APPARATUS FOR MAINTENANCE OF HARVESTED HEARTS FOR TRANSPLANTING

Abstract

An apparatus, a system, and methods for maintaining and monitoring an excised donor heart. The apparatus comprises a first component for receiving and submerging therein an excised heart in a constantly circulating perfusate solution and a second component comprising equipment for adjusting the temperature and oxygen content of the perfusate solution. The first component comprises an integral pair of defibrillating pads. A first conduit infrastructure interconnects the first module, the second module and an aorta of the excised donor heart pushing a perfusion solution from the first module through the second module into the aorta. The second conduit infrastructure connects the first module with the right atrium and the left atrium for pushing the perfusion solution from the first module into the atria. The third conduit infrastructure connects the first module with the pulmonary artery and provides an after pressure to the flow of the perfusion solution from the pulmonary artery.

Claims

1. A portable system for perfusing excised donor hearts, comprising: a first module and a second module; a first pump, a second pump, and a third, bi-directional pump; a plurality of conduits for interconnecting the first and second modules and the first, second and third pumps; a computer providing computer-controlled modulation of the third pump; and a transportable support configured for disengagably mounting thereon the first module, and for mounting thereon the second module, the first, second and third pumps, the plurality of conduits, and a reservoir of a perfusate; wherein the first module comprises a removable support for supporting an excised heart and conduits for connecting the excised heart to the reservoir; the second module comprises a heat-exchanger and an oxygenator for conditioning the perfusate; the plurality of conduits comprises a first conduit for connecting with an aorta of the heart, a second conduit for connecting with an atrium of the heart, and a third conduit for connecting with a pulmonary artery of the heart; the first pump is in communication with the first conduit; the second pump is in communication with the second conduit; the third pump is in communication with the third conduit; and the third pump is configured and the computer is programmed to modulate the third pump operable to apply an afterload pressure in the third conduit at the pulmonary artery to resist a flow of the perfusate in the third conduit from the pulmonary artery.

2. The portable system of claim 1, wherein at least one of the first, second and third pumps is a centrifugal pump.

3. The portable system of claim 1, wherein the transportable support comprises a housing for the first and second modules, the first, second and third pumps, and the plurality of conduits.

4. The portable system of claim 1, further comprising an ECG monitoring device and a cardiac pacemaker.

5. The portable system of claim 1, further comprising a pair of defibrillator pads mounted in the first module, and a defibrillator for communicating with the pair of defibrillator pads.

6. The portable system of claim 1, wherein the first pump is configured to apply an afterload pressure to the aorta.

7. The portable system of claim 1, wherein the second pump is connected to the second conduit for applying a preload pressure to the atrium.

8. The portable system of claim 1, wherein the atrium is the left or right atrium of the heart.

9. The portable system of claim 1, comprising the reservoir.

10. The portable system of claim 1, further comprising an occlusion clamp connected with the second conduit for regulating a rate of flow through the second conduit, or modulating a pressure therein.

11. The portable system of claim 10, wherein the occlusion clamp comprises a servo-actuated partial occlusion clamp.

12. The portable system of claim 10, wherein the atrium is the right atrium of the heart.

13. The portable system of claim 1, further comprising flow sensors for monitoring flow rates of the perfusate or measuring pressure in selected ones of the conduits.

14. The portable system of claim 1, further comprising one or more pressure ports in selected ones of the plurality of conduits.

15. A system for maintenance and assessment of hearts for implantation, comprising: a movable base module comprising a first sub-system configured to supply and condition a perfusate; a perfusion module removably connected to the base module, comprising a second sub-system configured to support a heart and connect the heart for circulating the conditioned perfusate through the heart; and a control and assessment sub-system connected to the first and second sub-systems and configured to control perfusate flows through the heart and to assess the heart during perfusion, wherein the control and assessment subsystem comprises a computer programed to provide computer-controlled modulation of an afterload pressure applied at a pulmonary artery of the heart to resist a flow of the perfusate egressing from the pulmonary artery.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Example embodiments of the present disclosure will be described in conjunction with reference to the following drawings in which:

[0017] FIG. 1 is a schematic illustration of an example maintenance apparatus for harvested donor hearts, according to one embodiment of the present disclosure;

[0018] FIG. 2 is a close-up partial view of an example embodiments of the pacemaker, ECG monitor, and defibrillator components of the harvested donor heart maintenance apparatus of the present disclosure; and

[0019] FIG. 3 is a schematic side view of a perfusion apparatus on a support, illustrating an example embodiment of the disclosure.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In order that the example embodiments of the present disclosure may be fully understood, the following terms and definitions are provided herein.

[0021] The word comprise or variations such as comprises or comprising will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers.

[0022] The term about or approximately means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0023] The term modulate as used herein means to regulate the operation of a device by increasing a signal to the device in order to increase an output by the device, or by decreasing a signal to the device in order to decrease an output by the device.

[0024] The term afterload means the mean tension produced by a chamber of the heart in order to contract. It can also be considered as the load that the heart must eject blood against. Afterload is therefore a consequence of aortic large vessel compliance, wave reflection and small vessel resistance (left ventricular afterload) or similar pulmonary artery parameters (right ventricular afterload).

[0025] The term preload refers to the stretching of a single cardiac myocyte immediately prior to contraction and is therefore related to the sarcomere length. Since sarcomere length cannot be determined in the intact heart, other indices of preload such as ventricular end diastolic volume or pressure are used. As an example, preload increases when venous return is increased.

[0026] The term cardiac myocyte means a cardiac muscle cell.

[0027] The term stroke volume (SV) means the volume of blood ejected by the right/left ventricle in a single contraction. It is the difference between the end diastolic volume (EDV) and the end systolic volume (ESV). Mathematically, SV=EDVESV. The stroke volume is affected by changes in preload, afterload and inotropy (contractility). In normal hearts, the SV is not strongly influenced by afterload whereas in failing hearts, the SV is highly sensitive to afterload changes.

[0028] The term stroke work (SW) refers to the work performed by the left or right ventricle to eject the stroke volume into the aorta or pulmonary artery, respectively. The area enclosed by the pressure/volume loop is a measure of the ventricular stroke work, which is a product of the stroke volume and the mean aortic or pulmonary artery pressure (afterload), depending on whether one is considering the left or the right ventricle.

[0029] The term ejection fraction (EF) means the fraction of end diastolic volume that is ejected out of the ventricle during each contraction. Mathematically, EF=SV/EDV. Healthy ventricles typically have ejection fractions greater than 0.55. Low EF usually indicates systolic dysfunction and severe heart failure can result in EF lower than 0.2. EF is also used as a clinical indicator of the inotropy (contractility) of the heart. Increasing inotropy leads to an increase in EF, while decreasing inotropy decreases EF.

[0030] The term end systolic pressure volume relationship (ESPVR) describes the maximal pressure that can be developed by the left ventricle at any given left ventricular volume, or alternatively, by the right ventricle at any given right ventricular volume. This implies that the PV loop cannot cross over the line defining ESPVR for any given contractile state. The slope of ESPVR (Ees) represents the end-systolic elastance, which provides an index of myocardial contractility. The ESPVR is relatively insensitive to changes in preload, afterload and heart rate. This makes it an improved index of systolic function over other hemodynamic parameters like ejection fraction, cardiac output and stroke volume. The ESPVR becomes steeper and shifts to the left as inotropy (contractility) increases. The ESPVR becomes flatter and shifts to the right as inotropy decreases.

[0031] The term preload recruitable stroke work relationship (PRSW) means a measure of cardiac contractility, and is the linear relationship between SW and EDV.

[0032] The term pressure-volume area (PVA) means the total mechanical energy generated by ventricular contraction. This is equal to the sum of the stroke work (SW), encompassed within the PV loop, and the elastic potential energy (PE). Mathematically, PVA=PE+SW.

[0033] The term Langendorff perfusion refers to a method of perfusing an excised heart with a nutrient-rich oxygenated solution in a reverse fashion via the aorta. The backwards pressure causes the aortic valve to shut thereby forcing the solution into the coronary vessels, which normally supply the heart tissue with blood. This feeds nutrients and oxygen to the cardiac muscle, allowing it to continue beating for several hours after its removal from the animal.

[0034] The term working heart as used herein, refers to clinical ex vivo coronary perfusion throughout a excised heart by ventricular filling via the left atrium and ejection from the left ventricle via the aorta driven by the heart's contractile function and regular cardiac rhythm. The excised heart is attached by cannulae to a perfusate reservoir and circulatory pumps in a Langendorff preparation. The flow of perfusate through the excised heart in working heart mode is in the direction opposite to the flow of perfusate during Langendorff perfusion.

[0035] The term ischemia means a condition that occurs when blood flow and oxygen are kept from the heart.

[0036] The term conduit as used herein means tubing and/or cannula.

[0037] The present disclosure pertains to apparatus, systems and methods for maintaining an excised heart under continuous Langendorff perfusion until transplantation. The apparatus and systems are communicable and cooperable with cardiac monitoring equipment and microprocessors for monitoring the physiological condition and functioning of the excised heart.

[0038] One embodiment of the present disclosure pertains to an exemplary modular apparatus for receiving and maintaining an excised heart under continuous Langendorff perfusion until transplantation. The exemplary apparatus comprises two modules. The first module comprises a hard-shell reservoir, also referred to herein as a reservoir, for housing therein an excised heart under constant bathing with a suitable perfusate solution. The excised heart is mounted onto a stand and submerged within the hard-shell reservoir. The hard-shell reservoir is provided with four ports (i.e., two egress ports and two ingress ports) that are sealingly engageable by conduits that have been interconnected to the excised heart's right atrium, left atrium, aorta, and pulmonary artery. The second module is a perfusate conditioning apparatus comprising: (i) a heat-exchanger for warming and maintaining the perfusate solution at a user-specified temperature (typically referred to as a normothermic temperature), and (ii) and oxygenator for maintaining the dissolved oxygen levels in the perfusate solution above 95% saturation, and maintaining the pH balance through addition of carbon dioxide. The two modules are interconnected by a conduit infrastructure that is engageable by a pump such as those exemplified by centrifugal pumps. Suitable centrifugal pumps are exemplified by ROTAFLOW centrifugal pumps (ROTAFLOW is a registered trademark of Maquet Cardiopulmonary AG Corp., Hirrlingen, Fed. Rep. Ger.), by Medtronic's centrifugal blood BIO-PUMPs BIO-PUMP is a registered trademark of Medtronics Bio-Medicus Inc., Minnetonka, Minn., USA), by Sorin's RevOlution 5 blood pump (Sorin Group USA, Arvada, Colo., USA). In operation, the centrifugal pump provides a constant flow of perfusate solution from the first module (i.e., the hard-shell reservoir) to the second module (i.e., the perfusate conditioning apparatus). The first module is additionally provided with ports for receiving therethrough leads from cardiac monitoring equipment for engaging specific sites on and/or in the excised heart. Each module can be separately assembled and prepared for use multiple units, thereby facilitating rapid assembly and configuration of the apparatus as needed to receive and maintain an excised heart.

[0039] An exemplary apparatus 10 according to one embodiment of the present disclosure is shown in FIG. 1. The apparatus comprises: (i) a first component which is a hard-shell reservoir 20 housing a removable support (not shown) for mounting thereon and therein an excised heart 100, and (ii) a second component which is a perfusate solution conditioning device 35 comprising a heat-exchanger 36 and an oxygenator 37. The hard-shell reservoir 20 may additionally have a level sensor (not shown) for monitoring the level of perfusate solution in the hard-shell reservoir 20. The two components are interconnected by a first conduit infrastructure comprising: (i) an egress line 30 that is sealably engageable at one end with a port 22 provided therefore near the bottom of the hard-shell reservoir 20 and is sealably engageable at its other end with the inlet of a first centrifugal pump 32. The outlet of the first centrifugal pump 32 is sealably engaged with a line 34 that is sealably engageable with an inlet to the heat exchanger 36 of the perfusate solution conditioning device 35. A line 40 is sealably engageable with an outlet from the oxygenator 37 of the perfusate solution conditioning device 35. The other end of line 40 is sealably engageable with a Y-connector 42 which diverts a portion of the flow of conditioned perfusion solution from the perfusion solution conditioning device 35 into a purge line 50 that is sealably engageable with a first ingress port 26 provided therefore on the hard-shell reservoir 20. The remainder of the flow of perfusate solution conditioning device 35 is diverted by the Y-connector 42 into a flow sensor 44 interconnected with an integrated pressure port 46 that is clampable into the aorta 150 of the harvested heart. In operation, the flow sensor 44 measures aortic flow of the conditioned perfusion solution from the perfusate solution conditioning device 35 into the aorta 150. Perfusion solution egressing from the hard-shell reservoir 20 into the perfusate solution conditioning device 35 is conditioned by heating in the heat exchanger 36 to a normothermic temperature from the range of about 20 C. to about 37 C. and then is oxygenated by oxygenator 37 prior to flowing into line 40 for conveyance into the aorta 150. The diastolic pressure in the aorta 150 can be specified and tightly regulated by computer controlled feedback to modulate the centrifugal pump 32. During assessment mode, with provision of flow into the left atrium the heart ejects the perfusion solution back through line 40 with the centrifugal pump 32 providing resistance (afterload). In this manner the heart can beat against an afterload pressure that is delivered by the flow of perfusate solution from the centrifugal pump 32.

[0040] A second conduit infrastructure comprises a line 60a sealably engageable at one end with a second egress port 24 provided therefore near the bottom of the hard-shell reservoir 20, and its other end sealably engageable with the inlet into a second centrifugal pump 62. The outlet of the second centrifugal pump 62 is sealably engageable with a line 60b that terminates in a Y-connector 64. Y-connector 64 splits the pressurized flow of perfusion solution into two lines 70, 80. Line 70 is interconnected with, firstly, an occlusion clamp 72, secondly, a flow sensor 74, and thirdly, an integrated pressure port 76. The terminal end of line 70 is insertable into the right atrium 130 of the harvested heart 100. It should be noted that occlusion clamp 72 is preferably a servo-actuated partial occlusion clamp whose variable positions enables regulation of the rate of flow of the perfusion solution into the right atrium 130 and therefore, can also be used to modulate pressure delivered to the harvested heart 100. Line 80 is interconnected with, firstly, a flow sensor 82, and secondly, an integrated pressure port 84. The terminal end of line 80 is insertable into the left atrium 140 of the harvested heart 100. It should be noted that lines 70, 80 are additionally provided with bubble detectors (not shown). During the assessment mode, pump 62 provides flow of the perfusate solution into the right atrium and left atrium (preload pressure) under a feedback loop from pressure ports 84, 76 with differential control of flow into the right atrium and left atrium being provided by modulation of clamp 72. In the event of overpressurization of the heart as a consequence, for example, of arrhythmia or poor cardiac function, the flow of perfusate solution from pump 62 is decreased thereby allowing decompression of the heart to occur through passive retrograde flow of the perfusate solution back through the pump 62.

[0041] A third conduit infrastructure comprises a line 96 that is clampable into the pulmonary artery 160 of the harvested heart 100. The line 96 is sequentially sealably engageable with an integrated pressure port 90, a flow sensor 92, and a third centrifugal pump 94. The terminal end of the line 96 is sealably engageable with the second ingress port 28 provided therefore on the hard-shell reservoir 20. Pump 94 provides resistance (afterload pressure) to the right ventricle, through computer-controlled modulation of the pump 94 in reference to feedback from pressure port 90.

[0042] FIG. 2 illustrates exemplary monitoring and maintenance equipment for maintaining a harvested heart in a functional condition during storage and transport in the exemplary apparatus of the present disclosure. Leads 172 from an ECG monitoring device 170 are engageable with, for example, the right ventricle 110 and the left ventricle 120 of a harvested heart 100 for monitoring the electrical activity of the harvested heart 100. Alternatively, the ECG leads may be integrally incorporated into the walls of the hard-shell reservoir 20. Leads 192 from a dual-chamber pacemaker 190 are engageable with the right atrium 130 and the right ventricle 110 of the harvested heart 100. Although a dual-chamber pacemaker is preferable for use with the apparatus 10 of the present disclosure, it is optional to substitute a single-chamber pacemaker having a single lead that is engageable with the right atrium or the right ventricle. Two defibrillator pads 184 are integrally provided opposite each other on the inner surfaces of the hard-shell reservoir 20 and are connected by leads 182 to a defibrillator. The ECG monitoring device 170, the pacemaker 190 and the defibrillator 180 may be mounted on a support provided therefore (not shown) that is an integral component of the hard-shell reservoir 20. Alternatively, the ECG monitoring device 170, the pacemaker 190 and the defibrillator 180 may be integrally incorporated into the housing of a transportation container configured to receive therein the hard-shell reservoir 20.

[0043] As soon as an excised heart 100 is mounted onto the removable support and placed into the hard-shell reservoir 20, the terminal end of line 40 is clamped into the aorta, 150, line 70 is inserted into the right atrium 130, line 80 is inserted into the left atrium 140, and line 96 is clamped into the pulmonary artery 160. Then, a suitable perfusion solution exemplified by whole blood, whole blood amended with citrate and/or phosphate and/or dextrose, modified Krebs solutions, Viaspan, modified Viaspan solutions, and the like, is added into the hard-shell reservoir 20 until the heart 100 is completely submerged. It should be noted that the hard-shell reservoir 20 may be additionally provided with a level sensor (not shown) and a supplementary supply of the perfusion solution (not shown) for conveyance into the hard-shell reservoir 20 as need to maintain the excised heart 100 fully submerged during storage and transport in the apparatus of the present disclosure.

[0044] When in operation, the pump 32 continuously draws the perfusate solution from the hard-shell reservoir 20 from egress port 22 into line 30 into the perfusate solution conditioning device 35 wherein the perfusate solution is conditioned by warming to a normothermic temperature and then, is oxygenated. The conditioned and pressurized conditioned perfusate solution is then conveyed to Y-connector 42 that diverts a portion of the conditioned perfusate solution into purge line 50 for conveyance through ingress port 26 back into the hard-shell reservoir 20 where it circulates about and baths the heart 100. The remaining flow of pressurized conditioned perfusate solution is conveyed through flow sensor 44 and integrated pressure port 46 into the aorta 150. It is to be noted that the purge line 50 is positioned to be the highest point in the assembled apparatus 10 when an excised heart 100 is mounted therein so that any air that is ejected by the heart immediately goes out via the purge line 50 and back to the hard-shell reservoir 20.

[0045] A preload centrifugal pump 62 draws the perfusion solution out of the hard-shell reservoir through egress port 24 into line 60b and then pushes the perfusion solution to Y-connector 64 where its flow is split into two lines 70,80. The perfusion solution is pushed through line 70 through a computer-controlled servo-actuated partial occlusion clamp 72, a flow sensor 74, and an integrated pressure port 76 into the right atrium 130. The variable positions of the servo-actuated partial occlusion clamp 72 enables precise regulation of the rate of flow of the perfusion solution into the right atrium 130. The perfusion solution is concurrently pushed through line 80 through a flow sensor 82, and an integrated pressure port 84 into the left atrium 140.

[0046] The pressurised perfusion solution flowing into the aorta 150, right atrium 130, and left atrium 120 flows into the right ventricle 140, and then out through the pulmonary artery 160 into line 96 through, firstly, an integrated pressure port 90, secondly, a flow meter 92, thirdly, an afterload centrifugal pump 94 to regulate the right ventricular afterload pressure (which is measured by the flow meter 92), and finally, back into the hard-shell reservoir 20 through ingress port 28. The pressurized flow of conditioned perfusion solution into the aorta 150 via line 40 is supplied by centrifugal pump 32 and is monitored by aortic flow sensor 44. The pressurized flow of conditioned perfusion solution into the aorta 150 and then out of the pulmonary artery 160 will maintain the heart 100 in a Langendorff, isolated root perfusion state. To maintain and assess the heart's function in working mode, tight regulation of preload is required. Therefore lines 70, 80 connected to the right atrium and left atrium, respectively, comprise tubing and receive pressurized flow of perfusion solution from the preload pump 62. Right atrial flow pressure is monitored by flow sensor 74 while left atrial flow pressure is monitored by flow sensor 82. The computer-controlled servo-actuated partial occlusion clamp 72 enables precise control over the rate of perfusion solution to the right atrium 130 and the left atrium 140, and therefore, the pressure applied to the receiving chamber. The flow meters 44, 74, 82, 92 and the integrated pressure points 46, 76, 84, 90 are connectable to and communicable with a computer for constant monitoring and integrating of the flow rates and pressures to enable constant assessment of cardiac function, i.e., the right ventricular stroke work and the left ventricular stroke work while varying resistance to the flow of perfusion solution (i.e., afterload). It should be noted that the levels of haematocrit, Ca.sup.++, K.sup.+, NaHCO.sub.3, Na.sup.+, pO.sub.2, CO.sub.2, and glucose in the perfusion solution must be balanced before perfusion starts. In the case of using bank CPD donor blood, deranged K.sup.+ and Ca.sup.++ concentrations may not allow for a homeostatic prime. This can be adjusted by haemofiltration using Ringers solution as the rinse. All these values should ideally start within normal physiological ranges and should be monitored by inline continuous blood gas analysis. The primary purpose for the perfusion solution is to avoid causing tissue edema and to maintain ion homeostasis to preserve cardiac function.

[0047] Another exemplary embodiment of the present disclosure relates to a support for mounting thereon and dismounting therefrom of the modules and the pumps, as illustrated in FIG. 3. As depicted in FIG. 3, a support 200 is provided for disengagably mounting thereon a first module 202, a second module 204, and pumps 206, 208 and 210. First module 202 includes the first component described above, and the second module 204 includes the second component described above. The support 200 may include a housing 212 for encasing or housing modules 202, 204 and pumps 206, 208, 210. The support 200 may be mounted on wheels 214. The modules 202, 204 are interconnected with respective conduit infrastructures 216, 218, 220, which include pumps 206, 208 and 210 respectively. Pump 206 may be similarly connected and operated as pump 32. Pump 208 may be similarly connected and operated as pump 62. Pump 210 may be similarly connected and operated as pump 94. The arrows besides pumps 206, 208 and 210 in FIG. 3 indicate the pumping directions, or the directions of the fluid pressure applied by the respective pumps, in a selected mode of operation. The support may additionally have mounts for installation of cardiac monitoring equipment and/or computer equipment and/or monitors for displaying the physiological condition and functioning of the excised heart. For example, as shown in FIG. 3, a computer 222 may be mounted on the support 200 and connected to the pumps and conduit infrastructures to control the pumps and for monitoring the conditions and functions of the excised heart. In particular, and as depicted in FIG. 3, the computer 222 may be programmed to provide computer-controlled modulation of pump 210 based on the feedback from conduit infrastructure 220. The support may be a racking system mounted on wheels so that the apparatus is transportable within a medical facility, for example between surgical theatres, staging rooms, assembly rooms and disassembly rooms. The support may be a cabinet with two opposing side walls and with other two sides having opening doors. Alternatively, the support may be a cabinet with three fixed side walls being opposing walls and having one side with opening doors. The side walls and doors may be insulated and/or cushioned. The support may be configured for transport by vehicles or by airplanes.

[0048] Another exemplary embodiment of the present disclosure relates to a system for receiving, perfusing and maintaining and assessing an excised donor heart. The system generally comprises the above-disclosed apparatus interconnected with: (i) a perfusate-processing system, (ii) a perfusate pumping system, (iii) flow sensors for monitoring the flow of perfusate to and from an installed heart's aorta, right atrium, left atrium, and pulmonary artery vena cava, (iv) an ECG apparatus interconnectable with the installed heart, (v) a pacemaker interconnectable with the installed heart, (vi) a defibrillator interconnectable with the pair of defibrillator pads integral with the inner surface of the hard-shell reservoir component of the apparatus, and (vii) probes interconnecting the installed heart with instruments for monitoring the heart's physiological functionality using load independent indices and load dependent indices. Suitable perfusion-processing systems are exemplified by heart-lung machines commonly used for coronary bypass surgeries.

[0049] An exemplary use of the apparatus, system and methods of the present disclosure generally comprises the steps of selection, preparation, and balancing of a perfusate solution, setting up the system by interconnecting the perfusate-processing system and the bi-directional perfusate pumping system with cannulae that are subsequently interconnected with the appropriate ports on the lid of the receiving, maintaining, and assessing apparatus, priming the interconnected system with the perfusate solution, installing an excised heart onto the support provided with the apparatus and then installing the appropriate cannulae into the heart's aorta, pulmonary artery, pulmonary vein, and vena cava, expressing all air from within the heart and the cannulae, and then commencing the Langendorff perfusion at a normothermic temperature from the range of about 25 C. to about 35 C.