WO2006118990A2

WO2006118990A2 - Method and device for organ perfusion

Info

Publication number: WO2006118990A2
Application number: PCT/US2006/016163
Authority: WO
Inventors: Marlin L. Alford; Robert M. Dowben
Original assignee: Transplan, Inc.
Priority date: 2005-04-29
Filing date: 2006-04-28
Publication date: 2006-11-09
Also published as: EP1879997A2; WO2006118990A3

Abstract

A portable organ perfusion device has an organ chamber for supporting an organ immersed in a perfusion fluid. A pump circulates the perfusion fluid around a circuit containing a pump, a heat exchanger for cooling the perfusion fluid, an oxygenator for oxygenation of the perfusion fluid, and a pressure head device for ensuring a constant head supply to the vasculature of the organ. A bypass channel forms a fluid connection from upstream of the oxygenator to the organ chamber allowing excess fluid to bypass the organ. The device also includes an oxygen source, sensors, a power source and a control unit for receiving information from the sensors and providing control instructions to control elements.

Description

METHOD AND DEVICE FOR ORGAN PERFUSION

BACKGROUND OF THE INVENTION

1. Field of the Invention

[0001] The invention relates to devices for perfusion of organs ex-vivo, in particular human organs such as the heart, liver, kidney and lungs for use in transplant operations. The invention further relates to methods of ex-vivo perfusion of such organs.

2. Description of the Related Art

[0002] Surgical transplantations of hearts and lungs, first performed in 1960, have become frequent procedures owing to the improvement of surgical techniques, the introduction of by- pctώa oiiwαlatiυii anu the development of drugs^'that suppress immune rejection of the donor organ. At the present time, the donor organ is conventionally harvested under sterile conditions, placed in a plastic bag submerged in a buffered salt solution, cooled to about 4°C and kept over crushed ice until the organ is finally transplanted into the recipient. The solution is not oxygenated and is not perfused through the organ blood vessels.

[0003] The lack of donor organ availability, particularly hearts, lungs, and livers, is a limiting factor for the number of organ transplants that can be performed. At the present time, less than 23% of patients who require a heart transplant ever receive a new heart, and less than 10% of patients who require a lung transplant receive one. In the United States the greatest number of heart transplants were performed in 1998, 2,334; the number has declined each year since, 2,182 were performed in 2002. A major consideration is the length of time that a donor organ will remain viable after it is harvested until the transplant surgery is completed. For hearts, using conventional storage techniques, this interval is about four hours. In 4 hours, 12% of the transported organs "die" or become unusable, and all the organs are damaged at the cellular level even during this short period. The donor heart must be harvested, transported to the recipient, and the transplant surgery completed within this four- hour time limit. Thus, at present donor hearts can be used only if they are harvested at a site close to the location where the transplant surgery will take place.

[0004] It has been found that donor hearts and lungs will survive ex-vivo for a longer time if they are cooled to 4°C and actively perfused through their vascular beds with a buffered salt solution containing nutrients. It has also been found that ex-vivo survival of an isolated organ can be further extended if the solution is oxygenated. Several factors play a role in the prolonged survival. At 4⁰C metabolism is greatly reduced, lowering the requirements for nutrients and oxygen, and the production of lactic acid and other toxic end products of metabolism is also greatly reduced. Circulation of the perfusion fluid provides oxygen, replenishes the nutrients available to the tissue, and removes the lactic acid and other toxic metabolites. The buffered solution maintains the intracellular pH and electrolyte concentrations of the tissue close to physiological.

[0005] There have been previous attempts to develop workable systems for low temperature preservation of organs but none has adequately achieved a significant increase in ex-vivo survival.

[0006] One device that has been proposed for maintaining organs in vitro is described in US Patent No. 3,545,221. The device comprises a pressurized organ preservation receptacle in which e.g. a kidney may be immersed in a preserving solution at a temperature of 0 to 4⁰C. The organ may be supplied with a perfusion solution by gravity feed from a suspended container.

[0007] According to an alternative device known from US Patent No. 3,995,444 a pulsatile flow of cold perfusate is circulated through a harvested organ. The organ is supported in an organ chamber on a perforated panel that allows perfusate draining from the organ to collect in a well at the bottom of the chamber.

[0008] Another organ preservation system is disclosed in US Patent No. 5,326,706 which discloses an outer insulated container containing an inner organ chamber for holding a donor organ. A perfusion solution is circulated around a circuit, passing through the organ. The perfusate pressure is measured and a pump control circuit adjusts the pump pulse rate in accordance with the pressure of the perfusate. The pump comprises a flexible membrane, actuated by a source of carbon dioxide gas.

[0009] A device that discloses a combined pumping and oxygenation function is known from US Patent No. 5,362,622. The organ is stored in a compliant chamber submerged in a perfusate. In this device, a cyclically pumped source of oxygen acts on a gas permeable membrane to simultaneously oxygenate the perfusate and pump the oxygen-enriched perflisate through the organ. The device may be placed in an insulated container provided with cool packs to maintain the organ a temperature of around 4⁰C.

[0010] An alternative device that can be used for static storage or perfusion of an organ is known from US Patent No. 5,586,438. The device comprises an organ container in which an organ may be supported between pads of soft sterile foam material. The organ is connected via a perfusion tube to a bubble trap. The bubble trap is in turn connected to an arterial line while a venous line has an open end at the bottom of the organ container. For the purpose of perfusion, the venous and arterial lines extend outside the container for connection to an appropriate pump. The entire device may be inserted into an insulated and cooled organ shipping box for transport.

[0011] More recently, further developments have attempted to maintain organs at a substantially normothermic temperature. One such device is described in US Patent No. 6,100,082 in which an organ is enclosed in a preservation chamber or pouch. In the described example, fluid connections to the aorta, pulmonary artery and left atrium are provided to permit various possible perfusion flows of warmed, oxygenated blood to a beating heart.

[0012] An alternative support system for maintaining an organ at a near normal metabolic rate is disclosed in US Patent No 6,642,045. The organ is supported on an organ support within an organ chamber. Perfusate is pumped to the organ through heat exchanger to bring the perfusate temperature to a value of 25 - 27⁰C and an oxygenator. Venous effluent from the organ is collected in an effluent reservoir below the organ and may be recirculated. Various sensors are provided in the circuit to monitor parameters of the perfusate. The specific problems related to normothermic preservation of organs are well documented and may at least be partially overcome by the reduced temperature operation of the device according to the present invention.

SUMMARY OF THE INVENTION

[0013] According to the invention there is provided a device that at least partially fulfils the considerations stated above. The device comprises a fluid circuit in which are arranged an organ chamber for receiving the organ and a quantity of a perfusion fluid; a pump for circulating the perfusion fluid around the fluid circuit; an oxygenator for oxygenation of the perfusion fluid; a pressure head device for maintaining a substantially constant pressure fluid supply to the organ; and an organ connector for forming a fluid connection from the pressure head device to the vasculature of the organ. Prior art devices have attempted to provide constant pressure supply using pumps calibrated to an appropriate flow rate. As perfusion continues however, stricture may occur in certain vessels of the organ leading to localized increase in pressure. As the pressure backs up, further edema may occur leading to further pressure increase. By ensuring that a constant pressure is supplied to the organ, damage due to edema can be substantially eliminated

[0014] According to an advantageous form of the invention, the organ chamber has an organ chamber inlet and an overflow connection is provided from the pressure head device to the organ chamber inlet. This is a particularly convenient way of ensuring that constant pressure is maintained. In use, excess fluid may be supplied to the pressure head device. The constant pressure supply to the organ vasculature determines the quantity of perfusion fluid that will follow this route via the organ connector. Fluid in excess of this quantity can pass through the overflow connection to the organ chamber.

[0015] In one embodiment of the device, the organ chamber is provided with a sealed closure with the organ connector passing through the closure. Preferably, the closure is of sufficient size and can be opened to allow insertion and removal of the organ from the organ chamber. This provides an extremely convenient arrangement for insertion of the organ into the chamber as e.g the arterial connection to the organ vasculature may be connected to the organ connector prior to inserting the organ into the organ chamber. Alternatively, the organ connector may be clipped in to a further connector passing through a wall of the organ chamber.

[0016] In a preferred embodiment of the invention, the organ may itself be suspended from the closure by the organ connector. This is considered preferable to certain prior arrangements in which the organ is supported from below on a solid platform. In particular, if the organ is immersed or substantially surrounded by the perfusion fluid, good protection against shocks is ensured while the organ is optimally bathed by the perfusion fluid.

[0017] The organ chamber may be provided with a number of additional connections. In particular, a filtered vent may be communicate with an upper part of the organ chamber allowing pressure equalization of the interior of the organ chamber. Furthermore, the organ chamber may comprise a chamber outlet located at an upper level of the organ chamber. The height of the chamber outlet may determine the level of the perfusion fluid within the organ chamber by acting as an overflow. As the fluid level rises above the chamber outlet, the perfusion fluid will flow out of the chamber and may be recirculated. The organ chamber may also preferably provide connections for insertion of various sensor probes for determining the condition of the perfusion fluid as will be explained in further detail below.

[0018] In a preferred form of the device, the pressure head device comprises a chamber having a free surface located at a vertical distance above the organ. Preferably, the pressure head device is itself located above the organ chamber but it is also within the scope of the invention inai only an upper portion of the pressure head device extends upwards and that the free surface is located in this upper portion. In this context, free surface refers to a surface of the perfusion fluid which determines the pressure head pressure with respect to the organ. Such a free surface is preferably achieved by providing an overflow outlet to the pressure head chamber such that excess fluid in the chamber will overflow through the overflow connection to the organ chamber. If the pressure head device also comprises a filtered vent allowing pressure equalization of an interior of the pressure head device, then both the free surface of the pressure head device and that of the organ chamber will be at atmospheric pressure. In such a case, the total head is the vertical distance between the two free surfaces. The location of these free surfaces may be adjustable to adjust the pressure of the fluid supply to the organ. This may be achieved by changing the relative heights of the respective chambers. Although the pressure head device has been described as a gravity feed, the skilled reader will readily understand that alternative forms of pressure head device, such as pressure responsive valves, may be used that ensure that excess fluid is circulated instead of being delivered to the vasculature.

[0019] According to a further aspect of the invention, the oxygenator may be located in the fluid circuit between the pressure head device and the organ. In this way, only the fluid that is actually perfused to the organ vasculature is directly supplied with oxygen prior to entering the organ. The remainder of the fluid in the circuit will have a lower oxygen content. This provides for a greater efficiency of oxygen usage and also allows oxygen usage across the organ to be more readily monitored. This may be achieved by providing an upstream oxygen sensor located in the fluid circuit between the oxygenator and the organ. The sensor may sense the oxygen saturation level of the oxygenated perfusion fluid or any equivalent indicator of this value.

[0020] According to another aspect of the invention, an oxygenator is provided comprising an oxygen inlet and an oxygen outlet connected by an oxygen permeable member. A source of oxygen connected to the oxygen inlet causes a flow of oxygen through the oxygen permeable member. The oxygen permeable member preferably comprises a plurality of small bore silastic tubing elements arranged in parallel. The oxygenator may further comprise an oxygenation chamber having an oxygenation inlet for perfusion fluid and an oxygenation ouiiel for oxygen enriched perfusion fluid, the oxygen permeable member being located within the oxygenation chamber between the oxygenation inlet and the oxygenation outlet. In this manner, perfusion fluid entering the chamber via the inlet is obliged to pass over the oxygen permeable member if it is to exit via the oxygenation outlet.

[0021] According to a still further aspect of the invention the oxygenator and the pressure head device may be combined as a single unit. In such an arrangement, an overflow outlet and a filtered vent will be arranged at an upper level within the oxygenation chamber providing an alternative outlet for perfusion fluid to exit without passing over the oxygen permeable member. Such a construction is understood to be in itself both novel and inventive.

[0022] In order to ensure a correct temperature operation of the device, there is preferably also provided a heat exchanger for cooling the perfusion fluid in the fluid circuit. The heat exchanger may comprise an insulated container containing a cooling medium such as ice or equivalent cooling packs. A heat exchange channel for the perfusion fluid can pass through the insulated container in heat exchanging relation with the cooling medium. A copper coil has been found suitable for use as a heat exchange channel although the skilled person will be well aware of further heat exchange elements that could ensure adequate heat transfer from the perfusion fluid to the cooling medium. For longer-term perfusion, circulation of the cooling medium or further refrigeration capability could also be provided.

[0023] In a preferred embodiment of the heat exchanger a bypass is provided allowing perfusion fluid to pass around the heat exchanger. A flow control member controls the amount of bypass flow. In this way, a freshly harvested organ may be rapidly chilled by routing all perfusion fluid through the heat exchanger. Once a correct temperature has been achieved, most of the perfusion fluid may be caused to bypass the heat exchanger. Further cooling is then only required to compensate for heat transfer into the device and for metabolic heat generated in the organ.

[0024] In order to provide correct and accurate operation of the device, a number of sensors may be provided at various locations in and around the fluid circuit. Thus a temperature sensor for monitoring the temperature of the perfusion fluid and/or monitoring the temperature within the device may be provided. Output of the temperature sensor or sensors may be used to control operation of the heat exchanger. Furthermore there may be provided: a downstream oxygen sensor for defecting an amount of oxygen entrained in the perfusion fluid after passing through the organ; a pH sensor for detecting a pH value of the perfusion fluid; a CO₂ sensor for detecting an amount OfCO₂ entrained in the perfusion fluid and further sensors for detecting other characteristics of the organ or parameters of the perfusion. The above sensors may preferably be located in the organ chamber but may also be located at any other convenient location where they can provide a reliable and accurate indication of the sensed value.

[0025] Preferably, the device comprises a monitoring unit operatively connected to receive sensor input from the above-mentioned sensors. The sensor input provides an indication of actual parameters of the operation of the device. A particularly important parameter for monitoring is the oxygen uptake across the organ which can be determined by monitoring the oxygen saturation level of the perfusion fluid upstream and downstream of the organ.

[0026] It is furthermore preferable that the device comprises a control unit operatively connected to receive user input from a user input device indicative of a desired value for certain parameters of operation of the device. The control unit may then control operation of the device to achieve such desired parameters. The control unit may control various elements of the device including but not limited to: the speed of the pump; the value of the constant pressure supply; the operation of the flow control member governing flow around the heat exchanger; the oxygen supply; and the addition of additional medication. [0027] In a preferred form of the device, there is provided an insulated housing and at least the organ chamber, the pressure head device and the oxygenator are located within the insulated housing. Preferably a source of cooling is also provided to the interior of the housing. The housing is preferably also provided with a dedicated source of energy making the whole device easily portable for transport of an organ.

[0028] The device is particularly suited to perfusion of a heart. Other organs such as the lungs, kidney and liver may also be treated as may other less conventionally transplanted organs and members such as severed limbs.

[0029] Various forms of perfusion solution may be used according to the nature of the organ to be oerfυsed. For perfusion of a heart, a Celsior type solution is preferred although adjustment of certain concentrations of elements may be required in order to optimize the preservation action at the chosen temperature.

[0030] In a most preferred embodiment of the invention, the device further comprises a medication infusion device operative to controllably introduce a quantity of medication into the perfusion fluid circuit. In this way intermediate addition or replenishment of certain components required for perfusion may be achieved. This manner is particularly important when dealing with medication that is unstable and which must be added shortly before delivery.

[0031] According to the invention there is also provided a method of ex-vivo perfusion of an organ, the method comprising: circulating a perfusion fluid at a first volumetric flow rate around the a fluid circuit; providing a supply of a perfusion portion of the perfusion fluid to the organ to realize a second volumetric flow rate through the vasculature of the organ, wherein the second volumetric flow rate is lower than the first volumetric flow rate; and supplying an overflow quantity of perfusion fluid, corresponding to the difference between the first and second volumetric flow rates, to a bypass line for bypassing the vasculature of the organ. The method may be performed as further herein described below.

[0032] By virtue of the invention as disclosed above there is provided a self-contained unit designed to maintain viability of a human heart for 16 hours or more. After the donor heart is removed, the aorta is sewn to an adapter that snaps into the organ chamber filled with oxygenated perfusion fluid. The pressure head device combined with oxygenation chamber is mounted atop the organ chamber and partially filled with perfusion fluid. The oxygenated fluid is maintained at 4° C throughout the holding period and continually circulated through the organ by gravity during storage. The fluid is oxygenated as it passes through the oxygenator just before the fluid enters the organ. Thus the organ is fed with the proper nutrients contained in the perfusion fluid, chilled to a temperature that sustains the organ for the longest period of time, and protected from bruising during a potentially bumpy ride. The result is an organ in pristine condition.

[0033] Perfusion that allows the transport of a harvested organ from a site distant from the location where the transplant surgery will be carried out requires the device to be lightweight, portable and to operates under sterile conditions for pumping the cold buffered nutrient salt perfusion soiuiion ihiough the organ blood vessels. In order for one person to carry the entire assembly without assistance, and to transport it in an auto or airplane, it is preferably compact, sturdy and light-weight. The system for loading the perfusion fluid is simple resulting in minimal spillage due in part to the large opening to the organ chamber. Appropriate arrangements are provided to ensure that sterility is maintained. To be completely portable, the device contains a source of oxygen, an energy source to operate the pump, sensors and alarms, and the housing is insulated, water tight and can be loaded with cold packs.

[0034] The insulated housing may be a single unit molded e.g. using polycarbonate and provided with the control panel and battery on the exterior. All fluid and gas lines may be color coded and supplied with quick connect closures. A "C" style oxygen bottle with regulator is preferred containing sufficient oxygen for 30+ hours of perfusion. Alarms may be included in the system in case of pump failure (loss of pressure), oxygen loss, or temperature spike, any of which could be fatal errors if not quickly corrected. The organ perfusion device can be easily loaded and unloaded by double-gloved surgical personnel and the fittings require minimal dexterity to assemble and disassemble.

[0035] The use of a light-weight, cooled, self-contained perfusion device according to the invention has a number of beneficial consequences:

[0036] (1) The organs are in better physiological condition at the time of transplantation. [0037] (2) Prolonging the survival time of donor organs will enlarge the pool of available organs by allowing organs to be harvested at a distance from the site of the transplant surgery in spite of a longer transport time.

[0038] (3) It allows more time for testing to rule out infection of the donor, for example with AIDS, hepatitis-C, herpes, or other viral or bacterial diseases.

[0039] (4) The pressure on transplant surgeons to complete the transplant procedure within a short time frame would be eased, providing surgical teams with more predictable scheduling and relieving transplant centers of crisis management. Transplant surgeons are often faced with unexpected surgical complications that prolong the time of surgery.

[0040] (5) Better preservation of the integrity of the heart and the endothelium of the coronary arteries at the time of transplantation may also lessen the incidence and severity of post-transplantation coronary artery disease.

[0041] (6) Perfusion with oxygenated fluid lessens reperfusion injury to the myocardium of the transplanted heart.

[0042] It is expected that the device according to the invention will deliver organs in better physiological condition, shorten recovery times, reduce overall cost, increase the available time to improve tissue matching and sizing of the organ, to perform clinical chemistries and diagnostic testing for infectious diseases prior to transplantation, enlarge selection of donor organs, and widen the range of available organs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The features and advantages of the invention will be appreciated upon reference to the following drawings of an exemplary embodiment, in which:

[0044] Figure 1 shows a schematic circuit diagram of the fluid circuit of an organ perfusion device according to the invention;

[0045] Figure 2 shows a perspective view of a preferred embodiment of an organ chamber according to the invention;

[0046] Figure 3 shows the organ chamber of figure 2 in plan elevation; [0047] Figure 4 shows a cross-section through the device of Figure 3 along line A-A;

[0048] Figure 5 shows a perspective view of a preferred embodiment of an oxygenator according to the invention;

[0049] Figure 6 shows a side elevation of the oxygenator of Figure 5;

[0050] Figure 7 shows a front elevation of the oxygenator of Figure 5,

[0051] Figure 8 shows a detailed view of the oxygen inlet of Figure 6;

[0052] Figure 9 shows a cross-section through the oxygen inlet of Figure 6 along line C-C;

[0053] Figure 10 shows a cross-section through the device of Figure 7 along line D-D;

W i ^I _{1- 1} Figure 1 ! -shows ά petspαUive view of a preferred embodiment of the organ perfusion device according to the invention;

[0055] Figure 12 shows a front elevation of the organ perfusion device of Figure 11 ; [0056] Figure 13 shows a side elevation of the organ perfusion device of Figure 11; [0057] Figure 14 shows a plan elevation of the organ perfusion device of Figure 11 ; and

[0058] Figure 15 shows an elevation of the organ perfusion device of Figure 11 in the direction F.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0059] According to Figure 1, there is disclosed a schematic representation of an organ perfusion device 1 for ex-vivo perfusion of an organ according to the present invention. The device comprises a number of individual components, connected together to form a fluid circuit 2 and a number of further components, peripheral to and interacting with the fluid circuit.

[0060] A first item in the fluid circuit 2 is an organ chamber 10. The organ chamber 10 has a chamber inlet 12 and a chamber outlet 14. Within the organ chamber 10, an organ 16 is suspended from an organ connector 18 and almost completely immersed in a perfusion fluid 20. The organ chamber 10 has a closure 22 in which the organ connector 18 is supported. The closure 22 also supports a filtered vent 24, an oxygen and temperature probe 26 and a pH probe 28. [0061] The chamber outlet 12 is connected to a pump inlet 30 of a pump 32 for circulating the perfusion fluid 20. The pump 32 may be any commercially available medical circulation pump. The pump also has a pump outlet 34.

[0062] The pump outlet 34 connects via a two-way valve 36 to a heat exchanger 38 for cooling the perfusion fluid 20. The heat exchanger 38 has a hx inlet 40, a hx outlet 42 and a hx bypass 44, the function of which will be described in further detail below.

[0063] A further component in the fluid circuit is an oxygenator 46 for oxygenation of the perfusion fluid. The oxygenator 46 has an oxygenation chamber 48 having a threaded cap 50 hermetically sealed thereto. The oxygenator 46 also comprises a number of ports for connection tn Φe fluid circuit 2 and to an oxygen source 52. At an upper part of the oxygenation chamber 48 an oxygenator inlet port 54 is connected in fluid communication with the hx outlet 42 of the heat exchanger 38 and an overflow port 56 is connected to the organ chamber inlet 12. At a lower part of the oxygenation chamber 48 are provided an oxygen inlet 58 and an oxygen outlet 60. The oxygen inlet 58 and oxygen outlet 60 are connected together internally of the oxygenation chamber 48 by an oxygen permeable member 62 as will be described in further detail below. The oxygen inlet 58 is connected to the oxygen source 52, the oxygen outlet 60 connects to a vent 64 Also, at the lower part of the oxygenation chamber 48, there is provided an oxygenator outlet port 66. The oxygenator outlet port 66 is connected in fluid communication to the organ connector 18 on the organ chamber closure 22.

[0064] The oxygenator 46 is also provided with a filtered vent 68 for equalizing pressure within the oxygenation chamber 48. The filtered vent 68 is connected through the threaded cap 50. Furthermore, an oxygen saturation probe 70 is provided in the oxygenator outlet port 66.

[0065] The entire perfusion fluid circuit 2 is contained in an insulated housing 72. The oxygen source 52 is exterior to the housing 72. Also located exterior of the housing 72 are a control unit 74 and alarm system 76. Control unit 74 comprises a display 78 and a number of input keys 80. It is connected to the alarm system 76 for generation of appropriate alarms to medical personnel in the event of malfunction etc. of the organ perfusion device 1. [0066] Figures 2 to 4 show a number of views of an organ chamber 10 according to a preferred embodiment of the invention for use in the perfusion and transport of a heart. As can be seen, the organ chamber 10 comprises a generally cylindrical body 81 having a removable secondary lid 82 in which the access opening 21 is provided. The closure 22 is retained in a closed position by a pair of fastening elements 84 of the quarter turn locking fastener type. The fastening elements 84 engage through the closure 22 into locking recesses 86. To prevent leakage of perfusion fluid, seals 88, 90 are located respectively between the organ chamber 10 and the secondary lid 82 and between the secondary Hd 82 and the closure 22. Hold-down clamps 92 retain the secondary lid 82 in position and also serve to immobilize the organ chamber 10 during transport.

|UU67] According to Figures 2 and 3, it can be seen that the filtered vent 24, oxygen and temperature probe 26 and pH probe 28 are all arranged in the secondary lid 22. In this manner, the closure 22 may be removed to allow insertion of an organ 16 without disturbing these connections. It can also be seen from Figure 4 that the organ chamber inlet 12 is provided at an upper part of the cylindrical body 81 and the chamber outlet 14 is provided at the lower part of the cylindrical body 81.

[0068] Figure 4 also illustrates the construction of the organ connector 18. The organ connector 2 comprises an organ portion 94 and a closure portion 96. The organ portion 94 may be connected to the organ by the surgeon by means of sutures. In the present case this is attached to the aorta, distal to the coronary artery for perfusion of the coronary vasculature. The closure portion 96 is connected through the closure 22. It is understood however that the closure portion 96 could also be integrally formed as part of the closure 22. The organ portion 94 and closure portion 96 are embodied as mating quick-connect connectors allowing a harvested organ 16 to be attached to the closure in an efficient fluid-tight manner. Any suitable medical grade quick-connect system e.g. luer lock, may be used for the organ connector.

[0069] Figures 5 to 10 show a number of views of the oxygenator 46 according to the preferred embodiment of the invention. As can be seen the oxygenator 46 has a generally cylindrical oxygenation chamber 48 having a base 100 and threaded cap 50 hermetically sealed thereto. According to the detail E of figure 10, a seal 98 is arranged between the oxygenation chamber 48 and the cap 50. Also visible in Figure 10 is the connection between the cap 50 and the filtered vent 68.

[0070] Figures 6 and 7 clearly show the location of the oxygenator inlet port 54 and overflow port 56 at the upper portion of the oxygenation chamber 48 and also the oxygenator outlet port 66 located centrally in the base 100. The oxygenator outlet port 66 is in the form of a standard Y-connector 1 12. The oxygen saturation probe 70 is inserted into a side branch 114 of the Y-connector 112. The oxygen inlet 58 and oxygen outlet 60 enter the oxygenation chamber 48 adjacent to the base.

[0071] The form of the oxygen inlet 58 is shown in further detail in Figures 8 and 9. The oxvgen oηtW 60 is essentially identical to the oxygen inlet 58 and will not be further described. The oxygen inlet 58 comprises a threaded plug 102 that can be secured hermetically into a mating socket in the wall of the oxygenation chamber 48. The plug 102 has a stub connector 104 for reception of tubing 106 leading to the oxygen source 52. A plurality of small bores 108 - in this case 8 - pass through the plug 102. Each bore 108 receives an end of a section of silastic tubing 1 10. The silastic tubing 110 is preferably .07" diameter medical grade tubing and has been found to be particularly suitable as an oxygen permeable member 62. The silastic tubing 110 is potted into the stub connector 104 using a suitable potting compound. As can be seen in Figure 7, the silastic tubing 110 extends from the oxygen inlet 58 to the oxygen outlet 60.

[0072] Figures 11 to 15 illustrate the configuration of the organ perfusion device 1 according to the preferred embodiment. According to Figure 11, insulated housing 72 is partially cut-away to reveal the organ chamber 10, oxygenator 46 and heat exchanger 38. These components are connected together by standard 3/16" internal diameter medical grade tubing (not shown) to form the fluid circuit 2 as described above. The tubing is color coded to ensure correct connection of the fluid circuit 2. A preferred material for the housing is polystyrene although other plastics materials or combinations of material offering good thermal insulation and the necessary rigidity may also be used. Figure 11 also shows a frame 1 12 providing additional rigidity to the structure and also serving as a mounting for the attachment of various components. [0073] In Figure 12, the insulated housing 72 is omitted for the sake of clarity. It can also be observed that the hold-down clamps 92 are attached at their lower ends 1 14 to a base plate 1 16 to prevent movement of the organ chamber 10. The oxygenator 46 is also supported from the base plate 116 by a pair of stands 118. Stands 118 maintain the oxygenator 46 at the correct height above the organ chamber 10. This height is adjustable by appropriate connections between the oxygenator 46 and the stands 118. Although not shown, it is understood that automatic height adjustment could also be provided.

[0074] Figures 13, 14 and 15 show respectively a side view, plan view and angled view of the organ perfusion device 1. In these figures the location of the oxygen source 52 on the rear face of the frame 1 12 is shown. There is also illustrated a pump drive 120, a battery pack 122 and a control unit 74. The pump drive 120 is located externally of the insulated housing 72 and communicates with the pump 32 (not shown) located inside the insulated housing 72 and forming part of the fluid circuit 2. By mounting these components external of the housing 72, easy access is ensured. As these components may furthermore generate heat, it is desirable that they should not be located within the insulated housing 72.

[0075] Operation of the device will now be described with reference to Figures 1 to 15.

[0076] Prior to use, the organ perfusion device 1 is prepared for receipt of an organ: the organ chamber 10 is partially filled with a quantity of perfusion fluid 20, cooled to around 40 C; ice packs are inserted into the heat exchanger 38; the battery 122 is fully charged; and a fresh source of oxygen 52 is connected. The device 1 may then be primed by operation of the pump 32 for circulation of the perfusion fluid 20 around the fluid circuit 2 to remove any unwanted air that may be present.

[0077] An organ 16 is removed from a donor according to the applicable protocol. The present example will be given in relation to a heart but it is to be understood that the principles of the procedure may be adapted for the ex vivo perfusion of any organ. The harvested organ 16 is then attached to the organ portion 94 of the organ connector 18 by suturing of the aorta such that the interior of the organ portion 94 communicates with the entry to the coronary artery. In order to insert the organ 16 into the organ chamber 10, the fastening elements 84 are unlocked and the closure 22 is removed. The organ 16 may then be lowered into the organ chamber 10 and the two parts of the organ connector 18 are joined together. Prior to closing of the closure 22, a further quantity of perfusion fluid 20 may be added if necessary to bring the level in the organ chamber 10 up to the required point such that the organ 16 is effectively immersed in the fluid 20. The closure 22 is then firmly and hermetically closed and the fastening elements 84 locked into position.

[0078] Once the organ 16 is correctly installed in the organ chamber 10, the overseeing surgeon or medical technician initiates operation of the organ perfusion device 1. Using the input keys 80 on the control unit 74, the device is set to an "INITIATE PERFUSION" mode. In this mode, the pump 32 commences to circulate perfusion fluid 20 from the organ chamber 10 via the organ chamber outlet 14 to the heat exchanger 38. The two-way valve 36 is initially set to direct the flow through the heat exchanger 38. Here it is rapidly cooled by heat exchange to tne ice packs contained in the heat exchanger.

[0079] The cooled perfusion fluid 20 leaves the heat exchanger 38 via the hx outlet 42 and is directed to the oxygenator 46 via the oxygenator inlet port 54. Although reference is made to oxygenator 46 it is to be understood that this component is in fact a combined oxygenator and pressure head device. These distinct functions could also be separately provided in two different items. Perfusion fluid 20 entering the oxygenator 46 causes the level of fluid in the oxygenation chamber 48 to rise above the level of the overflow outlet 56. As a consequence, flow takes place through the overflow outlet 56 back to the organ chamber via the organ chamber inlet 12.

[0080] Consecutively with the flow through the overflow outlet 56, perfusion fluid 20 also passes through the oxygenator outlet 66 to the organ connector 18 and into the organ 16. In the illustrated example, the perfusion fluid 20 enters the heart via the aorta and passes to the coronary artery of the heart for perfusion through the circulatory system of the heart. The perfusion fluid 20 exits from the cardiac system via the coronary vein and/or vena cava directly into the interior of the organ chamber 10.

[0081] According to the present invention, the flow rate of perfusion fluid 20 through the coronary system (coronary flow) is determined by the pressure at the inlet to the coronary artery (with respect to the outlet). This pressure is determined effectively by the difference in height H between the fluid level in the oxygenator 46 and the fluid level in the organ chamber. By maintaining a constant height difference H, a substantially constant coronary flow is achieved. By ensuring that the pump 32 circulates perfusion fluid 20 at a rate greater than the coronary flow, there will always remain a flow through the overflow port 56 and the level in the oxygenation chamber 48 will remain substantially constant. In the present embodiment, the height H is set to 12.6" which equates to a pressure across the coronary system of around 3100 N/m2. This pressure has been found substantially adequate for maintaining an ideal perfusion flow of 7-9 ml/min per 100 grams of weight.

[0082] Of particular importance to the present invention, it may be noted that should restriction occur in the coronary system, the coronary flow may be reduced. In known systems, such restrictions may lead to increases of pressure within the coronary system and possible damage to the organ. According to an aspect of the present invention, should restriction occur in the coronary system, more perfusion fluid 20 may circulate from the oxygenation chamber 48 via the overflow port 56 to the organ chamber 10. The pressure to which the coronary system is exposed remains constant however, based on the pressure head H. Due to the substantially constant circulation by pump 32, the cooling effect of the perfusion fluid remains substantially constant. Only the volume of fluid passing through the coronary system will be slightly reduced.

[0083] During the "INITIATE PERFUSION" mode, the control unit 74 monitors the temperature of the perfusion fluid 20 using the oxygen and temperature probe 26. Initially, this temperature will have risen on immersion of the freshly harvested warm heart in the organ chamber. As perfusion of chilled fluid through the coronary system takes place, the temperature measured by the oxygen and temperature probe 26 drops. Once the temperature reaches 4° C (or another desired value) the control unit 74 controls the two-way valve 36 to direct flow of the perfusion fluid 20 via the hx bypass 44. During steady-state operation of the device, the control unit 74 intermittently controls the two-way valve 36 to alternately direct flow through the hx inlet 40 or through the hx bypass. In this way temperature control of the perfusion fluid 20 and the organ 16 is achieved. It is of course clear that other control methods could also be adopted e.g. by using a mixer valve instead of two-way valve 36.

[0084] Oxygenation of the perfusion fluid 20 takes place in the oxygenator 46. Specifically, as described above, the oxygenation chamber 48 contains an oxygen permeable member 62 comprising a number of lengths of silastic tubing 110. On commencing perfusion, control unit 74 controls the oxygen source 52 to supply oxygen gas to the oxygen inlet 58. The oxygen supply is regulated at the oxygen inlet 58. The oxygen passes via the oxygen inlet 58 and is distributed to the eight lengths of silastic tubing 1 10. Because of the permeable nature of the silastic material and the driving pressure difference between the interior of the tubing 1 10 and the oxygenation chamber 48, oxygen can permeate through the wall of the tubing 110 into the perfusion fluid 20. Residual oxygen gas exits from the silastic tubing 110 via the oxygen outlet 60 to the vent 64.

[0085] The arrangement of the silastic tubing 110 in the base of the oxygenation chamber 48 ensures that all of the perfusion fluid 20 passing to the oxygenation outlet 66 comes into close contact therewith. The perfusion fluid 20 supplied to the organ 16 via the organ connector 18 is thus rich in oxygen. The oxygen saturation level is measured on exit from the oxygenator 46 by the oxygen saturation probe 70. The control unit 74 controls the oxygen supply 52 to ensure the perfusion fluid is saturated.

[0086] The control unit 74 also determines the oxygen saturation level as measured by the probe 26. By analyzing both values, the oxygen uptake across the heart may be determined and recorded, providing an indication of the level of metabolic activity in the tissue of the heart. Although according to the above embodiment the probe 26 is located in the organ chamber 10, it is also contemplated that such sensors may be located directly at the exit from the coronary vein in order to more closely determine the characteristics of the perfusion fluid 20 on exit from the organ 16.

[0087] Of significance in the performance of the perfusion device, the perfusion fluid must be carefully selected to optimize organ survival. Whereas other solutions described in the literature and in patents are for "preservation" of organs; wherein the organ is immersed in the solution passively, the solution described below is for perfusion of the organ. Although this solution was developed principally for heart perfusion, it represents a significant advantage particularly for the perfusion of kidneys, and also liver, lung and other organs.

[0088] The perfusion fluid, in stable form prior to use, may comprise physiologically acceptable concentrations of NaCl, CaCl₂ * 2H₂O, MgCl₂, K₂HPO₄, L-histidine, lactobionic acid as a lactone, D-mannitol, D-glucose, sodium L-glutamate, and adenosine. [0089] In the preferred embodiment, the perfusion fluid, in stable form prior to use, may comprise about 25 mM NaCl, about 0.5 mM CaCl₂ • 2H₂O, about 14.5 mM MgCl₂, about 15 mM K₂HPO₄, about 25 mM L-histidine, about 80 mM lactobionic acid, about 30 mM D- mannitol, about 12 mM D-glucose, about 15 mM Sodium L-glutamate, and about 5 mM Adenosine. The pH of the perfusion fluid is adjusted to about 7.6 at 22.5⁰C. This slightly alkaline pH neutralizes the lactic acid produced by the organ carrying out glycolysis. The following unstable components may be added to the perfusion fluid immediately before use: about 1 unit/50 ml regular insulin, about 0.2 mM amiloride, about 3 mM L-glutathione (reduced), and about 5 mM D-fructose bisphosphate.

[0090] A principal aim during perfusion is to maintain the integrity of the cardiac cell membrane, and the integrity of the cardiac cell membrane potential. The cardiac cell normally has a high concentration of potassium and a low concentration of sodium, while the extracellular fluid has a low potassium concentration and high sodium concentration. The intracellular cardiac ion concentrations are maintained by pumping sodium ions out of the cell by an energetically driven process. When the heart is cooled, energy production by oxidative phosphorylation stops, and sodium ions are no longer pumped out. The intracellular sodium concentration then increases. The sodium overload produced is accompanied by an abnormally high calcium influx that causes muscle cell injury and death by several different mechanisms. Therefore, the object during perfusion is to minimize the leakage of sodium ions into the heart cells, and the loss of potassium ions outward.

[0091] The formulation of the perfusion fluid described above confers several advantages contributing to the preservation of the organ. For example, high magnesium and low calcium keeps the heart in hyperpolarized arrest and helps preserve the heart muscle cell membrane so that membrane excitability is better restored after transplantation. The use of histidine allows a greater buffering capacity while holding the phosphate concentration down. Lactobionate does not permeate the cardiac muscle cell membrane and acts to reduce swelling. Mannitol is another impermeant and along with glutathione, is also a free radical scavenger. Fructose bisphosphate stabilizes heart muscle cell membranes, reduced oxygen free radical formation, reduces lipid peroxidation, and reduces damage to the coronary artery epithelium. Glutamate helps functional recovery after transplantation. Adenosine prevents peripheral vasoconstriction in the coronary circulation during long term perfusion; it increases the store of high energy phosphates in heart muscle; there is better recovery of the heart after transplantation.

[0092] The survival of cardiac muscle at 4° C depends on glycolysis that utilizes the muscle glycogen stores and produces lactic acid. Glucose is a nutrient energy source. Its use preserves the cardiac muscle glycogen. While glucose has often been used in Krebs- Henseleit perfusion solution for Langendorff perfusions, it has not been used in "preservation" solutions. This solution uses a higher glucose concentration than in plasma, which is protective. See e.g. Saupe, K., et al. J. Molec. Cell. Cardiol. 2001, 33:261-269. Insulin enhances the uptake of glucose into heart muscle cells, and promotes glucose utilization, lnsuiin also inhibits programmed cell death (apoptosis).

[0093] Amiloride is an inhibitor of the sodium-hydrogen ion-exchanger (NHE), a membrane transporter involved in the movement of sodium ions into the cardiac muscle cell in exchange for hydrogen ions moving outward. Six isoforms of NHE are known; NHE-I is the principal isoform in cardiac muscle cells. Normally NHE acts as a protective mechanism against acidosis. At 4° C, however, muscle cells survive only by glycolysis that produces lactic acid. Outward movement of these hydrogen ions is accompanied by a large influx of sodium ions that is detrimental to heart cells. Inhibition of NHE-I reduces sodium influx; the hydrogen ions then diffuse out of the cells without the influx of sodium ions. Thus, NHE-I inhibition reduces damage from anoxia, and the damage from reperfusion injury after transplantation. Three more powerful and more specific NHE-I inhibitors, eniporide, cariporide,and zoniporide, may be also be used for the perfusion solution.

[0094] Thus, the invention has been described by reference to certain embodiments discussed above. It will be recognized that these embodiments are susceptible to various modifications and alternative forms well known to those of skill in the art. Many modifications in addition to those described above may be made to the structures and techniques described herein without departing from the spirit and scope of the invention. Accordingly, although specific embodiments have been described, these are examples only and are not limiting upon the scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A device for ex-vivo perfusion of an organ, the device comprising in a fluid circuit: an organ chamber for receiving the organ and a quantity of a perfusion fluid; a pump for circulating the perfusion fluid around the fluid circuit; an oxygenator for oxygenation of the perfusion fluid; a pressure head device for maintaining a substantially constant pressure fluid supply to the organ; and an organ connector for forming a fluid connection (from the pressure head αcv lvOj iu iho /asυulature of the organ.

2. The device as claimed in claim 1, further comprising an organ chamber inlet and wherein an overflow connection is provided from the pressure head device to the organ chamber inlet.

3. The device as claimed in claim 1 or claim 2, further comprising a sealed closure to the organ chamber, the organ connector passing through the closure.

4. The device as claimed in claim 3, wherein the closure may be opened to allow insertion and removal of the organ from the organ chamber.

5. The device as claimed in any preceding claim, wherein the organ is suspended from the organ connector.

6. The device as claimed in any preceding claim, wherein the organ is substantially surrounded by the perfusion fluid.

7. The device as claimed in any preceding claim, wherein the organ chamber comprises a filtered vent allowing pressure equalization of the interior of the organ chamber.

8. The device as claimed in any preceding claim, wherein the organ chamber comprises a chamber outlet located at an upper level of the organ chamber, the chamber outlet determining a substantially constant level of perfusion fluid within the organ chamber.

9. The device as claimed in any preceding claim, wherein the pressure head device comprises a chamber having an overflow outlet for defining a free surface located at a vertical distance above the organ.

10. The device as claimed in claim 9, wherein the location of the free surface is adjustable to adjust the pressure of the fluid supply to the organ.

11. The device as claimed in any preceding claim, wherein the pressure head device comprises a filtered vent allowing pressure equalization of an interior of the pressure head device

12. The device as claimed in any preceding claim, wherein the oxygenator is located in the fluid circuit between the pressure head device and the organ.

13. The device as claimed in any preceding claim, wherein the oxygenator comprises an oxygen inlet and an oxygen outlet connected by an oxygen permeable member.

14. The device as claimed in claim 13, wherein the oxygen permeable member comprises a plurality of small bore silastic tubing elements arranged in parallel.

15. The device as claimed in claim 13 or claim 14, wherein the oxygenator comprises an oxygenation chamber having an oxygenation inlet for perfusion fluid and an oxygenation outlet for oxygen enriched perfusion fluid and wherein the oxygen permeable member is located within the oxygenation chamber between the oxygenation inlet and the oxygenation outlet.

16. The device as claimed in any preceding claim, wherein an upstream oxygen sensor for sensing the oxygen saturation level of the oxygenated perfusion fluid is located in the fluid circuit between the oxygenator and the organ.

17. The device as claimed in any preceding claim, wherein the oxygenator comprises part of the pressure head device.

18. The device as claimed in any preceding claim, further comprising a source of oxygen.

19. The device as claimed in any preceding claim further comprising a heat exchanger for cooling the perfusion fluid.

20. The device as claimed in claim 19, wherein the heat exchanger comprises a bypass allowing perfusion fluid to pass around the heat exchanger and a flow control member for controlling an amount of bypass flow.

21. The device as claimed in claim 19 or claim 20, wherein the heat exchanger comprises a insulated container containing a cooling medium and a heat exchange channel for the perfusion fluid to pass through the insulated container in heat exchanging relation W itli iht; cooling medium.

22. The device as claimed in any preceding claim, further comprising a downstream oxygen sensor for detecting an amount of oxygen entrained in the perfusion fluid after passing through the organ, the downstream oxygen sensor being preferably located in the organ chamber.

23. The device as claimed in any preceding claim further comprising a pH sensor for detecting a pH value of the perfusion fluid, the pH sensor being preferably located in the organ chamber.

24. The device as claimed in any preceding claim, further comprising a CO₂ sensor for detecting an amount of CO₂ entrained in the perfusion fluid, the CO₂ sensor being preferably located in the organ chamber.

25. The device as claimed in any preceding claim, further comprising a temperature sensor for monitoring the temperature of the perfusion fluid and/or monitoring the temperature within the device.

26. The device as claimed in any preceding claim, further comprising a monitoring unit operatively connected to receive sensor input from device sensors, indicative of actual parameters of the operation of the device.

27. The device as claimed in any claim 26, wherein the monitoring unit monitors the oxygen uptake across the organ.

28. The device as claimed in any preceding claim, further comprising a control unit operatively connected to receive user input from a user input device indicative of desired parameters of operation of the device, the control unit controlling operation of the device to approximate such desired parameters.

29. The device as claimed in any preceding claim, further comprising an insulated housing, at least the organ chamber, the pressure head device and the oxygenator 'uJmg located within the insulated housing.

30. The device as claimed in any preceding claim, wherein the organ is selected from the group consisting of: a heart, a heart and lungs, lungs, a kidney, a liver.

31. The device as claimed in any preceding claim, wherein the perfusion fluid comprises a modified Celsior solution.

32. The device as claimed in any preceding claim, further comprising a medication infusion device operative to controllably introduce a quantity of medication into the perfusion fluid circuit.

33. A portable organ perfusion device comprising: an organ chamber having a fluid-tight closure, the closure being openable for insertion of an organ into an interior of the organ chamber, the organ chamber having a chamber inlet and a chamber outlet for perfusion fluid; a pump for circulating the perfusion fluid, the pump having a pump inlet in fluid communication with the chamber outlet and further having a pump outlet; a heat exchanger for cooling the perfusion fluid, the heat exchanger having a hx inlet in fluid communication with the pump outlet and further having a hx outlet; an oxygenator for oxygenation of the perfusion fluid, the oxygenator having an oxygenator inlet in fluid communication with the hx outlet and further having an oxygenator outlet; an oxygen source, the oxygen source being connected to the oxygenator for supply of oxygen thereto; an organ connector for forming a fluid connection from the oxygenator outlet to an inlet of the vasculature of the organ, an outlet of the vasculature of the organ being in fluid communication with the interior of the chamber; a bypass channel forming a fluid connection from upstream of the oxygenator to the chamber inlet; a first oxygen sensor for sensing the oxygen saturation level of the perfusion fluid flowing from the oxygenator to the organ and a second oxygen sensor for sensing the oxygen saturation level of the perfusion fluid downstream of the organ; a temperature sensor for measuring the temperature of the perfusion fluid; a pH sensor for measuring the pH of the perfusion fluid; a control unit for receiving information from the respective sensors and providing control instructions to the pump; and a thermally insulated housing, at least the organ chamber, the oxygenator and the heat exchanger being located within the housing.

34. A method of ex-vivo perfusion of an organ, the method comprising:

circulating a perfusion fluid at a first volumetric flow rate around a fluid circuit;

supplying a perfusion portion of the perfusion fluid to the organ to realize a second volumetric flow rate through the vasculature of the organ, wherein the second volumetric flow rate is lower than the first volumetric flow rate; and supplying an overflow quantity of perfusion fluid, corresponding to the difference between the first and second volumetric flow rates, to a bypass line for bypassing the vasculature of the organ.

35. The method as claimed in claim 34, wherein the perfusion portion is supplied to the organ by a pressure head device providing a constant pressure supply of the perfusion fluid.

36. The method as claimed in claim 34 or claim 35, wherein the organ is contained in an organ chamber and the overflow quantity is supplied to the organ chamber for bathing ci the orgar\

37. The method as claimed in any of claims 34 to 36, further comprising oxygenating the perfusion portion.

38. The method as claimed in any of claims 34 to 37, further comprising cooling the perfusion fluid, preferably to below 1O⁰C and more preferably to between 1 and 4⁰C.

39. The method as claimed in any of claims 34 to 38, wherein the perfusion portion is supplied to the vasculature of the organ at a pressure of between 10" and 20" of water, more preferably at around 12.6".

40. A solution for the perfusion of an organ comprising physiologically acceptable concentrations of:

sodium chloride; calcium chloride; magnesium chloride; potassium phosphate dibasic; L-histidine; a lactone; D-mannitol; D-glucose; sodium L-glutamate; and adenosine.

41. The solution of claim 40 wherein the pH is adjusted to 7.6 at 22.5⁰C.

42. The solution of claim 40, further comprising:

insulin at a concentration of about 20 units; amiloride at a concentration of about 0.2 mMolar; L-glutathione (reduced) at a concentration of about 3 mM; and D-fructose bisphosphate at a concentration of about 5 mM

43. The solution of claim 42 wherein the insulin, amiloride, L-glutathione (reduced), and D-fructose bisphosphate are added immediately before use.

44. The solution of claim 40 with concentrations comprising:

sodium chloride at a concentration of about 25 mMolar;

calcium chloride at a concentration of about 0.5 mMolar;

magnesium chloride at a concentration of about 14.5 mMolar;

potassium phosphate dibasic at a concentration of about 15 mMolar;

L-histidine at a concentration of about 25 mMolar;

Lactobionic acid as a lactone at a concentration of about 80 mMolar;

D-mannitol at a concentration of about 30 mMolar;

D-glucose at a concentration of about 12 mMolar; sodium L-glutamate at a concentration of about 15 mMolar; and adenosine at a concentration of about 5 mMolar.