Normothermic ex-SITU organ perfusion and assessment system

Abstract

A system and method for prolonged organ preservation and real-time ex-vivo organ assessment. The organ perfusion and assessment system includes three components: A full cardiac output circuit, a resuscitation and low flow circuit, and an organ indexing stack to perform real-time hemodynamic ex-vivo organ assessment. A containment vessel maintains sterility and allows for complete echo-analysis. The slope of preload recruitable stroke work (PRSW) and other load-independent and load-dependent assays are used to stratify donor heart function. The best assessment of systolic function and therefore transplant suitability appears to be PRSW; the best systolic function is seen with full support of a donor heart. The best assessment of diastolic dysfunction appears to be Tau; the best diastolic function is also seen with full support of a donor organ. Load independent assessments appear to be highly repeatable and should identify currently discarded human donor hearts that are actually suitable for transplantation.

Description

Docket No. 30275 / 70725UM2025-025-02NORMOTHERMIC EX-SITU ORGAN PERFUSION AND ASSESSMENT SYSTEMSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under 5R01 HL161139 - 03 awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority benefit, under 35 USC § 119(e), to US Provisional Application No. 63 / 696,710, filed September 19, 2024, the entirety of which is hereby incorporated by reference.FIELD

[0003] This disclosure relates generally to organ transplant procedures and, more specifically, to a system and method for maintaining viability of an organ ex vivo and assessing the condition of the organ in real time.BACKGROUND

[0004] There is a severe shortage of donor organs for transplantation. In the case of particular organs, including the heart, the donor must be deceased before the organ can be harvested for transplant. Often several organs will be harvested from the same donor, and unless the heart and lungs are procured as a single block for transplantation in the same recipient, which is extremely rare, lung procurement necessitates leaving the pulmonary veins intact and in continuity with the rest of the lung tissue. As such, when the lungs are harvested, all four pulmonary veins, which in vivo normally drain into the left atrium, are typically removed from the left atrium. This creates a large defect in the left atrium.

[0005] While efforts have been made to prolong viability of organs ex vivo, these efforts have heretofore suffered from a lack of real-time diagnostics of the organ ex vivo.

[0006] Death is declared either by circulatory criteria (DCD) or determination of brain death (DBD). Currently, the vast majority of hearts for transplantation are from DBD donors because heart function is known immediately prior to procurement. This effectively serves as a constraint on the size of the pool of viable donor hearts, as most deaths are pronounced by circulatory criteria (DCD). Potential donor hearts are declined (according to information from organ procurement organizations (OPO’s) such as the United Network for Organ Sharing (UNOS) and Organ Procurement Transplantation Network (OPTN)) for reasons such as neurological function (related to concerns of a prolonged dying process andDocket No. 30275 / 70725UM2025-025-02 organ preservation), prolonged warm ischemic time, and other concerns over organ preservation.

[0007] Our analysis of DCD and DBD heart transplant rates and characterized organ refusal using UNOS and OPTN data indicated DCD hearts were declined 3.37 times more often than DBD hearts. In 2022, 92% (1 ,329 / 1 ,452) of all DCD refusals in the United States were attributed to neurological function. UNOS refusal codes analysis demonstrates that the primary reason for discarding both DBD and DCD hearts is concern over hemodynamic dysfunction following transplantation. However, most donor hearts are discarded with no hemodynamic assessment of function.

[0008] Following DBD, donor heart function can be assessed while the heart is still in the donor. This is why DBD donor hearts are considered ideal for transplantation. The heart function is known so the donor heart is simply procured and transplanted. Further assessment is not required. However, many marginal DBD hearts are refused and subsequently discarded with incomplete assessment and / or no attempt to improve function. Following death, DCD donor heart function can be assessed 1) while the heart is still in the donor, 2) ex vivo (aka ex situ), or 3) after transplantation. Currently, the first hemodynamic assessment of donor heart function following DCD donor heart transplantation is typically performed after the heart is already in the recipient.

[0009] When the first assessment of DCD donor heart hemodynamic function will occur following transplantation, recipient surgeons are understandably cautious and the American Association for Thoracic Surgery (AATS) consensus recommends that the functional warm ischemic time (FWIT), the time from a substantial drop in blood saturation or blood pressure until reperfusion of the donor heart, should be 30 minutes or less. This serves to maintain the current strict DCD donor criteria and contributes to the low DCD heart transplantation rate (5%). Normothermic regional perfusion (NRP) allows assessment of donor heart function following death by using cardiopulmonary bypass or extracorporeal membrane oxygenation to resuscitate the heart and restore cardiovascular function while the heart is still in the donor. Donor heart hemodynamic function can then be evaluated after death but prior to transplantation. Results have been promising. However, there are significant ethical and legal concerns surrounding restoration of donor cardiovascular function after declaration of circulatory death (DCD requires "irreversible cessation of all cardiac and respiratory function"). These concerns limit the availability of NRP and may also contribute to low DCD transplantation rates. Given the FWIT restrictions imposed on cardioplegic arrest and direct procurement where the first DCD post-mortem hemodynamic assessment of the donor heart is performed after transplantation and the ethical and legal concerns associated with NRP, itDocket No. 30275 / 70725UM2025-025-02 appears that ideally donor heart assessment should be done following procurement but prior to transplantation (Dann et al).

[0010] The best marker or parameter for assessing suitability for heart transplantation is not known. Our UNOS refusal code analysis suggests that assessments of donor heart function could identify currently discarded hearts that were actually suitable for transplantation.

[0011] Inasmuch as the heart is a pump, it stands to reason the best assessments of transplantation suitability would be hemodynamic assessments. The most sensitive, accurate, and comprehensive assessments of cardiac performance have long been known to be pressure-volume loop (PVL) derived direct measures of heart function. PVL-derived direct measurements of hemodynamic performance are direct indices of cardiac function (as opposed to surrogates, such as lactate levels, blood pressure, oxygen consumption, coronary vascular resistance, or FWIT’s). PVL-derived direct measurements of hemodynamic performance would be ideal for assessing hemodynamic function and therefore transplantation suitability.

[0012] Clinically, PVL indices are rarely acquired because it requires a prolonged invasive procedure to acquire them and the loading conditions (heart filling pressures, blood pressure, volume status, patient position, etc.) and inotropic state (endogenous or therapeutic chemical milieu) almost continuously change and therefore change the PVL derived indices. PVL indices result from both the intrinsic and extrinsic properties of the heart. Intrinsic properties are unique to each individual heart and are therefore ideal for comparing one heart to the next and for determining donor heart suitability for transplantation. Extrinsic properties influence cardiac function but are not specific to each heart. They include preload, afterload, and endogenous and exogenous inotropic milieus. Unfortunately, intrinsic properties are routinely overshadowed by extrinsic properties. It appears that load-independent indices such as preload recruitable stroke work (PRSW) and Tau are required for assessment of intrinsic properties. Ideally, these are acquired ex situ. NEHP in a full cardiac output working mode offers complete control of loading conditions that cannot be reliably accomplished in situ.

[0013] Most PVL indices and other measurements of heart function are loading conditiondependent and all are affected by inotropic state. PVL-derived measurements typically require very invasive procedures such as puncturing the apex of the heart or cannulation of the carotid artery and crossing the aortic valve with a stiff catheter. Therefore, their use is largely limited to small animal research in non-recovery models. Transcatheter intracardiac lines may also introduce infection or damage intracardiac structures, such as the donor heartDocket No. 30275 / 70725UM2025-025-02 valves. Obviously, those techniques are not appropriate for human donor hearts that are suitable for transplantation.

[0014] Existing organ perfusion systems used for maintaining a heart ex-vivo use pumps to actively infuse perfusate into the aorta (Langendorff mode) or the left atrium. These existing systems present several disadvantages. For instance, active flow-controlled systems that use a pump can force blood down the coronary arteries. This can result in overwhelming of the microvasculature of the coronary bed, prevent coronary autoregulation, rupture the microvasculature, and increase myocardial edema. This problem is especially prevalent in Langendorff mode when there is no circulatory system simulation. It can also occur in working heart (atrial infusion) systems when the afterload (systemic circulation resistance) cannot be adequately controlled. Forcing blood down the coronaries and failure to allow autoregulation can cause gross hemorrhage in the myocardium and rapid deterioration of cardiac function.

[0015] Using pumps to pressurize the atrial inflow is also problematic. Pump-driven infusion into the atrium can lead to the appearance of a functioning heart even though the heart is obviously dead as seen via echocardiogram. In other words, the pump itself creates a cardiac output as measured by aortic flow, even though the heart is not pumping any blood itself. In this situation, echocardiogram shows that the hearts inflow and outflow valves are always open, the heart does not contract sufficiently to contribute to cardiac output and, when the pump is turned off, there is no forward flow. Therefore, the pump has the potential to contribute to cardiac output, create erroneous measurements, and thereby deceptively make it appear the heart is better than its actual condition.

[0016] Donor heart procurement typically requires that the heart is arrested. Although hearts can be procured without cardioplegic arrest (Krishnan A., Kasinpila P., Wang H., Ruaengsri C., Shudo Y., Jackson E., Woo Y.J., First-in-human beating-heart transplant. JTCVS Tech. 2023 Mar 2; 19:80-85. Doi: 10.1016 / j.xjtc.2023.02.015. PMID: 37324334; PMCID: PMC10267812), it is technically burdensome and not typically performed. Regardless of the techniques used for procurement, surgical removal of the heart is associated with the suboptimal milieu of the donor blood and the production of microscopic and macroscopic surgical debris (also referred to as procurement waste products), which tend to impair donor heart function.

[0017] Open rigid reservoirs are typically used for cardiopulmonary bypass and other perfusion systems. Open rigid reservoirs necessitate an air-blood interface which is known to be detrimental to the perfusate. Current Langendorff and artificial circulatory systems have chambers that include air-perfusate interfaces.Docket No. 30275 / 70725UM2025-025-02

[0018] The above-referenced shortcomings of conventional organ perfusion and assessment systems and methodologies limit the potential transplantation of viable organs into a wider pool of recipients.SUMMARY

[0019] In view of our observations and analysis, we determined that there exists a critical need for reliable real-time, minimally- or non-invasive, hemodynamic organ assessment ex- vivo. \Ne developed, and disclose herein, a full cardiac output (CO) normothermic ex vivo (ex situ) heart perfusion (NEHP) coupled with an organ indexing system (OIS), referred to herein as an organ perfusion and assessment system, that can completely control inotropic states and acquire all direct indices across the full spectrum of loading conditions that may be encountered in a transplant recipient, and control and sustain loading conditions at any point in that spectrum.

[0020] To achieve this, an artificial circulatory system was developed and constructed, capable of handling full cardiac output, with direct indices of hemodynamic function. As direct indices of hemodynamic function are only accurate when heart valve function is preserved, the system advantageously facilitates real-time non-invasive epicardial echocardiography. However, sustained organ submersion in fluid suitable for epicardial echocardiography can result in epicardial edema, which could interfere with heart function and compromise visual assessment of heart function, as well identification of bleeding of the ex-vivo organ that may require surgical intervention. We therefore constructed a pericardial fluid infusion system that allows filling of the donor heart vessel with sterile, normothermic fluid for the duration of an echocardiogram and subsequent draining of the fluid from around the heart following echocardiography.

[0021] Identifying optimal suitability for transplantation requires direct comparison of one potential donor heart to another. Stratification of hearts may also allow suboptimal recipients who currently do not qualify for transplantation to acquire suboptimal hearts that may not be suitable for the best recipients but are in fact suitable for those will life expectancy limited by age, such as potential recipients over the age of 65 years.

[0022] We determined that preload recruitable stroke work and exponential decay of differential change in pressure (dP / dT) during isovolumic relaxation (“Tau (T)”), which are the indicators that are most loading condition agnostic of known indicators, are the best indicators of systolic and diastolic function, respectively. As an artificial circulatory system, the organ perfusion and assessment system of the present disclosure facilitates measurement of stroke volume, afterload, stroke work, and atrial filling pressures, and end systolic and end diastolic pressures. This, combined with echocardiography, allowsDocket No. 30275 / 70725UM2025-025-02 computation of all other direct indices including overall cardiac efficiency and documentation of valvular function, and retains the ability to implement all preservation techniques, including cold, sub-thermic, and normothermic preservation, or a combination thereof, without requiring ventricular puncture or traversal of the aortic valve.

[0023] An artificial circulatory system for maintaining and assessing an organ ex vivo includes a preload controller, a vessel for containing the organ, and an afterload simulator. The vessel for containing the organ includes a containment vessel top, which has a plurality of apertures therethrough and a sealing membrane associated with each of the apertures, and a base. The base of the vessel defines a fluid-tight chamber having a mouth at a first end thereof that is selectively engageable with the containment vessel top, and the base is provided with an inflow / outflow port.

[0024] The organ perfusion and assessment system of the present disclosure includes an improved perfusion circuit that overcomes problems associated with existing organ perfusion systems which use pumps to infuse perfusate into the aorta (Langendorff mode) or the left atrium. In Langendorff mode, aortic inflow is pressure controlled and not volume controlled. Myocardial perfusion is facilitated by normal perfusion pressure, coronary vascular autoregulation is preserved, and blood is never forced down the coronaries. Similarly, in all working heart modes, the preload controller is never pressurized - atrial filling is passive and not pump driven.

[0025] In one embodiment, gravity fed preload control is used, and the afterload simulator, also referred to herein as an afterload chamber, is always higher than the preload chamber. This combination ensures that the artificial circulatory measurement system never contributes to cardiac output. If heart function is declining it will immediately be recognized even if echocardiography is not immediately available. The artificial circulatory system must not overwhelm autoregulation or contribute to forward flow in any mode. Servoregulation is important to optimize organ preservation, and contributes to reproducible assessments of organ function, safety, and ease of use.

[0026] The organ indexing system of the present disclosure is operated in parallel with the maintenance perfusion system. Isolation of these systems allows for low priming volume resuscitation of the donor heart, perfusate exchanges, ease of organ treatment, storage, and transportation, implementation of various preservation techniques, and permits intermittent full cardiac output organ assessment.

[0027] Also disclosed herein is a technique for perfusate exchange that allows for the removal of the initial perfusate that contains waste products and replacement with fresh perfusate that does not. This removes the majority of procurement waste products.Docket No. 30275 / 70725UM2025-025-02

[0028] Because full cardiac output (CO) mode requires both an afterload simulator and a preload controller, larger priming volume is required as compared to the initial Langendorff resuscitation mode. Exposing the full CO circuit to the donor heart immediately after procurement would contaminate the entire circuit with waste products and necessitate a full circuit perfusate exchange to eliminate waste products.

[0029] A dual parallel circuit design, disclosed herein, allows the larger full CO prime to be isolated from the circuit when the donor heart is initially connected. This avoids contamination of the larger full CO circuit with waste products. The full CO circuit is not exposed to the donor heart until after perfusate exchange has been performed.

[0030] The dual reservoir systems of the present disclosure employ passive infusion and replication of physiologic afterload. Utilizing this passive system allows for non-invasive titration of pre-load and after-load conditions to replicate diverse recipient physiology for targeted hemodynamic assessment.

[0031] The systems of the present disclosure exclusively use closed collapsible venous reservoirs for both preload control and afterload simulation. These can be completely deaired and maintained free of air even if air is inadvertently introduced.

[0032] Collapsible reservoirs are very compliant and allow frequent or continuous air removal to minimize the risk of pumping air into the coronary arteries. Pumping air into the coronary arteries effectively blocks blood flow and can result in myocardial ischemia and terminal injury to the donor organ. Afterload simulation chambers that allow for adjustment of the afterload across the full spectrum that might be encountered in the recipient must be nearly rigid. If afterload compliance needs to be increased it should be done with a separate sterile chamber with little or no air-fluid interface. Ideally, the afterload simulator should have very low priming volumes. Small priming volumes require that changes in afterload volume status be quickly recognized and corrected to optimize index assessments.

[0033] The systems of the present disclosure utilize a very flat, non-compliant low-volume afterload chamber. This chamber is rigid, contains no air-perfusate interface, can be continuously de-aired, and allows for rapid identification of incomplete filling. It can be directly connected to an adjustable compliance chamber.

[0034] As a result of the typical removal of all four pulmonary veins from the left atrium when lungs are harvested from a donor, a large defect is imparted to the left atrium. This defect prevents left atrial infusion and assessment of cardiac function in all left-heart working modes. To address this issue, the system of the present disclosure may be provided with a left atrial infusion dome that can be sewn in place to effectively reconstruct the atrial wall ofDocket No. 30275 / 70725UM2025-025-02 the donor heart and permit sterile access to the left heart chambers for perfusate infusion, pressure monitoring, and transcatheter assessments.

[0035] The organ containment vessel is designed to support a suspended or semisuspended heart while allowing access with necessary cannulas for perfusate infusion and pressure monitoring. The containment vessel additionally allows for ease of echocardiography for assessment of the heart function during full cardiac output assessment, by virtue of the vessel being constructed of optically-transparent, seamless walls.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is a perspective view of an organ containment vessel of an organ perfusion and assessment system of the present disclosure;

[0037] FIG. 2 is a top view of the organ containment vessel of the organ perfusion and assessment system of the present disclosure;

[0038] FIG. 2A is a cross-sectional view of a containment vessel top, taken along lines 2A-2A of FIG. 2;

[0039] FIG. 3 is an exploded view of the organ containment vessel of the organ perfusion and assessment system of FIG. 1 ;

[0040] FIG. 4 is a top perspective view of a left atrial dome, for use in conjunction with a donor heart which may be used to compensate for a defect in the left atrium of the donor heart resulting from removal of all four pulmonary veins when lungs are harvested for transplant separately from the donor heart;

[0041] FIG. 5 is a top perspective view of the left atrial dome of FIG. 4, as sewn into place in a donor heart;

[0042] FIG. 6 is a perspective view of a donor heart, suspended by the aorta, from the containment vessel top of the organ perfusion and assessment system of the present disclosure;

[0043] FIG. 7 is a semi-schematic illustration of the organ perfusion and assessment system of the present disclosure, operating in a Langendorff mode with hemofiltration, in which a full cardiac output circuit is recirculating to prevent stagnant perfusate;

[0044] FIG. 8 is a semi-schematic illustration of the organ perfusion and assessment system of the present disclosure, with hemofiltration, and operating in an intermittent or continuous low flow working heart (coronary flow working heart) mode, in which a full cardiac output circuit is recirculating to prevent stagnant perfusate;Docket No. 30275 / 70725UM2025-025-02

[0045] FIG. 9 is a semi-schematic illustration of the organ perfusion and assessment system of the present disclosure operating in full cardiac output mode, in which a resuscitation / low flow working heart circuit is recirculating to prevent perfusate;

[0046] FIG. 10 is a semi-schematic illustration of the organ perfusion and assessment system of the present disclosure capable of working in all modes;

[0047] FIG. 11 is a plot of preload recruitable stroke work (PRSW) over time (t), for potential donor porcine hearts receiving minimal support, partial support, and full support, illustrating how differences in slopes of PRSW / t can be used in stratification of systolic function of different donor hearts;

[0048] FIG. 12 is a plot of PRSW / t, for potential donor porcine hearts receiving minimal support, partial support, and full support, similar to FIG. 11 , plus a plot of Tau against time, illustrating Tau stratification of diastolic dysfunction and preservation of potential donor hearts with full support;

[0049] FIG. 13 is a collection of plots of PRSW / t and Tau at 24 hours, with an illustration of a potential donor heart connected to the organ perfusion and assessment system of the present disclosure, summarizing an indication of preliminary results that preload recruitable stroke work slope must be greater than 38° for transplant suitability;

[0050] FIG. 14 is a collection of plots of PRSW / t and Tau at 24 hours, summarizing an indication that in utilization of the organ perfusion and assessment system of the present disclosure, load-independent assessments may be used to identify currently discarded potential donor hearts that are actually suitable for transplant;

[0051] FIG. 15A is a semi-schematic illustration of the organ perfusion and assessment system of the present disclosure, illustrating overall circuit design and crystalloid-based (saline fluid based) primed circuit prior to addition of blood-based perfusate;

[0052] FIG. 15B is a semi-schematic illustration of the organ perfusion and assessment system of the present disclosure, in a Langendorff mode of operation;

[0053] FIG. 15C is a semi-schematic illustration of the organ perfusion and assessment system of the present disclosure, in a Working Heart Coronary Flow Only mode of operation

[0054] FIG. 15D is a semi-schematic illustration of the organ perfusion and assessment system of the present disclosure, in a Full Cardiac Output Working Heart mode of operation;

[0055] FIG. 16 is a semi-schematic illustration of the full cardiac output normothermic ex situ heart perfusion (NEHP) and organ indexing system (OIS) assessment of the present disclosure;Docket No. 30275 / 70725UM2025-025-02

[0056] FIG. 17A is a plot of Preload Recruitable Stroke Work, reflecting systolic function after 1 , 12, and 24 hours for porcine hearts having received minimal support (MS), partial support (PS), and full support (FS);

[0057] FIG. 17B is a plot of Tau, reflecting diastolic dysfunction after 1 , 12, and 24 hours for porcine hearts having received minimal support (MS), partial support (PS), and full support (FS);

[0058] FIG. 18A is a collection of plots of load-independent indices, including Preload Recruitable Stroke Work and Tau, and load-dependent indices, including Cardiac Index and Maximum dp / dt, for porcine hearts having received minimal support (MS), partial support (PS), and full support (FS);

[0059] FIG. 18B is a plot of recipient hemodynamic trends following transplantation, graphically displaying systolic blood pressure as a function of time for transplanted porcine hearts having received partial support (PS) and full support (FS); and

[0060] FIG. 19 is a collection of plots of PRSW / t and Tau at 24 hours, similar to FIG. 14.DETAILED DESCRIPTION

[0061] With reference to the accompanying drawings, an organ perfusion and assessment system 10 of the present disclosure provides an artificial circulatory system, capable of handling, in the case of a donor heart DH, full cardiac output, with direct indices of hemodynamic function. The system 10 includes a containment vessel 20 in which the organ, such as the donor heart DH, is received. The containment vessel 20 includes a containment vessel top 22 having a generally planar portion with a plurality of apertures 25 therein, and an annular wall depending downwardly from the generally planar portion. As best illustrated in FIG. 2A, immediately beneath the generally planar portion of the containment vessel top 22, a silicone sealing disk 24 is provided, which is held firmly in place within the annular wall of the containment vessel top 22 by an acrylic disk 26. A containment vessel cylinder 30 extends downwardly from the containment vessel top 22. All cannulas, ECG leads, and pacing wires (if needed) are introduced through the apertures 25 in the containment vessel top 22.

[0062] The organ, such as the donor heart DH, is received in a sterile and compliant containment vessel base 32, having an infusion / drainage port, referred to herein as an inflow / outflow port 34, at or near a bottom thereof. The containment vessel base 32 is secured to, and suspended from, the containment vessel cylinder 30, such as by a vessel base clamp ring 36. The flexible containment vessel base 32 is easily and quickly removed from the containment vessel cylinder 30 and containment vessel top 22 if sterile surgicalDocket No. 30275 / 70725UM2025-025-02 access to the donor heart DH is required. The flexible containment vessel base 32 may then be easily reconnected following intervention. A new sterile containment vessel base 32 can be used if it is required to preserve sterility.

[0063] The containment vessel base 32 is optically and acoustically transparent, and is preferably seamless, such that when filled (via the inflow / outflow port 34) with an acoustically conductive fluid to a sufficient depth to submerge the donor heart DH in the fluid, the donor heart is surrounded with pericardial (or other suitable) fluid so that direct epicardial contact with the heart is avoided. It is possible to reliably perform analysis by echocardiography on the ex-vivo donor heart DH. The fluid may be a synthetic perfusate, a natural perfusate, or a mixture. The fluid may even be pericardial fluid. However, it is recognized that prolonged submersion of an organ ex-vivo result in epicardial edema and other problems, such as interference with heart function, compromised visual assessment of heart function, and diminished ability to identify bleeding of the ex-vivo organ that may require surgical intervention. The inflow / outflow port 34 can therefore be used to drain the fluid from around the donor heart DH following echocardiography. The inflow / outflow port 34 port can also be used to capture and rapidly reinfuse perfusate into a circuit if significant bleeding occurs.

[0064] Advantageously, the donor heart DH may be assessed ex-vivo, from 360°, at any time there is access to echocardiography equipment, an important aspect of hemodynamic ex-vivo organ assessment.

[0065] In an effort to determine which direct indices are optimal for comparing one potential donor heart to another and thereby stratify hearts to determine transplantation suitability, it is necessary to preserve and assess full heart valve function ex-vivo.

[0066] We used our full cardiac output organ perfusion and assessment system to acquire the direct indices in 3 donor hearts at 3 times over 24 hours. At each time, numerous echocardiograms and indice assessments were performed over a variety of controlled loading conditions and controlled inotropic states. Each of the 3 hearts received different management protocols to stratify cardiac performance.

[0067] The organ perfusion and assessment system must be tested against known controls. The industry standard for currently assessing function and durability for human artificial heart valves is called a pulse duplicator (aka artificial heart) that is calibrated with its own Windkessel (aka artificial circulation) system. Current industry standard Windkessel systems are not set up to measure PVL derived direct indices in ex vivo hearts. They are simply set up to confirm that their pulse duplicators are generating the digitally programed cardiac performance parameters. We have contacted and evaluated a number of companies that currently manufacture pulse duplicators for assessment of human valve prosthesis. ToDocket No. 30275 / 70725UM2025-025-02 the best device of our ability to determine, the best pulse duplicator and accompanying Windkessel device for our purposes is made by a Canadian company called ViVitro. Their pulse duplicator can be digitally programmed to simulate a broad spectrum of hemodynamic cardiac function and validate it through their Windkessel device. We have already some of their components.

[0068] Turning to FIGs. 4 and 5, it is recognized that when the lungs and left pulmonary veins are removed from the left atrium, a defect remains in the left atrium of a potential donor heart. To address this issue, a left atrial infusion dome 40 is provided, which includes an atrial dome base 42 that may be sewn in place in the left atrium of the donor heart DH, effectively reconstructing the atrial wall and permitting sterile access to the left heart chambers of the donor heart DH for perfusate infusion, pressure monitoring, and transcatheter assessments. The left atrial infusion dome further includes a left atrial infusion connector 44, a transcatheter access port 44, and a left atrial pressure port 46, extending outwardly from, and in fluid communication with an interior of, the atrial dome base 42.

[0069] As illustrated in FIG. 6, the donor heart DH is suspended, via the aortic arch, from the containment vessel top 22. A perfusion circuit for the organ perfusion and assessment system of the present disclosure employs an aortic cannula 52, in fluid communication with an aortic low flow resuscitation connector 52a, an aortic high flow full cardiac output connector 52b, and a pressure monitoring port 52c. Also provided is a left ventricular vent cannula 54, in fluid communication with a pulmonary vein (left atrial) infusion cannula with low flow connector 54a, a pulmonary vein (left atrial) high flow connector 54b, and a pressure monitoring port 54c. Additionally, a pulmonary artery cannula 56 and a left ventricular vent 58 are provided, as well as a pulmonary arterial branch outflow vent (or PA outflow cannula) 60 for pressure monitoring.

[0070] The perfusion circuit for the organ perfusion and assessment system of the present disclosure can operate in a variety of modes. For instance, the system may be operated in a Langendorff mode, as illustrated semi-schematically in FIG. 7. In this mode, perfusate is infused through the aortic cannula 52, the heart is in a non-working, resting mode, and a pump P dictates coronary blood flow.

[0071] As illustrated semi-schematically in FIG. 8, the perfusion circuit for the organ perfusion and assessment system of the present disclosure may operate in an intermittent working heart mode, with hemofiltration, in the range of 1 mL / h / gm cardiac tissue. In this mode, the aortic cannula 52 is occluded so all cardiac output is into the coronaries. A left ventricular vent monitors pressure, a right ventricular cannula drains the coronary sinusDocket No. 30275 / 70725UM2025-025-02 return. A left atrial cannula infuses perfusate in this working heart mode, and left atrial pressure is also monitored.

[0072] As illustrated semi-schematically in FIG. 8, the perfusion circuit for the organ perfusion and assessment system of the present disclosure may also operate in a coronary flow only working heart mode. It may be noted that this circuit is similar to that of a Langendorff mode, but with an extra circuit branch added. As illustrated semi-schematically in FIGs. 8 and10, the perfusion circuit for the organ assessment system of the present disclosure may also operate in a coronary flow working heart mode, under which there is partial working heart, not full cardiac output, a pump P dictates coronary blood flow. It is noted that this mode may be less favorable, as it is not conducive to direct indices of organ function, and ability to obtain reliable echocardiogram scans is limited.

[0073] As illustrated semi-schematically in FIG. 9, the perfusion circuit for the organ perfusion and assessment system of the present disclosure may also operate in full cardiac output (CO) working heart mode. This involves the larger full circuit and allows full assessment of the donor heart under the conditions that it will encounter in the recipient. The heart is primarily a pump. PV loop derived indices are considered the “gold standard” for hemodynamic assessment of heart function. Therefore, assessment of hemodynamic function is essential for determining transplantation suitability. Unlike in vivo (in situ) assessments such as normothermic regional perfusion (NRP - donor heart is still in the donor), during normothermic ex-vivo heart perfusion (NEHP) a broad spectrum of loading conditions can be created and maintained and the inotropic state (both endogenous and therapeutic) can be completely controlled. The full CO circuit configuration is ideal for acquiring all of the PV loop derived indices including loading condition independent preload recruitable stroke work (PRSW) and Tau (exponential decay of dP / dT during isovolumetric relaxation). Of equal importance, complete echocardiography can be obtained in full working heart mode across a broad spectrum of loading conditions. Again, complete echocardiography can only be obtained in full CO working heart mode. Ex vivo hemodynamic assessment avoids the ethical or legal issues associated with NRP.

[0074] Current clinical ex situ extracorporeal machine perfusion is limited to heart reanimation and basic metabolic assays and cannot perform hemodynamic assessments. Hemodynamic assessment of donor heart suitability for transplantation immediately prior to transplantation should relax donor selection criteria, facilitate identification of currently discarded hearts that are suitable for transplantation, and thereby substantially increase the number of hearts available for transplantation. Using PV derived direct indices, especially the load independent assessments PRSW and Tau, in a normothermic ex vivo heart perfusion system that replicates the recipient circulatory system allows optimal stratificationDocket No. 30275 / 70725UM2025-025-02 of donor heart function and therefore the ability to compare the donor heart to known indices that predict transplantation suitability. This can only be accomplished in full CO working heart mode as described. All other assessments are simply surrogates.

[0075] The organ perfusion and assessment system of the present disclosure may advantageously be configured, as illustrated semi-schematically in FIG. 10, to selectively operate in any one of a variety of modes, such as Lagendorff (a conventional aortic perfusion mode where perfusate flows directly into the coronary arteries), low flow (or low volume), and high volume, full cardiac output modes, referred to herein working heart or working mode perfusion, including left atrial perfusion. A passive (non-pump driven) preload control 70 is provided upstream of, and in fluid communication with, the containment vessel 20, with a caval simulator 72 intermediate the preload control 70 and the aortic cannula 52. An afterload simulator 74 is provided downstream of the containment vessel 20.

[0076] The afterload simulator 74 includes a priming volume sterile container 76, which is preferably flat and optically transparent. The priming volume sterile container 76 may be constrained between transparent flat plates 78, 80 to maintain non-compliance (rigidity). A lower end region provided with at least one inflow port and at least one outflow port 82. An air removal port 84 may also be provided in a top region of the afterload simulator 74. The afterload simulator 74 may further include an adjustable rigid diaphragm 86. The afterload simulator 74 may also be provided with a variable resistance regulator.

[0077] A dual loop architecture, also referred to herein as a dual parallel circuit design, allows small prime prior to perfusate exchange, as well as a full cardiac output, and pressure-volume loop indexing. Perfusate exchange is desirable, as it promotes removal of residual cardioplegia, surgical debris, and other potential toxins following resuscitation. A full cardiac output afterload simulator 90 is provided with a collapsible venous reservoir, which may be secured, for instance on an IV pole (not shown), at a height above a level of the preload control. A Hoffman clamp can be used to control systolic blood pressure, and a compliance chamber is provided for controlling diastolic blood pressure.

[0078] A thermal control unit 100, such as a heat exchanger, may serve as a heater / cooler to maintain the fluid in the circuit at the desired temperature. For instance, if it is desired to operate the organ perfusion and assessment system at normothermic conditions, the thermal control unit 100 may be operated to maintain both circuits at 37°C. Alternatively, if desired to maintain a donor heart DH or other organ in a hypothermic condition, the thermal control unit 100 may be operated to lower the temperature of the circuits to the desired temperature.Docket No. 30275 / 70725UM2025-025-02

[0079] A full cardiac output compliance chamber is optically transparent (preferably made of glass or acrylic), with access via a plurality of luers / stopcocks, at least one for fluid control and one for air. The full cardiac output compliance chamber is preferably mounted in an inverted orientation and above an afterload controller.

[0080] The artificial circulatory system of the present disclosure facilitates intermittent equal volume exchange between the Langendorff circuit (used for resuscitation) and the full cardiac output circuit (used for non-invasive assessments) while maintaining perfusate composition and temperature in the two circuits. In particular, the organ perfusion and assessment system facilitates real-time full epicardial echocardiographic assessment (unlike conventional donor organ maintenance systems which, due to mechanical obstructions and / or organ orientation, lack complete circumferential acoustic access). By providing a system and methodology for real-time ex-vivo hemodynamic donor organ assessment, the organ perfusion and assessment system can prolong the time that organs, such as donor hearts, may be maintained in a viable condition, and advantageously expand the pool of viable donors and recipients.EXAMPLESExample 1

[0081] An adult porcine hemofiltration circuit was adapted to accommodate two modes of operation, namely a first beating resting NEVHP (Langendorff) mode and a second working heart mode. To convert modes of operation from the first beating resting NEVHP (Langendorff) mode to the second working heart mode, the aortic cannula was occluded so all cardiac output flows into the coronaries. A left ventricular vent was used to monitor pressure. A right ventricular cannula drained the coronary sinus return. A left atrial cannula was used to infuse perfusate in the working heart mode, and left atrial pressure was monitored.

[0082] Five adult porcine donor hearts were resuscitated in non-working heart mode and maintained ex-vivo (ex situ) for 24 hours. Three of the adult porcine donor hearts were randomly assigned to minimal support, atrial support, full support (vasoactive support and serum supplementation). At 1 hour, 12 hours, and 24 hours, pressure-volume loop (PVL) parameters were assessed and echocardiograms acquired, in full cardiac output mode across a broad loading spectrum. Two of the adult porcine hearts in the partial support group were assessed at 24 hours for transplant with load dependent parameters. All PVL data was acquired. All assessments were blinded to preload recruitable stroke work (PRSW) and Tau (exponential decay of dP / dt during isovolumic relaxation made loading condition independent).Docket No. 30275 / 70725UM2025-025-02

[0083] Results of this series of tests are illustrated graphically in FIGS. 11-14. Load independent assays of both systolic (preload recruitable stroke work (PRSW)) and diastolic (Tau) function were observed to be suitable for objectively comparing potential donor hearts, and are therefore considered to be ideal for determining donor heart transplant suitability. PRSW was used to stratify systolic function at times of 1 hour, 12 hours, and 24 hours. Tau was used to stratify diastolic dysfunction, and the best diastolic function was observed for the donor hearts that received full support. In analyzing the PRSW slope data from the donor hearts, it was determined that in this model a PRSW slope of above 38° is necessary for transplant suitability.

[0084] All adult porcine hearts that received full support ex situ, as well as those adult porcine hearts that received partial support ex situ, were functional at 24 hours. The adult porcine hearts that received minimal support developed gross valvular incompetency and a refractory / terminal wide-complex arrythmia. Donor adult porcine hearts on partial support were assessed for transplant (Tx) after 24 hours. For hearts with a preload recruitable stroke work (PRSW) slope of 38° or less, poor cardiac output and echocardiogram results, no transplant attempts were made. For donor adult porcine hearts with a PRSW slope of 37°, although cardiac output and echocardiography results were satisfactory, transplant attempts failed. Based on these tests, we concluded donor heart function can be reproducibly stratified using PV loop derived parameters. PRSW and Tau are load independent and appear optimal for use in stratifying potential donor hearts, and thereby provide a sound basis for determining transplantation suitability, while load dependent assessments may be unreliable. While all model hearts were initially identified as transplant suitable, those without full support became unsuitable over 24 hours.Example 2

[0085] A novel NEHP system was constructed using two circuits in parallel: a clinically equivalent Langendorff maintenance circuit (MC) and a full cardiac output circuit (FCOC) with passive left atrial preload infusion and an afterload chamber with adjustable systemic vascular resistance and compliance. Following baseline in situ echocardiography, three porcine donor hearts were procured, canulated, suspended in an acoustically optimized containment vessel, resuscitated, and maintained using our 24-hour NEHP model. At hours 1 , 12, and 24, perfusion was transitioned to the FCOC with atrial preload pressures 6-12 mmHg, and afterload adjusted to replicate physiologic LV conditions. The containment vessel was filled with synthetic pericardial fluid to facilitate imaging. A short-axis view was first obtained to establish ex situ orientation based on identification of the papillary muscles. ASE directed assessment was completed in the following sequence: SAX, LAX, A4C, A5C, A2C, and A3C. The mitral and aortic valves were assessed with color flow Doppler andDocket No. 30275 / 70725UM2025-025-02 pulsed and continuous wave Doppler, and LV function was assessed with tissue Doppler imaging in the appropriate guideline directed views. Following completion of echo assessments, the containment vessel was drained, and perfusion was transitioned back to the MC.Example 3

[0086] Porcine hearts were procured and maintained in Langendorff mode. Coronary perfusate flow rate was maintained at 0.5-0.6 cc / g of cardiac tissue / min. Perfusate electrolyte balance was maintained with 22 kDa hemofiltration and cc / cc replacement. Hearts were randomized to: minimal support (MS, perfusion and hemofiltration only); partial support (PS, hemofiltration and vasoactive infusions); or full support (FS, hemofiltration, vasoactive infusions, and plasma infusions). At NEHP hours 1 , 12 and 24, hearts were transitioned to full CO for 30 minutes using passive left atrial infusion and were exposed to 3 pre-determined preload pressures (2-4, 6-10, and 12-22 mmHg). Aortic pressures and vascular compliance were adjusted to replicate normal afterload physiology. Data collection included continuous flow (atrial, aortic, coronary outflow) and pressure monitoring (atrial, ventricular, aortic), and echocardiography. Donor hearts procured for transplantation were managed using the above protocols. Standard bicaval techniques were used for implantation. Transplantation success criteria required cardiopulmonary bypass (CPB) cessation within 2 hours of allograft reperfusion and subsequent hemodynamic for 4 hours off CPB. Early end criteria included refractory arrythmias, asystole, and mechanical arrest.

[0087] Results: Five adult porcine donor hearts were procured (58.1 ± 3.6 kg). At 24 hours, the MS heart developed valvular incompetency, pressure equalization, and dysrhythmias refractory to intervention. The PS and FS hearts all remained functional. When compared to the PS group, the FS group (n=2) demonstrated a trend towards greater inotropic preservation by cardiac index, Dp / Dt max, and PRSW at hour 24. The FS group demonstrated improved lusitropy (Tau) when compared to PS at hour 24. Comprehensive validation by transplantation of our graduated NEHP series was completed separately. The MS hearts were not viable at hour 24 by any metric and therefore transplantation was not attempted. The PS (n=3) group developed severe arrythmias following implantation, 2 could not be weaned from CPB and 1 had an asystolic arrest refractory to pacing immediately following discontinuation of CPB. Two consecutive FS hearts remained hemodynamic stable while weaning vasoactive infusions during the entire 4-hour observation period.

[0088] Conclusions: Donor heart function can be reproducibly stratified using full CO ex situ load-independent assessments. PRSW and Tau appear to be ideal for assessingDocket No. 30275 / 70725UM2025-025-02 transplant suitability prior to implantation. These findings were validated by orthotopic transplantation.Example 4

[0089] Fifteen adult pigs (53.0 ± 5.8 kg) were anesthetized and instrumented. Hearts were procured and placed on our prolonged NEHP system. On circuit, hearts were resuscitated and maintained in Langendorff mode. Electrolytes were managed with 22 kDa hemofiltration and cc / cc replacement. Hearts were randomly assigned to one of three perfusate flow rates: 1 .5 cc / g of cardiac tissue / min ("high flow" (HF), n=5), 0.75 cc / g / min ("medium flow" (MF), n=5), and 0.25 cc / g / min ("low flow" (LF), n=5). Data acquisition included: hemodynamics Q30min, ABGs / VBGs Q1 h, antegrade LA (iLA) perfusion testing (fixed flow rate 0.75cc / g / min) Q6h, perfusate biochemical markers Q12h, echocardiography Q24h, and histopathology.

[0090] Results: Hemodynamic Performance: The MF group displayed significantly decreased aortic regurgitation (Al) compared to the LF and HF groups at hr24 (LF 12.9 ± 8.9%, MF 7.9 ± 3.4%, HF 14.0 ± 16.3%; LF vs MF p < 0.001 , LF vs HF p = 0.888, MF vs HF p < 0.023). LAEDP / LVEDP ratio during hour 24 iLA testing was >50% in 3 / 5 LF hearts, 1 / 5 ME hearts, and 3 / 5 HF hearts.

[0091] Metabolic Performance: Lactate remained significantly lower in the MF group when compared to the LF and HF groups at all time points.Example 5

[0092] Consecutive orthotopic transplants were planned with n=2 in each of four graduated preservation protocols. 1) cold static storage (CS): hearts submerged in Custodial preservation solution at 4°C 2) non-supplemented NEHP (NS): hearts maintained via NEHP implementing Langendorff perfusion with fixed flow rates of 0.6-0.75 cc / g of cardiac tissue / min and managed with hemofiltration, electrolyte replacement, vasoactive management, and q12hr perfusate exchanges 3) SF65 supplemented NEHP (SF): hearts managed per NS group with supplementation of serum fractionated to include components <65 kDa or 4) plasma exchange treated NEHP (PE): hearts managed per NS group with continuous perfusate plasma exchange. Following 24 hours of ex situ storage, hearts were implanted into adult sized pigs (50.2±5.7 kg) via bicaval technique. Post-implantation success criteria required cessation of cardiopulmonary bypass (CPB) support within 2 hours of allograft reperfusion and subsequent stabilization of arterial pressures with clinically appropriate vasopressor support for 4 hours. Post implantation end criteria included ventricular arrythmia refractory to cardioversion and / or asystole with mechanical arrest.Docket No. 30275 / 70725UM2025-025-02Recipient data collection included q15min hemodynamics, q1 hr echocardiography, and final histopathology.

[0093] Results: Eight donor hearts were procured from adult pigs (53.2±4.7 kg). There was no difference in implantation warm ischemic times across groups (CS 48±1 .4 min, NS 56±8.5 min, SF 55 min, PE 66.5±0.7 min). The CS group was characterized by echocardiographically observed flow stagnation at the left ventricular outflow tract with failure of aortic valve opening during partial CPB wean attempts. Both CS hearts developed asystole within 15 minutes of cardiopulmonary bypass (CPB) cessation. The NS group was characterized by recurrent ventricular fibrillation requiring multiple electrical cardioversions. The first SF was successfully weaned from CPB with maintenance of arterial pressures for 1 .25 hours however developed refractory ventricular fibrillation related to a Swan-Ganz catheter adjustment. The second SF heart was aborted prior to implantation due to development of a pericardial effusion with clot during NEHP. Both PE hearts were successfully weaned from CPB support within 1 .25 hours of allograft reperfusion and subsequently maintained stable arterial pressures for >4 hours before elective termination. Both PE hearts displayed decreasing vasopressor requirements during the 4-hour observation period.

[0094] Conclusions: NEHP with plasma exchange optimally preserved donor heart function for 24 hours. Suitability for transplantation following 24 hours ex situ was validated by successful consecutive orthotopic transplantations.Example 6

[0095] Animals were placed under general anesthesia prior to instrumentation. Hearts were recovered following standard pericardial preserving procurement methods as previously described. Briefly, the descending aortic, left subclavian, and distal innominate artery were occluded prior to infusion of 50 cc / kg of cold (6°C) del Nido cardioplegia (CAPS Inc., Detroit, Ml) into the coronary arteries via the proximal innominate artery. Following procurement, hearts were flushed with chilled CoStorSol preservation solution (Preservation Solutions, Elkhorn, Wl) and weighed.

[0096] All back-table preparation was performed with the heart submerged in chilled Costorsol preservation solution. A 12-Fr venous drainage cannula (Medtronic, Minneapolis, MN) connected to a pressure transducer was inserted through a right pulmonary vein and positioned in the left ventricular (LV) apex. A 20-Fr venous drainage cannula (Medtronic Inc, Dublin, Ireland) was inserted through the right pulmonary artery (PA) and positioned at the coronary sinus. A 10-Fr venous drainage cannula connected to a pressure transducer was inserted through a left pulmonary vein and positioned in the left atrium (LA). A modified 3 / 8”Docket No. 30275 / 70725UM2025-025-02 x 3 / 8” Luer Lock connector (Medtronic, Minneapolis, MN) connected to a pressure transducer was secured in the aortic root (Ao). Similarly, a modified 3 / 8” x 3 / 8” Luer Lock connector connected to a pressure transducer inserted through a left pulmonary vein and positioned superficially in the LA. All remaining branches were ligated for hemostasis.

[0097] Ex vivo Heart System, Perfusate, and Re-Animation:

[0098] The perfusion system was composed of two circuits ran in parallel: the NEHP maintenance circuit (MC) and the full cardiac output circuit (FCOC). The MC (Figure 1 A) consisted of commercially available components including a reservoir (Terumo, Ann Arbor, Ml), an FX05 Baby Capiox Oxygenator / Heat Exchanger (Terumo, Ann Arbor, Ml), an M- pump (proprietary roller-style pump, University of Michigan, Ann Arbor, Ml), and a Prisma flex HF1000 Hemoconcentrator (Baxter, Deerfield, IL). The FCOC (Figure 1A) consisted of commercially available components including a reservoir (Terumo, Ann Arbor, Ml), an Affinity Fusion Oxygenator / heat exchanger (Terumo, Ann Arbor, Ml), a Sarns Centrifugal pump (Medtronic, Dublin, Ireland), a Capiox HC05 hemoconcentrator (Terumo, Ann Arbor, Ml), and passive MVR1600 pre-load and after-load soft-shell chambers (Medtronic, Dublin, Ireland).

[0099] Donor blood was collected in CPDA-1 . Platelet and leukocyte reduction was achieved via centrifugation of whole blood at 3600 RPM at 25°C for 20 minutes followed by collection of plasma and red blood cells. Red blood cells were further leukocyte reduced with RS1 leukocyte reduction filters (Haemonetics, Boston, MA) and were washed prior to use. Perfusate was composed of 110 cc pRBCs, 90 cc plasma (target Hgb 8.5-10.5 g / dL), 250 mg solumedrol, 45 mg magnesium, 50 mg calcium (perfusate target 1.15-1 .3 mmol / L), 2,500 U heparin, 40 mg Gentamicin, and 250 mg Nafcillin.

[0100] Prior to heart connection, both the MC and FCOC were primed and de-aired with perfusate that did not contain calcium or bicarbonate supplementation. All tubing connections were made to their respective cannulas and de-aired. The inflow and outflow lines of the FCOC were occluded with tubing clamps so the hearts received prefusion from the MC only. Just prior to heart connection, 3 mg adenosine was added to the MC perfusate. Perfusate flow was initiated through the Ao cannula to achieve Langendorff perfusion at an initial coronary flow rate of 0.5 cc / g / min. Once PA return flow was achieved indicating tissue reperfusion, a 5-minute stand-off period was completed prior to progressive perfusate rewarming to 37°C and progressive bicarbonate and calcium correction to physiologic levels. After physiologic perfusate conditions were achieved and the heart displayed return of spontaneous contraction, coronary flow rates were maintained at 0.5-0.75 cc / g / min and titrated as needed to ensure SvC>2 >75%. Thirty minutes after return of spontaneousDocket No. 30275 / 70725UM2025-025-02 contraction, the perfusate was exchanged to eliminate residual cardioplegia and ischemiareperfusion toxins that may have accumulated during re-animation.

[0101] Maintenance Circuit Management and Treatment Groups

[0102] Hearts were maintained on the NEHP circuit for 24 hours via Langendorff maintenance perfusion. Coronary artery perfusion rate was measured by PA flow rate and maintained at 0.60 cc / g / min and titrated as needed to ensure SvC>2 >75%. Perfusate temperature was maintained at 37°C. Sweep gas (50% O2, 45% N2, and 5% CO2) was adjusted to maintain pCC>2 at 40 ± 5 mmHg. Hemoglobin was maintained 8.5-10.5 g / dL. A perfusate exchange was performed every 12 hours. Hemofiltration, electrolyte supplementation and glucose supplementation were titrated as needed to maintain physiologic perfusate conditions (K+3.0-5.5 mmol / L, Ca2+1.15-1.30 mmol / L, HCO3- 22-28 mEq / L, and Glucose 100-300 g / dL). Perfusate was supplemented with adenosine as needed to maintain coronary resistance (CR) < 0.5 mmHg / mL / min.

[0103] Hearts were randomly assigned to one of three treatment groups: minimal support (MS), partial support (PS), and full support (FS). The MS group was maintained on the NEHP circuit as described without additional interventions. The PS group was maintained on the NEHP circuit as described with active titrations of adenosine to maintain coronary resistance (CR) < 0.5 mmHg / mL / min, and epinephrine or dobutamine to maintain aortic insufficiency (Al) <15%. The FS group included all interventions of the PS group with the addition of serum supplementation. For the PS group, donor serum was leukocyte reduced with RS-1 leukocyte reduction filters (Haemonetics, Boston, MA) and supplemented with 250 mg / L solumedrol. Serum supplementation was initiated at a rate of 20-40 cc / hr immediately after the initial perfusate exchange.

[0104] Transition to Full Cardiac Output and Data Collection:

[0105] Assessment by full cardiac output was completed at hour 1 following perfusate exchange, hour 12, and hour 24. Flow was first transitioned to a coronary-only antegrade LA perfusion strategy (iLA perfusion)(FIG. 15C). Baseline hemodynamic measurements were acquired during iLA perfusion. The FCOC inflow chamber was positioned roughly 2 cm above the left atrium and the outflow chamber was positioned roughly 10 cm above the aorta. The FCOC aortic outflow line was unclamped quickly followed by a simultaneous clamping of the MC LA infusion line and unclamping of the FCOC LA infusion line. PA outflow was then diverted to the FCOC for completion of the transition to FCOC perfusion (FIG. 15D).

[0106] With continuous monitoring of chamber pressures, the heights of the FCOC inflow and outflow chambers were adjusted to replicate the desired inflow and afterload conditions.Docket No. 30275 / 70725UM2025-025-02Hearts were allowed to equilibrate to the FCOC for at least 10 minutes prior to data collection. Data were acquired at a low preload condition (LA pressure 2-6 mmHg), medium preload condition (LA pressure 6-12 mmHg), and a high preload condition (LA pressure 12- 22 mmHg). At each preload condition, data acquisition included continuous hemodynamic data (LA pressure, LV pressure, Ao pressure), continuous flow rate data (LA inflow, Ao outflow, PA outflow), and echocardiography. Lab Chart 7.0 software (ADInstruments, Dunedin, New Zealand) was used for all data acquisition and post-acquisition processing.

[0107] For additional hemodynamic evaluation, inflow occlusion assessments were performed to replicate a physiologic inferior vena cava occlusion study. The inflow chamber was positioned to maintain a medium preload condition. During continuous data acquisition, the LA infusion line was then progressively occluded over the course of ~3 seconds resulting in a progressive reduction of preload pressures. Once occluded, hearts were allowed to equilibrate for an addition 5-10 seconds before removing the LA infusion occlusion.

[0108] Orthotopic T ransplantation :

[0109] Recipient porcine (52.5 ± 6.5 Kg) were induced under general anesthesia and a median thoracotomy was performed. Lidocaine (2.5mg / kg) and heparin (initial dose 400 U / kg, ACTs maintained >400 sec) were administered. The transverse aorta was cannulated with a 7.0mm Soft-Flow extended aortic cannula (MC3 Inc, Dexter, Ml) proximal to the innominate artery. The superior vena cava and inferior vena cava were cannulated with a 20Fr and a 22Fr DLP single-stage venous cannulas (Medtronic, Inc, Minneapolis, MN), respectively. The aorta was cross clamped proximal to the aortic reinfusion cannula, the superior and inferior vena cavae were occluded with Rumels proximal to the drainage cannulas, and cardiopulmonary bypass (CPB) was initiated at 4 L / min. Vasoactive medications were initiated and titrated to maintain MAP > 55mmHg. A recipient cardiectomy was performed in preparation for standard bicaval heart implantation.

[0110] Donor hearts were prepared for implantation. NEHP-PE hearts were arrested with 3.0 cc / g of cardiac tissue of cold (6°C) del Nido cardioplegia into the coronary arteries via retrograde infusion through the Ao cannula at a rate of 0.75cc / g / min. NEHP-PE hearts were then removed from the NEHP circuit, placed in chilled CoStorSol preservation solution, and decannulated. CSS and NEHP-PE hearts were weighed and anatomically prepared for implantation in chilled preservation solution.

[0111] Standard bicaval implantation was performed. Briefly, the left atrial anastomosis, inferior vena cava anastomosis, and aortic anastomosis were completed in succession. The aortic cross clamp was then removed to achieve donor heart reperfusion. For all implantations, ischemic time was maintained under 70 minutes and did not require re-dosingDocket No. 30275 / 70725UM2025-025-02 of cardioplegia. Finally, the pulmonary artery anastomosis and superior vena cava anastomosis were completed in succession. A Swan-Ganz catheter was appropriately placed for post-implantation hemodynamic monitoring. Pacing leads were placed in the right atrium and right ventricle for DDD pacing as needed.

[0112] Post-Implantation Cardiopulmonary Bypass Weaning

[0113] Animals were maintained on CPB for at least 30 minutes from allograft implantation completion prior to weaning attempts. During this time, clinically appropriate vasoactive medications (epinephrine, dobutamine, vasopressin, norepinephrine, and / or inhaled nitric oxide (iNO)) were titrated to hemodynamic effect. Hgb was maintained >8.5 g / dL, and electrolytes were adjusted to physiological levels with hemofiltration and electrolyte supplementation. Protective ventilation was initiated at a tidal volume 3 cc / kg, PEEP 7 mmHg, 100% FiO2. Internal DDD pacing was initiated as appropriate. In the event of hemodynamically stable atrial arrythmia without rapid ventricular response (RVR), amiodarone was administered (50-100 mg). In the event of hemodynamically unstable atrial arrythmia, atrial arrythmia with RVR, or ventricular arrythmia, limited electrical cardioversion was attempted with internal paddles. Three attempts at electrical cardioversion were attempted at 5 joules, 10 joules, and 10 joules until sinus rhythm was achieved. If sinus rhythm was not achieved following three successive attempts at electrical cardioversion or if more than three attempts at electrical cardioversion were required within 15 minutes, the donor heart met the predetermined end-criteria and the experiment was terminated.

[0114] Following 30 minutes of post-implantation CPB support, epicardial echocardiography (echo) was performed for assessment of wall motion. Caval Rumels were then removed and progressive CPB weaning attempts were initiated. CPB flow rates were reduced from 4 L / min to 2 L / min to 1 L / min to 0 L / min in succession. Each successive CPB flow rate was maintained for at least 15 minutes. With each weaning attempt, if systolic blood pressure (SBP) was not maintained >55 mmHg, CPB flow rate was increased to the prior flow rate and maintained for an additional 15 minutes prior to further weaning attempts. If CPB cessation was not achieved within 2 hours of allograft return of spontaneous contraction, the heart met end-criteria was the experiment was terminated.

[0115] Once CPB cessation was achieved, allografts were clinically managed and observed for an additional 4 hours. Allograft viability and implantation success was defined as hemodynamic stability (SBP >55 mmHg) for the 4-hour observation period with unchanging or decreasing vasoactive medication requirements. Data collection during the observation period included q15 min hemodynamics, q1 hr arterial blood gas (ABG), andDocket No. 30275 / 70725UM2025-025-02 q1 hr echo. At the conclusion of the experiment, hearts were weighed and sectioned for histopathology.

[0116] Pressure-volume loop and echocardiographic derived calculations

[0117] Ejection fraction (EF)= left ventricular end diastolic volume (LVEDV) - left ventricular end systolic volume (LVESV)

[0118] Cardiac index (Cl)= cardiac output (CO) / post-operative weight of heart

[0119] Left ventricular developed pressure (LVDP) = mean arterial pressure (MAP) - left ventricular end diastolic pressure (LVEDP)

[0120] Stroke Work (SW) = stroke volume (SV) * LVDP

[0121] Preload recruitable stroke work (PRSW): Pressures and flow rates were recorded at various preload load conditions or inflow occlusion conditions. Echocardiography was obtained at each condition for determination of LVEDV and LVESV. From the raw data collected, stroke work was calculated at each preload condition. LVEDP (x-axis) was plotted against SW (y-axis) and the linear regression was calculated; the resulting linear regression slope equates to PRSW. PRSW derived slopes were plotted over identical physiologic preload conditions for each donor heart and the slopes were superimposed for stratification and comparative analysis.

[0122] Results

[0123] Nine healthy adult sized Yorkshire pigs (54.1 ± 5.2kg) were used as heart donors and an additional four were used as transplantation recipients (48.6 ± 3.3). Donor hearts were randomly assigned to MS (n=1 ), PS (n=4) or FS (n=4) treatment groups. All were maintained on the MC for 24 hours implementing the previously described protocols. All donor hearts were functional at hour 24 although, as expected, the MS heart was unstable and marginally viable (FIGs. 17A, 17B, 18A, and 18B).

[0124] The NEHP-OIS was performed on 6 donor hearts. Both intrinsic and loaddependent indices confirmed that the MS donor heart was not suitable for transplantation. Transplantation was not attempted. Four additional donor hearts appeared to be functionally viable and underwent OIS assessments (3 PS and 1 FS-I with a significant ischemic injury). Intrinsic indices, load-dependent hemodynamic assessments, echocardiography, oxygen dynamic, and other metabolic assays were acquired. Transplantation was performed on 2 consecutive PS hearts of which both failed. Load-independent indices and echocardiographic findings demonstrated that the remaining PS donor heart and injured FS heart were equivalent or worse than the 2 failed PS transplant attempts and that transplantation would be futile. NEHP-OIS on a FS donor heart established baseline intrinsicDocket No. 30275 / 70725UM2025-025-02 indices associated with transplant suitability. Transplant suitability of FS donor hearts was confirmed by two consecutive successful orthotopic transplantations. When compared to the FS heart, PRSW of all 3 PS hearts, the injured FS heart, and the MS heart indicated that the intrinsic function of all five was compromised and they were not suitable for transplantation. FIG. 19 depicts the potential of NEHP-OIS load-independent indices for reliably stratifying intrinsic indices and thereby predict transplant suitability prior to transplantation.

[0125] Load-dependent assessments, echocardiography, and other surrogate assays were inconclusive.METHODS OF USE

[0126] A method of use of the organ perfusion and assessment system of the present disclosure may include the following. Procurement of the donor heart DH is completed in the standard fashion. Back-table organ instrumentation includes reconstruction of the left atrium with the left atrial infusion dome 40 and placement of an aortic cannula 52, left ventricular cannula 54 for pressure monitoring and venting, left atrial pressure monitor, pulmonary artery cannula (PA), and temporary electrocardiograph (ECG) and pacing leads.

[0127] The donor heart DH is placed in the organ containment vessel 20 suspended vertically or horizontally by the aortic arch. We prefer vertical suspension because it optimizes imaging but the organ perfusion and assessment system of the present disclosure can accommodate other positions. Perfusate infusion and return lines are connected to their respective cannulas. Resuscitation perfusate is infused through the left atrial and ventricular cannulas and the system is de-aired.

[0128] Perfusion is initiated via a retrograde Langendorff perfusion and perfusate is subsequently treated with progressive re-warming and electrolyte correction for controlled organ re-animation. Once re-animation has been achieved, perfusate is exchanged with a physiologic cellular perfusate.

[0129] Perfusion is maintained at a goal flow rate of 0.5-0.6cc / g of cardiac tissue / min and titrated to ensure SvO2 >75%. Maintenance perfusion strategies include implementation of a heat exchanger for temp 37° C, oxygenator for physiologic respiration conditions, titration of vasoactive medications, continuous infusion of modified serum, maintenance of Hgb > 8.0 g / dL, active hemodialysis, and corrections of electrolytes to physiologic conditions. All systems are controlled and automated via a servoregulator. Perfusate is exchanged at a rate of q18 hr.

[0130] Intermittent Organ Assessment.Docket No. 30275 / 70725UM2025-025-02

[0131] The organ perfusion and assessment system is connected to the maintenance circuit including an infusion line to the left atrial cannula and a perfusate return line to the aortic cannula and is subsequently de-aired. During connection, the system inflow and outflow lines are occluded and remains isolated from the maintenance circuit. System perfusate is adjusted to physiologic conditions.

[0132] The organ perfusion and assessment system is configured to transition the system from a maintenance circuit mode to a coronary-only left atrial infusion circuit mode with a perfusate flow rate of 0.75cc / g of cardiac tissue / min to pre-condition the donor heart DH to atrial infusion. The containment vessel base 32 of the organ containment vessel 20 is filled with a synthetic pericardial fluid to permit detailed echocardiography.

[0133] At this time, the heart is transitioned to the organ perfusion and assessment system of the present disclosure via sequential occlusion of the maintenance circuit inflow, unclamping of the system’s inflow and outflow lines, and redirection of pulmonary artery flow from the maintenance circuit to the coronary-only left atrial infusion circuit mode.

[0134] Once transition to the coronary-only left atrial infusion circuit mode has been completed, the heights of the collapsible pre-load controller reservoir and semi-rigid afterload simulator reservoir are adjusted relative to the donor heart DH to titrate preload and afterload conditions to the desired parameters. The afterload simulator is always slightly higher than the preload controller.

[0135] With pre-load and afterload parameters set, data acquisition is completed including continuous hemodynamic monitoring (left atrial inflow flow rate, aortic outflow flow rate, pulmonary artery outflow flow rate, left atrial pressure, left ventricular pressure, aortic root pressure) and echocardiography assessment. IVC / SVC occlusion studies may be completed via progressive occlusion of the left atrial infusion line with continuous data acquisition. All data acquisition utilizing the organ perfusion and assessment is able to be completed within approximately 30 minutes of the transition to the coronary-only left atrial infusion circuit mode.

[0136] Once data acquisition has been completed, organ perfusion can be transitioned back to the maintenance circuit by completion the transition process in reverse.

[0137] While exemplary systems, circuits, operating modes, and methods of use have been described herein, it is understood that variations may be made thereto that are still within the scope of the appended claims.

Claims

Docket No. 30275 / 70725UM2025-025-02What is claimed is:1 . An artificial circulatory system for maintaining and assessing an organ ex vivo, comprising: a preload controller; a vessel for containing the organ, including a containment vessel top, the containment vessel top including a plurality of apertures therethrough and a sealing membrane associated with each of the apertures; a base, the base defining a fluid-tight chamber having a mouth at a first end thereof that is selectively engageable with the containment vessel top, and the base provided with an inflow / outflow port; and an afterload simulator.

2. The system of claim 1 , wherein the preload controller is compliant and collapsible.

3. The system of claim 1 , wherein the preload controller includes at least one inflow port, at least one out flow port, and at least one air removal port.

4. The system of claim 1 , wherein the preload controller is configured such that passive preload pressures are adjusted by gravity.

5. The system of claim 1 , wherein the preload controller is provided with means for providing digitally modulated outflow resistance linked to afterload.

6. The system of claim 1 , wherein the base is selectively tillable with an acoustically- conductive fluid to a level that submerges an organ contained in the base.

7. The system of claim 1 , wherein a sidewall of the base is seamless.

8. The system of claim 1 , wherein a sidewall of the base is acoustically transparent.

9. The system of claim 2, wherein the base is drainable through the inflow / outflow port.

10. The system of claim 2, wherein the fluid is a sterile, normothermic fluid.Docket No. 30275 / 70725UM2025-025-0211 . The system of claim 1 , wherein the containment vessel top is secured to a containment vessel cylinder, the containment vessel cylinder including an axially-extending portion about which the base is removably secured.

12. The system of claim 1 , wherein the mouth at the first end of the fluid-tight chamber is provided with a tube clamp to facilitate the selective engagement with the containment vessel cylinder.

13. The system of claim 1 , wherein the afterload simulator includes: a priming volume sterile container, the priming volume sterile container being flat, optically transparent; a lower end region provided with at least one inflow port and at least one outflow port; and a top region including at least one additional port permitting air removal and attachment of a compliance chamber.

14. The system of claim 13, and the afterload simulator further including an adjustable rigid diaphragm.

15. The system of claim 13, further including a detachable sterile rigid compliance chamber in communication with one of the one or more additional ports in the top region.

16. The system of claim 13, wherein the outflow port of the afterload simulator is provided with a variable resistance regulator.

17. The system of claim 1 , further comprising a left atrial dome, including an atrial dome base; a left atrial infusion connector; a transcatheter access port; and a left atrial pressure port.

18. A system for obtaining data from an ex vivo organ and communicating the data to a controller to facilitate hemodynamic assessment of the ex vivo organ, comprising: a vessel for containing the organ, the vessel being a seamless, radiotransparent, fluid- tight chamber selectively engageable with a containment vessel top, the containmentDocket No. 30275 / 70725UM2025-025-02 vessel top including a plurality of apertures therethrough and a sealing membrane associated with each of the apertures; the vessel further containing a volume of an echocardiogram-compatible fluid sufficient to substantially submerge an organ received in the vessel; and an afterload simulator.

19. A method of assessing an organ ex vivo, comprising: securing the organ in a vessel for containing the organ, the vessel including a preload controller; a containment vessel top, the containment vessel top including a plurality of apertures therethrough and a sealing membrane associated with each of the apertures; a base, the base defining a seamless, radiotransparent, fluid-tight chamber having a mouth at a first end thereof that is selectively engageable with the containment vessel top, and the base provided with an inflow / outflow port; and an afterload simulator; filling the base with an acoustically-conductive fluid; and acquiring, via at least one of a group consisting of measurement, sensing, detection, and observation, one or more parameters of the organ indicative one or more load conditionagnostic indices.

20. The method of claim 19, wherein at least one of the one or more parameters is acquired by a hemodynamic assessment of the organ.21 . The method of claim 19, wherein at least one of the one or more parameters is a pressure-volume loop derived measure of organ function.

22. The method of claim 19, wherein the one or more parameters includes preload recruitable stroke work.

23. The method of claim 19, wherein the one or more parameters include at least one of a group consisting of stroke volume, afterload, stroke work, exponential decay of dP / dt during isovolumic relaxation (T), atrial pressure, ventricular pressure, atrial volume, ventricular volume, end systolic pressure, end diastolic pressure, left ventricle end diastolic pressure, left ventricle end diastolic volume, pulmonary capillary wedge pressure, cardiac output, ejection fraction, dP / dt for systolic function, dP / dt for diastolic function, myocardialDocket No. 30275 / 70725UM2025-025-02 oxygen consumption, myocardial oxygen demand, myocardial oxygen extraction, mixed venous oxygen saturation, vascular resistance, and vascular compliance.

24. The method of claim 23, wherein at least one of the atrial pressure and the ventricular pressure includes at least one of end systolic pressure and end diastolic pressure, throughout a cardiac cycle.

25. The method of claim 23, wherein at least one of the atrial volume and the ventricular volume includes at least one of end systolic volume and end diastolic volume, throughout a cardiac cycle.

26. The method of claim 23, further comprising measuring at least one of myocardial fiber length, blood viscosity, and lactate production.

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