermittently to prevent distension due to left ventricular filling.Vent the left ventricle through the open left atrium during this period. Start data acquisition and initiate the adenosine drip at 333 microliters per minute. Then, connect the pacing wires to the pacing box and set it to 60 beats per minute for backup pacing.If fibrillation is present, defibrillate the heart using 30 JUULs paddles. Once an organized rhythm is present, discontinue manual venting and place EKG leads directly on the heart using hook needles. Connect the right angle cannula to the atrial port of the organ chamber.Once connected, clamp the cannula and release the Hoffman clamp in the line between the oxygenator and the loading reservoir to allow fluid into the loading reservoir. Then, fill the loading reservoir until the pressure reaches 15 to 20 millimeters of mercury. Increase the pump output to maintain both aortic and atrial pressure.Next, insert half of the right angle metal tip into the left atrium with the tip pointing toward the appendage to promote mitral valve competence. Insert the pressure sensor inside the left ventricle to record left ventricular pressure. After that, release the clamp on the cannula to allow the left atrium to fill.Once de-aired, use the previously placed sutures and tourniquet snares to close the left atrium opening completely. Adjust the cannula and snares as needed to minimize fluid leakage. After securing the cannula in the left atrium, clamp the line from the oxygenator to the windkessel bag to completely stop retrograde perfusion to the aorta.Finally, move the adenosine drip from the line leading to the windkessel bag to the line between the loading reservoir and the atrial port. Vascular resistance, oxygen uptake rate, lactate accumulation, glucose consumption, and potassium accumulation showed no statistical difference throughout the perfusion period. Similarly, no difference was seen in the weight gained by the grafts.However, failing grafts heart rate steadily declined after two hours of loaded time with the area under the curve showing no statistically significant difference until three hours of perfusion. Additionally, loading dependent metrics suggested a loss of cardiac function as early as 30 minutes into loading time with the measure for contractility being the first metric to indicate a loss of function. Like heart rate, left ventricular pulse pressure steadily declined after two hours of loaded perfusion with statistically significant differences between grafts, pulse pressure, and relaxation seen after 1.5 hours of perfusion.

276 Views.  Massachusetts General Hospital, Harvard Medical School.  The protocol describes a porcine ex vivo heart perfusion system in which direct loading of the left ventricle may serve as an assessment technique for graft health while simultaneously providing a holistic evaluation of graft function. A discussion of the system design and possible assessment metrics is also provided.

Enhanced Cardiac Assessment in Ex Vivo Perfusion with Left Ventricular Loading

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