Dynamic organ storage system

Abstract

A dynamic organ storage system includes an insulating container with a lid enclosing an internal volume. Within the internal volume, an organ container with a lid that houses an organ submerged in perfusate, the lid includes at least one inlet port and at least one outlet port. At least one pressure bag, storing more of the perfusate, is tilted at an angle above the organ container. At least one infusion line enters the organ container via the at least one inlet port and connecting the at least one pressure bag to the organ to gravity flow the perfusate into the organ. A sampling line connected to the organ exits the organ container through the at least one outlet port for a user to monitor a condition of the organ. At least one cooling mechanism maintains the organ at a hypothermic temperature for a time without requiring power.

Description

DYNAMIC ORGAN STORAGE SYSTEMCross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 606,000, entitled “DYNAMIC ORGAN STORAGE SYSTEM,” filed 13 December 2023 and U.S. Provisional Application No. 63 / 538,471 , entitled “DYNAMIC ORGAN STORAGE,” filed 14 September 2023. These provisional applications are hereby incorporated by reference in their entirety for all purposes.Technical Field

[0002] The present disclosure relates generally to extracorporeal organ support and, more specifically, to a dynamic organ storage system that enables cold storage of an extracorporeal organ with perfusion and monitoring capabilities without requiring an external power supply during operation.Background

[0003] Organ transplantation is the only curative therapy for many end-stage diseases. The lack of viable donor organs for transplantation is enhanced due to significant bottlenecks arising from insufficient organ preservation and recovery and transportation strategies when a donor organ does become available. Static cold storage is a common method of organ preservation, but the use of static cold storage is limited by a number of issues, including tissue damage induced by prolonged hypothermic preservation, difficulty in assessing donor organ function and viability, inevitability of ischemia-reperfusion injury, and limited opportunity for organ repair.Alternatively, machine perfusion systems have promised to revolutionize organ storage, allowing for greater organ perfusion and having monitoring capabilities. However, machine perfusion systems are expensive, bulky, heavy, require significant external power, and can include many, or all, of the other limitations of static cold storage.Summary

[0004] Described herein is a dynamic organ storage system that enables cold storage of an extracorporeal organ in a stable condition for a longer period of time than traditional storage mechanisms. The dynamic organ storage system is equipped withperfusion and monitoring capabilities (e.g., ensuring temperature stability) without requiring an external power supply during operation. Moreover, the dynamic organ storage system is highly transportable, significantly reducing the costs of storing and / or transporting a potentially life-saving extracorporeal organ.

[0005] In an aspect, the present disclosure can include an organ preservation system for dynamic storage of an extracorporeal organ. The system can include an insulating container with a securable lid for enclosing an internal volume of the insulating container. The internal volume of the insulating container can further house an organ container, at least one pressure bag, at least one infusion line, a sampling line, and at least one cooling mechanism. The organ container can house an organ at least partially submerged in a volume of a perfusate and can have a lid to fully enclose the organ container. The lid can include at least one inlet port and at least one outlet port. The at least one pressure bag can store another volume of the perfusate and can be positioned above the organ container and tilted at an angle relative to the lid of the organ container. The at least one infusion line can enter the organ container via the at least one inlet port and connect the at least one pressure bag to the organ to gravity flow the perfusate from the at least one pressure bag into the organ. The sampling line can be connected to the organ and can allow a user to monitor a condition of the organ. The sampling line can exit the organ container via the at least one outlet port. The at least one cooling mechanism can maintain the organ at a hypothermic temperature for a time without requiring power.

[0006] In another aspect, the present disclosure can include a method for hypothermic organ preservation by dynamically storing an extracorporeal organ. The steps can include the following. Placing an organ in an organ container so that the organ is at least partially submerged within a volume of perfusate, wherein the organ container is within an internal volume of an insulating container. Connecting at least one infusion line and a sampling line to the organ. Enclosing the organ container with a lid having at least one inlet port that accepts the at least one infusion line and at least one outlet port that accepts the sampling line. Placing the organ container in an insulating container having an internal volume, a securable lid for enclosing the internal volume, and at least one cooling mechanism positioned in the internal volume. Connecting the atleast one infusion line to at least one pressure bag. Positioning the at least one pressure bag, storing another volume of the perfusate, above the organ container in the insulating container and tilted at an angle relative to the lid of the organ container so that the other volume of the perfusate gravity flows to the organ. Cooling the internal volume of the insulated container with the one or more cooling mechanisms to maintain the organ at a hypothermic temperature for a time, without requiring power. Perfusing the organ with perfusate gravity fed from the at least one pressure bag through the at least one infusion line to the organ for the time, without requiring power, where the at least one infusion line is cannulated into at least one vein and / or artery of the organ. A user can be allowed to monitor a condition of the organ using at least one sensor accessible with the sampling line with the securable lid closed.Brief Description of the Drawings

[0007] The foregoing and other features of the present disclosure will become apparent to those skilled in the art to which the present disclosure relates upon reading the following description with reference to the accompanying drawings, in which:

[0008] FIGS. 1 and 2 are schematic diagrams of an example of a dynamic organ storage system;

[0009] FIG. 3 is schematic diagram of a system including the dynamic organ storage system of FIG. 2 and an external device;

[0010] FIGS. 4-10 are pictorial views of setting up and using an example dynamic organ storage system to the one shown in FIGS. 1 and 2;

[0011] FIG. 11 shows illustrations of example organ container configurations;

[0012] FIG. 12 is a graphical representation of the temperature of an example organ and the insulated container (Yeti®) in a dynamic organ storage system similar to the one shown in FIGS. 1 and 2 over a course of 12 hours;

[0013] FIG. 13 is an illustration of an example pressure bag;

[0014] FIG. 1 is an illustration of an example insertion port;

[0015] FIGS. 15 and 16 are sketches of kidney containers for use with a dynamic organ storage system of FIGS 1 and 2;

[0016] FIGS. 17 and 18 are sketches of heart cannulation for use with the systems described above;

[0017] FIGS. 19-21 are pictorial examples of different cannulas that can be used by one or more of the systems described above;

[0018] FIG. 22 is a process flow diagram illustrating a method for hypothermic preservation of an organ with a dynamic organ storage system;

[0019] FIG. 23 shows illustrations of an experimental schematic and pictures of an example system set-up;

[0020] FIGS. 24-27 are graphical representations of experimental results; and

[0021] FIGS. 28 and 29 are pictorial and graphical representations of experimental results.Detailed DescriptionI. Definitions

[0022] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0023] As used herein, the singular forms “a,” “an”, and “the” can also include the plural forms unless the context clearly indicates otherwise.

[0024] As used herein, the terms “comprises” and / or “comprising,” can specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups.

[0025] As used herein, the term “and / or” can include any and all combinations of one or more of the associated listed items.

[0026] As used herein, the terms “first,” “second,” etc. should not limit the elements being described by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure. The sequence of operations (or acts / steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

[0027] As used herein, the terms “dynamic organ storage”, “dynamic organ preservation” or the like refer to a technology that enables storage of an extracorporeal organ with perfusion and monitoring capabilities. In a dynamic organ storage system, aperfusate is flowed through an organ, the organ can be oxygenated to maintain the organ at or near physiological stability, one or more health factors of the organ can be monitored, or the like.

[0028] As used herein, the term “perfusate” refers to any fluid flowed through an extracorporeal organ to assist in preserving the physiological stability of the organ. A perfusate can be blood and / or a man-made solution. A perfusate can include one or more added nutrients and / or added oxygen. In some instances, the perfusate can be a liquid and / or primarily a liquid.

[0029] As used herein, the term “pressure bag” refers to a specialized container designed to use air pressure to increase an infusion speed of a volume of liquid (e.g., perfusate) stored in the container into an infusion line and to an organ.

[0030] As used herein, the term “infusion line” refers to a path that a perfusate (e.g., blood, a man-made perfusate, or the like), flows through from a pressure bag to supply oxygen and nutrients to an organ.

[0031] As used herein, the term “effluent” refers to liquid waste from an organ and / or an organ container (e.g., used perfusate).

[0032] As used herein, the term “condition (of an organ)” refers to a state of the organ regarding appearance, quality, working order, and the like of the organ. The condition of the organ can be determined based on one or more measured biomarkers. Such as, but not limited to (depending on the organ type): venous oxygen saturation (SvO2) and arterial oxygen saturation (SaO2) (outflow & inflow saturations); lactate concentration; mitochondrial markers; glucose concentration; pH; hematocrit and / or hemoglobin (Hgb) level(s); organ specific markers such as troponin for hearts; organ specific exosome markers; temperature (inflow, outflow, and / or container); organ specific metabolites; inflow pressure of perfusate; line pressure of an infusion line; and organ specific venous and / or arterial flow (e.g. measured at a portal vein and / or hepatic artery for a liver).

[0033] As used herein, the term “hypothermic temperature” refers to an environmental temperature or temperature of an organ below at least 32 degrees Celsius, preferably between approximately 0 and 14 degrees Celsius. Lowering the temperature of a euthermic organ to a temperature below that normally maintained byhomeostasis of an organ reduces the organ’s metabolic rate, and thus the demand for oxygen and substrates by the tissues to protect against the effects of ischemia and hypoxia.

[0034] As used herein, the term “organism” can refer to any warm-blooded organism including, but not limited to, a human being, a pig, a rat, a mouse, a dog, a car, a goat, a sheep, a horse, a monkey, an ape, a rabbit, a cow, etc.

[0035] As used herein, the terms “extracorporeal organ” or “organ” refers to any organ situated outside the body of an organism (e.g., an organ provided by an organ donor, a lab grown organ, or an organ detached (voluntarily or involuntarily) from the host organism). An extracorporeal organ can include, but is not limited to, an internal organ (e.g., heart, liver, lungs, kidney, pancreas, small intestine, gut, etc.), an external organ (e.g., skin), tissue, a bioengineered graft, a xenogeneic organ graft or a limb (e.g., arm, leg, hand, foot, finger, toe, etc.).II. Overview

[0036] Donor organs begin to lose functionality upon a donor’s death or removal from a live donor’s body. Preservation can extend the functionality of donor organs for transplant to a recipient. Traditionally, donor organs have been preserved through static cold storage or machine perfusion systems, both of which have significant limitations with regard to duration, transportability, and / or quality of preservation. Static cold storage, the most common method of preserving solid organs, refers to cooling an organ to a hypothermic state for a limited amount of time to slow metabolic processes and decomposition. The time limit for static cold storage can be organ dependent but is generally limited to about four to eight hours before ischemia or other irreversible damage occurs. In fact, the combination of ischemia and static cold storage has been associated with an increased risk of graft dysfunction, graft failure, and posttransplant complications such as ischemic cholangiopathy. Machine perfusion systems, either hypothermic or normothermic, can deliver a perfusate through an organ’s vasculature in a continuous or pulsatile manner to elongate the preservation time of the organ, in some cases by as much as about ten hours. Traditionally, however, machine perfusion systems are heavy, bulky, and extremely difficult to transport due to size and externalpower requirements to run all the system components. For instance, machine perfusion systems can include, rotor or centrifugal pumps, oxygenators, heat exchangers, and bulky monitoring systems. The cost per organ being transplanted can be prohibitive, running from around $25,000 (for single use static cold storage) to around $95,000 for machine perfusion systems. Moreover, specialized transport vehicles (planes or automobiles) are required to transport machine perfusion systems (able to hold the weight and with sufficient power capabilities and space for the systems and the technicians).

[0037] Time, logistics, and costs of transporting organs to where they are needed are the largest roadblocks for successful transplantation of otherwise available donor organs. A dynamic organ storage system, as described herein, that does not require external power for operation can significantly remedy each of these roadblocks. Using the dynamic organ storage system can increase the time the organ is safely and effectively preserved. Additionally, the dynamic organ storage system can be easily transported at least due to (1 ) relatively small size and (2) lack of external power needed to run. The dynamic cold storage system can uniformly cool the organ to a desired hypothermic temperature and maintain the organ at that temperature for at least 12 or 24 hours and can simultaneously perfuse the organ using a pressure and gravity fed system. In some instances, sensors (e.g., with built-in reusable batteries) can interface with an external device for monitoring the condition of the organ and effluent can be sampled in real time.III. Systems

[0038] An aspect of the present disclosure can include a system 100 (FIG. 1 ) that can dynamically (while perfusing the organ) store an organ at a hypothermic temperature for a time. It should be noted that, the system 100 can be sized, shaped, and weighted to be easily transportable and to fit any type and / or number of organs. As shown in FIG. 1 , the dynamic organ storage system 100 can include an insulating container 102 (e.g., a temperature insulating container) having an internal volume 104 and a securable lid 106. The securable lid 106 can be positioned over an opening of the insulating container 102 to enclose the internal volume 104 of the insulating container. The securable lid 106 can be positioned over the opening and secured with one or moreresealable attachment mechanisms (not shown) such as, but not limited to, at least one of one or more hinges, one or more toggles, one or more snaps, a pressure top, a screw top, or the like. The securable lid 106 and the walls of the insulated container 102 can be filled with one or more types of temperature insulative materials, such as, one or more of metal, ceramic, fiberglass, polystyrene, cellulose, foams, mineral wool, or the like. For instance, the securable lid 106 and the walls of the insulated container 102 can be implemented as a double walled vacuum insulation structure.

[0039] The internal volume 104 of the insulating container 102 can hold an organ and the elements and components necessary for perfusing and hypothermically preserving the organ. For example, the internal volume 104 of the insulating container 102 can house an organ container 108 (housing an organ 110), at least one pressure bag 124 connected to the organ by at least one infusion line 120, a sampling line 122 connected to the organ, and at least one cooling mechanism 126. The organ container 108 can be shaped and sized to house an organ 110 submerged in a volume of a perfusate 112a. The organ container 108 can be shaped and sized generically to fit more than one type of organ from one or more donors (e.g., based on size, complexity, or the like) and / or specifically to fit one type of organ from one or more donors. For instance, the organ container 108 can be shaped and sized to fit a heart, a liver, one or more lungs, one or more kidneys, a pancreas, at least a portion of a small intestine, a gut, a portion of skin, a portion of tissue, a bioengineered graft, a xenogeneic organ graft, a limb, such as an arm, a leg, a hand, a foot, a finger, a toe, or the like, or the like. The amounts, concentrations, and types (e.g., blood, man-made solution, or a combination thereof) of the volume of the perfusate 112a in the organ container 108 can be based on the type of the organ 110 and / or the starting condition of the organ. The organ container 108 can have a lid 114 that can be positioned over an opening of the organ container to enclose the organ 110 in the organ container. The lid 114 of the organ container 108 can include at least one inlet port 116 and at least one outlet port 118.

[0040] At least one infusion line 120 can enter the organ container 108 through the at least one inlet port 116 and be connected to the organ 110 via a cannulation at a vein and / or an artery. A sampling line 122 can also be connected to the organ (e.g.,cannulated to a vein and / or an artery) and can exit the organ container 108 through the at least one outlet port 118. The sampling line 122 can be configured to allow a user of the system 100 to monitor a condition of the organ 110. For instance, if the organ 110 is a liver, then the system 100 can include a first infusion line and a second infusion line to flow perfusate into the liver. The first infusion line can be connected to (cannulated to) a hepatic artery of the liver and the second infusion line can be connected to (cannulated to) the portal vein of the liver. The sampling line can be connected to an inferior vena cava of the liver.

[0041] At least one pressure bag 124 can be connected to the at least one infusion line 120. For instance, one pressure bag 124 can be connected to each of the at least one infusion line 120 (only one is shown for easy of illustration). Each of the at least one pressure bag 124 can store another volume of perfusate 112b, which can be the same or different amounts, types, and / or concentrations. The other volume of perfusate 112b can be the same type as the volume of perfusate 112a in the organ container 108 or can be different. Both the volume of perfusate 112a in the organ container 108 and the other volume(s) of perfusate 112b in the at least one pressure bag 124 can be preoxygenated before use in the system 100. The at least one pressure bag 124 can be positioned above the organ container 108 inside the internal volume 104 of the insulating container 102 and tilted at an angle (6) relative to the lid 114 of the organ container to gravity flow the other volume(s) of the perfusate 112b from the pressure bag(s) to the organ 110 thought the infusion line 120. The angle (9) of the tilt of the at least one pressure bag 124 relative to the lid 114 of the organ container 108 can be, for example, between 1 and 90 degrees, between 2 and 65 degrees, between 3 and 45 degrees, between 4 and 30 degrees, between 5 and 20 degrees, or the like. In one instance the angle (©) of the tilt of the at least one pressure bag 124 relative to the lid of the organ container can be between 5 and 90 degrees. In another instance the angle (©) of the tilt of the at least one pressure bag 124 relative to the lid of the organ container can be 15 degrees.

[0042] The at least one cooling mechanism 126 can maintain the organ at a hypothermic temperature for a time without requiring power to operate (the cooling mechanism 126 and / or any other part of system 100 may need to be recharged or re-cooled using line power, but line power is not required for operation). The at least one cooling mechanism 126 can be positioned at different positions within the internal volume 104 (e.g., uniformly spaced around at least a portion of the organ container 108) to uniformly control the temperature of the organ for the time. The cooling mechanism(s) 126 can be positioned on at least one side (e.g., bottom, top, sides, or the like) of the organ container 108. The cooling mechanism(s) 126, in an instance, can be “no power required” cooling mechanisms including, for example, gel cooling pack(s), chemical coolant(s), and / or ice packs. For example, the cooling mechanisms(s) 126 can be based on a chemical coolant (e.g., including sodium polyacrylate) and can be, for instance, eutectic gel packs. In another instance the cooling mechanism(s) 126 can include long lasting self-powered and rechargeable cooling mechanism(s). The cooling mechanism(s) 126 can cool at least a portion of the internal volume 104 of the insulated container 102 and / or the organ 110 within the organ container 108 to a hypothermic temperature and maintain the hypothermic temperature for a time, without requiring power. The time can be, for example, 10 hours or less, 12 hours or less, 15 hours or less, 20 hours or less, 24 hours or less, 48 hours or less, or more. The hypothermic temperature can be, for example, between 0 and 20 degrees Celsius, between 0 and 14 degrees Celsius, between 0 and 12 degrees Celsius, between 0 and 11 degrees Celsius, between 0 and 10 degrees Celsius. The hypothermic temperature can be maintained within, for example, 10 degrees, 5 degrees, 1 degree, 0.5 of a degree, 0.1 of a degree, or the like, from a desired hypothermic temperature. In one instance the desired hypothermic temperature can be about 10 degrees Celsius for 24 hours or less. The desired hypothermic temperature can be pre-determined for each organ (e.g., for each organ specifically and / or for a general type of organ) to optimally slow organ damage and degradation, such as damage and degradation due to ischemia.

[0043] While the hypothermic temperature is maintained by the cooling mechanism(s) 126 and the insulation of the insulated container 102, the organ 110 can be perfused with other volume of perfusate 122b from the at least one pressure bag 124 through the at least one infusion line 120 cannulated into the at least one vein and / or artery of the organ for the time, without requiring power. The perfusion flow rate can be controlled by gravity (e.g., by changing the tilt angle 0 of the pressure bag 124 and / orone or more flow regulator and / or valves connected to the infusion line (not shown in FIG 1 , discussed in more detail in FIG. 2). In some other instances, the perfusion control rate can be controlled by external pressure applied to the at least one pressure bag.

[0044] Referring now to FIG. 2, illustrated is a more complex version of the dynamic organ storage system 200. The more complex dynamic organ storage system 200 includes all of the components of the dynamic organ storage system 100 (numbered as 200s) and can include additional components for improving the hypothermic preservation of the organ and overall use of the system.

[0045] Flow regulators 228, briefly discussed above, can be connected to the at least one infusion line 220 and / or the sampling line 222. The flow regulator 228 connected to the infusion line 220 can regulate the rate of the other volume of the perfusate 212b flowing from the pressure bag 224 to the organ 208 in addition to the gravity flow. The flow regulator 228 connected to the sampling line 222 can regulate a rate of sampling of the perfusate (from the volume the organ is submerged in 212a and / or the other volume at a point after the other volume has been flowed into the organ 208). The flow regulators 228 can be automatically and / or manually adjustable. The flow regulators 228 can be any type of valve capable of controlling flow of a fluid. At least one removable attachment mechanism 230 (shown as two attachment mechanisms for ease of illustration) can secure the organ container 208 to the insulating container 202. The removable attachment mechanism(s) 230 can be hooks, snaps, buttons, Velcro, or the like, with a first portion connected to the organ container 208 and a second portion attached to the insulating container 202. An effluent vessel 232 can be connected to the organ 210 and / or the organ container 208 by a drainage tube 234 and can receive effluents from the organ through the drainage tube 234. The effluent vessel 232 can be positioned beneath the organ container 208, for example as shown with the cooling mechanism(s) 226 in between. The drainage tube 234 can exit the organ container 208 through an outlet port 218 in the lid 214, on the side, or on the bottom of the organ container 208.

[0046] The system 200 can also include one or more sensors 236 that can detect one or more biomarkers related to the condition of the organ 210 and / or detect achange in the one or more biomarkers indicative of a change in the condition of the organ. For example, one or more sensors 236 can be positioned in the sampling line 222 or the organ container 208, as shown (or in any position in the lines). While not shown, the sensors 236 and / or one or more additional sensors can be positioned in the at least one infusion line 220, the sampling line 222, the effluent vessel 232, in the internal volume 204 of the insulating container 202, or the like. For example, one or more sensors 236 can be positioned in a sampling line 222 for a liver and can sample effluent directly from at least a portion of the inferior vena cava. The one or more sensors 236 can detect for example, temperature or biomarkers such as, but not limited to, chemical analyte concentration(s) (e.g., glucose, lactate, etc.), venous and / or arterial oxygen saturation, or the like. For instance, the one or more sensors 236 can detect flavin mononucleotide (FMN) levels, which are markers of mitochondrial complex I injury in real time, lactate levels, which can indicate metabolic function, bile production levels and / or bicarbonate and sodium secretion, which can indicate cholangiocyte viability. As another example, the one or more sensors 236 can detect levels of Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and / or lactate dehydrogenase (LDH) to predict transplant success and / or organ prognosis.

[0047] Additionally, the one or more sensors 236 may be visible (e.g., through a translucent window), accessible by briefly removing at least a portion of the securable lid 206 of the insulating container 202, and / or wirelessly connected to an external device. The one or more sensors 236 can require no power and / or can include rechargeable batteries (so no external power is required during use of the system. The system 200 can also include one or more transportation mechanisms 238 to improve the transportability of the system. The one or more transportation mechanisms can include one or more handles, straps, wheels, etc. The system 200 can be mobile and at least partially re-usable (e.g., the insulated container 202 and lid 206, the removable attachment mechanism(s) 230, the sensor(s) 236, etc.).

[0048] FIG. 3 shows a system 300 that can include the system 200, with one or more sensors 236 in wireless connection with an external device 340. The one or more sensors 236 can include a wireless transmitter that can be in wireless connection with a wireless transmitter (neither shown) of the external device 340 to communicaterecorded biomarker data and / or detected changes in one or more biomarkers. The external device 340. The external device 340 can be any device including at least a controller 342 and a display 344. For instance, the external device 340 can be a computer, a smartphone, a tablet, a dedicated device, or the like. The controller 342 of the external device 340 can include a non-transitory memory and processor (not shown for storing and executing instructions, respectively. The controller 342 can analyze recordings from the one or more sensor 236 (such as the one or more sensors in the sampling line) and produce read outs of at least one of the biomarkers of the organ, oxygen consumption of the organ, and effluent composition. The external device 340 can alert and / or notify a user of a change in a biomarker and / or a condition of the organ 210 with a visual, audio, and / or haptic alarm. The external device 340 can display (on the display 344) organ health data and / or biomarkers or the like recorded by the one or more sensors 236 and / or analyzed by the controller 342.

[0049] FIGS. 4-10 show example(s) of how to set up a dynamic organ storage system for a donor liver. FIG. 4 shows an internal volume of an example insulated container with double walled insulation. The internal walls of the insulated container were modified to include a plurality of hooks, four are shown but any number greater than one is contemplated (e.g., removable attachment mechanisms) for holding the organ container in place. A plurality of gel packs (e.g., cooling mechanisms) were positioned in the internal volume of the insulated container. Four gel packs are shown lining the bottom and part of the side of the inside of the insulated container. Eight total gel packs were used, with others added later (before the securable lid was attached).

[0050] FIG. 5 shows the liver chamber with the liver (e.g., organ container with the organ) partially submerged in a volume or perfusate and positioned on hooks in the insulating container. In this example, the hooks connect to holes in an outer lip of the liver container. While not visible, an effluent vessel is positioned between the gel packs and the liver chamber. FIG. 6 shows a close up of the liver chamber and the liver. A real-time sample line is positioned to extend into the infra hepatic inferior vena cava (IVC). A drainage tube is positioned in the organ container. And infusion lines are shown cannulated into the hepatic artery and portal vein of the liver. FIG. 7 shows a further close up of the liver and the liver chamber to show the drainage tube ispositioned to receive effluent from the hepatic vein of the liver. FIG. 8 shows steps for inserting and positioning the real-time sample tube. In element A of FIG. 8, the infra hepatic IVC was identified and an extension tube (e.g., the sampling tube) was inserted into the infra hepatic IVC. Element B of FIG. 8 shows fixing the edge of the infra hepatic IVC to hold the sampling tool in place. Element C of FIG. 8 shows an example sample collection tool (a syringe) for drawing sample perfusate from the liver.

[0051] FIG. 9 shows the example system with the lid positioned on the liver chamber and additional gel packs positioned around the sides of the interior volume of the insulating container. The lid can be translucent for easier visual inspection of the liver. A number of ports can be included in the lid for extending the infusion lines, the sampling line, and the drainage tube out of the liver chamber. FIG. 10 shows the two infusion lines each connected to a pressure bag with a pressure scale and flow regulator for measuring and controlling flow rate out of the pressure bags. A temperature probe was positioned in the insulating container and another temperature probe (not shown) was positioned in the liver to record temperatures over time. The pressure bags are shown tilted at an angle relative to the lid of the liver chamber (approximately 15 degrees) to gravity flow the perfusate into the liver. The lid can then be shut over all the internal components to insulate the system for the time - e.g., 12 hours for the experiment shown here. The sampling line can, for instance, extend out of the insulating container to enable an outside observer to check the condition of the organ without needing to open the lid of the insulating container. FIG. 11 shows illustrations of other, non-limiting, example liver chambers (it should be understood that further configurations of the liver chamber can be conceptualized). Element A shows a rounded bottom chamber with a drainage tube in the body of the organ container, rather than the lid, positioned such that effluent can be drained when the perfusate reaches the level of the opening of the drainage tube. Elements A and B additionally show a tube suspension system to support and / or organize the infusion lines. Element B shows a flat-bottomed chamber where the sampling line can extend out of the drainage tube rather than an outlet port in the lid (otherwise similar to Element A).

[0052] FIG. 12 shows a graphical representation illustrating how the temperature of the organ and the organ container is maintained to be relatively constant for at least 12hours. The graph shows that after cooling down the insulated container (in this example, a Yeti cooler, line starts at 20 degrees Celsius) to 11 degrees Celsius in 20 minutes the temperature of the insulated container is maintained within approximately 4 degrees of 10 degrees Celsius for 12 hours. The graph also shows that the liver temperature (line starting at just under 10 degrees Celsius) was maintained at approximately 10 degrees Celsius for 12 hours with an approximately less than ±1 degree Celsius fluctuation.

[0053] FIG. 13 shows an example pressure bag 324 that can be used with system(s) 100 and / or 200 (it should be understood that the pressure bag can be shaped differently and still work with system(s) 100 and / or 200). The pressure bag 324 can utilize gravity and / or pressure to flow perfusate to an organ. The pressure bag 324 can include an outer housing that can be a hard shell 350 that can protect the perfusate bag from exterior forces with an interior pressure bladder 352 that can apply pressure to the perfusate bag 354. For example, the hard shell 350 can act as a clam shell - with a top half and a bottom half that can open and close around the perfusate bag 354 using a hinge 356 and a locking mechanism 358. When locked into place the pressure bladder 352 can apply a given amount of pressure to the perfusate bag 354 to flow perfusate out of the perfusate outflow 360 to an infusion line at a given rate, which can be relative to the applied pressure. The applied pressure can be controlled and / or changed using the port for the pressure bladder 362. FIG. 14 shows an example insertion port 370 for connecting a sampling line 322 to an organ 310 (it should be understood that the insertion port can be any shape and still work with system(s) 100 and / or 200). The insertion port 370 can be incorporated into an already existing opening in the organ 310 and / or can be placed in a fenestration made in the organ 310. The insertion port 370 can include flaps 372 that can hold the sampling line 322 in position in the organ 310.

[0054] The following FIGS. 15-18 show various portions of the dynamic organ storage systems 100, 200 and / or 300 having configurations for use with organ’s other than the liver. FIGS. 15 and 16 shows example illustration of portions of the dynamic organ storage systems 100, 200 and / or 300 for use with a kidney or a pair of kidneys. FIGS. 17 and 18 shows example illustration of a cannula for use with a heart.

[0055] FIG. 15 shows a kidney basin container 402 for use in the dynamic organ storage systems 100, 200, and / or 300. The kidney basin container 402 of FIG. 15 canbe shaped and sized to hold a single kidney 406 in a kidney basin 404 (also shaped and sized to fit a kidney of a subject). The kidney 406 can be cannulated at the renal artery to input the perfusate into the kidney with a renal artery cannula 408 (e.g., connected to an infusion line). Additionally, connectors and / or cannulations connecting to the sampling port 410 and connectors and / or cannulations connecting to the effluent drainage tube 412 can also be connected to the kidney 406 (e.g., at the renal vein) for monitoring physiological parameters and value and removing effluent, respectively. A uterine stent 414 can be positioned in the kidney 406 and can send urine from the kidney into a urine collector 416.

[0056] FIG. 16 shows another version of a kidney basin container 402 for use in the dynamic organ storage systems 100, 200, and / or 300. The kidney basin container 402 of FIG. 16 can be shaped and sized to hold two kidneys 406(1 ) and 406(2) in a kidney basin 404 (shaped and sized to fit two kidneys and a portion of the renal vein(s) and artery(ies)). The renal vein and artery can be clamped distal of the connections to the kidneys. A single renal artery cannula 408 and connector to effluent drainage tube 412 can be used for flowing perfusate into and effluent out of both kidneys, respectively. Effluent sampling ports 410 can, for instance be positioned in a portion the renal vein connections to each of the kidneys 406(1 ) and 406(2) for separate sampling of each kidney. Urine can be collected separately from each kidney in urine collector 416(1 ) and (2). In some instances, a single urine collector 416 may be used.

[0057] FIGS. 17 and 18 show example illustrations of a flexible dual lumen cannula 500 for use when the organ in the cold, dynamic organ storage system (e.g., systems 100, 200, and / or 300) is a heart. If the organ is a heart, then one of the infusion lines for flowing perfusate into the heart can be connected to the heart with an innominate cannulation or a direct aortic cannulation. A flexible dual lumen cannula 500 can be used with an outer cannula (coronary flow cannula 502) to flow perfusate into the heart and an inner cannula 503 to vent and / or flow perfusate and / or effluent out of the heart. The outer cannula (coronary flow cannula 502) can be a coronary flow cannula for flowing perfusate into the area of the aortic valve near (shown as above) the sinotubular junction so perfusate can flow into the left coronary artery (LCA) and the right coronary artery (RCA). The inner cannula 503 can be positioned into the left ventricle and caninclude a sub-annular balloon 504 that can hold the inner cannula in the left ventricle and can act as a back-up valve if needed. The inner cannula 503 can use suction to act as a vent and / or to pull perfusate and / or effluent from the inside of the left ventricle using left ventricle vent 506.

[0058] FIG. 18 shows the flexible dual lumen cannula 500 in greater detail. FIG. 18, element A shows a side view of the flexible dual lumen cannula 500 with the anterior and posterior sides marked. FIG. 18, element B, shows an anterior view of the flexible dual lumen cannula 500 and additionally notes the flow port 508 connected to the coronary flow cannula 502, the suction port 510 connected to the inner cannula 503, and the balloon port 512 for inflating and / or deflating the sub-annular balloon 504. FIG. 18, element C shows an interior view of the flexible dual lumen cannula 500 with the flow lumen 514 (of the coronary flow cannula 502) around the suction lumen 516 (of the inner cannula 503). It should be noted that any shapes, sizes, and / or positions of components of the flexible dual lumen cannula are shown for illustrative purposes only and can have other shapes, sizes, and / or positions as known to persons having ordinary skill in the art.

[0059] FIGS. 19-21 show illustrations of different example cannula designs for use with any combination of the above described systems and devices. These examples are not meant to be limiting. FIG. 19 shows views of an example drainage cannula design (e.g., for connecting drainage tube 234 into the organ container 208) for use in the cold, dynamic organ storage systems described above (e.g., 100, 200, 300, etc.). The drainage cannula can have a perforated tip designed to optimize perfusate drainage, which can be positioned up or down depending on the system embodiment. The drainage cannula can include one or more grooves circumscribing at least a portion of the body to facilitate securing the cannula within the vessel. For instance, the one or more grooves can be positioned adjacent to the perforated tip and can by indented a sufficient amount to accept string ties or the like without slipping. The opposite end of the drainage cannula from the perforated tip can include a tapered end with one or more ridges to securely connect the drainage cannula to tubing connected to the effluent vessel. FIG. 20 shows different views of an example infusion cannula design with an elongated tip extending from a tapered body. The elongated tip can have various tiplengths and widths to accommodate an array of vessel and / or organ sizes. Similar to the drainage cannula design of FIG. 19 the infusion cannula design can also include one or more grooves to facilitate securing the cannula within the vessel and / or the organ using ties and a tapered other end with one or more ridges to securely connect the cannula with an infusion line. FIG. 21 shows an example of a vessel cannula design similar to the design of FIG. 20 and including a luer connector (shown extending from the widest part of the body of the cannula but it should be understood that the luer connector can extend from any portion of the body) and a tapered connector to facilitate connection with cold, dynamic organ storage system and / or machine perfusion without need for cannula exchange.IV. Methods

[0060] Another aspect of the present disclosure can include method 600 (FIG. 22) for improved preservation of an extracorporeal organ (e.g., for transplantation). The method 600 can be executed using the system 100, 200, or 300 shown in FIGS. 1 , 2, and 3. For purposes of simplicity, the method 600 is shown and described as being executed serially; however, it is to be understood and appreciated that the present disclosure is not limited by the illustrated order as some steps could occur in different orders and / or concurrently with other steps shown and described herein. Moreover, not all illustrated aspects may be required to implement the method 600, nor are methods 600 limited to the illustrated aspects.

[0061] FIG. 22 shows a method 600 for preserving an extracorporeal organ with a cold, dynamic organ storage system that does not use external power for up to 10 hours, up to 12 hours, up to 15 hours, or up to 24 hours or more. The dynamic organ storage system can increase the time the organ is safely and effectively preserved through maintaining an optimal preservation temperature and perfusion of the organ. The dynamic cold storage system can uniformly cool the organ to a desired hypothermic temperature and maintain the organ at that temperature for at least 12 or 24 hours and can simultaneously perfuse the organ using a pressure and gravity fed system. In some instances, sensors with built in reusable batteries can interface with an external device for monitoring the condition of the organ.

[0062] At 602, an organ (e.g., an organ from a donor for transplantation and / or scientific research or the like) can be placed in an organ container with a volume of perfusate. The organ container can be shaped and sized to fit one or more types of organs (e.g., a generic shape and size for small, medium, or large organs) and / or can be particularly shaped and sized for a type of organ (e.g., heart, liver, lungs, kidney, etc.). The volume and content of the perfusate placed in the organ container with the organ can be based on the specific needs of the organ in question. The organ can be at least partially submerged within the volume of the perfusate. At 604, at least one infusion line and a sampling line can be connected to (e.g., cannulated to) the organ (e.g., to at least one vein and / or artery of the organ). For example, if the organ is a liver, then the at least one infusion line can be a first infusion line connected to a hepatic artery of the liver and a second infusion line connected to the portal vein of the liver. The sampling line can be connected to an inferior vena cava of the liver.

[0063] At 606, a lid of the organ container can be closed to secure the organ within the organ container. The lid can include at least one inlet port and at least one outlet port. The at least one infusion line can be fed through the inlet port(s) and the sampling line can be fed through the outlet port(s) of the lid. A drainage tube for the effluent of the perfusion can also be fed through an outlet port of the lid. The drainage tube can be connected to the organ at a “downstream” portion of the circulation and / or in fluid communication with the volume of perfusate in the organ container to remove contaminants and / or waste. At 608, the organ container can be placed inside the insulating container (e.g., in the internal volume of the insulating container) with at least one cooling mechanism. The organ container can be secured into position in the insulating container using one or more removable attachment mechanisms (e.g., one or more hooks, snaps, buttons, Velcro, or the like with a first portion connected to the organ container and a second portion attached to the insulating container). The organ container can be positioned over top an effluent vessel (which can be connected to the organ and / or the organ container via the drainage tube). The cooling mechanism(s) can be positioned on at least one side (e.g., bottom, top, sides, or the like) of the organ container. The cooling mechanism(s) can be no power required cooling mechanisms including, for example, gel cooling pack(s), chemical coolant(s), and / or ice packs. Inanother instance the cooling mechanism(s) can include long lasting self-powered and rechargeable cooling mechanism(s).

[0064] At 610, the at least one infusion line can be connected to at least one pressure bag containing another volume of perfusate (can be the same or different perfusate as the perfusate in the organ container or in the other pressure bags if more than one pressure bag is utilized). The other volume(s) of perfusate can be preoxygenated. At 612 the at least one pressure bag can be positioned above the organ container inside the internal volume of the insulating container and tilted at an angle (9) relative to the lid of the organ container to gravity flow the other volume(s) of the perfusate from the pressure bag(s) to the organ. The angle (9) relative to the lid of the organ container can be, for example, between 5 and 90 degrees, between 5 and 80 degrees, between 5 and 70 degrees, between 5 and 60 degrees, between 5 and 50 degrees, between 5 and 40 degrees, between 5 and 30 degrees, or the like. In one instance the angle (9) relative to the lid of the organ container can be 15 degrees. A flow regulator and / or valve can be coupled to the at least one infusion line (e.g., along the length of the infusion line, at the inlet port, or the like) to control and adjust the flow of the other volume(s) of the perfusate into the organ in addition to gravity flow. The securable lid of the insulating container can then be secured after all of the components and elements described above are positioned, secured, and / or activated to begin dynamic hypothermic preservation of the organ.

[0065] Dynamic hypothermic preservation of the organ can include the following additional steps. At 614, the internal volume of the insulated container and the organ within the organ container can be cooled by the at least one cooling mechanism(s) to a hypothermic temperature and maintained at the hypothermic temperature for a time, without requiring power. The time can be, for example, 10 hours or less, 12 hours or less, 15 hours or less, 20 hours or less, 24 hours or less, or 48 hours or less, etc. The hypothermic temperature can be, for example, between 0 and 14 degrees Celsius, between 0 and 12 degrees Celsius, between 0 and 11 degrees Celsius, between 0 and 10 degrees Celsius. The hypothermic temperature can be maintained within, for example, 1 degree, 0.5 of a degree, 0.1 , of a degree from a desired hypothermic temperature. In one instance the desired hypothermic temperature can be 10 degreesCelsius for 24 hours or less. The desired hypothermic temperature can be predetermined for each organ (e.g., for each organ specifically and / or for a general type of organ) to optimally slow organ damage and degradation, such as ischemia. While the hypothermic temperature is maintained, at 616, the organ can be perfused with at least one perfusate (the other volume(s) of perfusate) from the at least one pressure bag through the at least one infusion line cannulated into the at least one vein and / or artery of the organ for the time, without requiring power. The perfusion flow rate can be controlled by gravity, external pressure applied to the at least one pressure bag, and / or the one or more flow regulator and / or valve connected to the infusion line. The flow regulator can be automatically and / or manually adjustable. Optionally, the concentration of oxygen and / or one or more nutrients of the perfusate can be manually adjusted by removing the lid from the insulating container for a short time or automatically adjusted.

[0066] Throughout the time of the perfusing and the maintaining the hypothermic temperature, at 618, a user (e.g., a technician, a nurse, a doctor, or another qualified professional) can be allowed to monitor a condition of the organ using at least one sensor positioned within the sampling line, the internal volume of the insulating container, and / or the internal volume of the organ container. When the at least one sensor is positioned within the sampling line, the at least one sensor can be accessible with the securable lid of the insulating container closed (e.g., viewed through a transparent window, exiting the insulating container through a port, or the like). For instance, a user can check, by the sampling line, at least one biomarker of the organ to monitor the condition of the organ. The temperature and / or the perfusion flow rates, for example, can be adjusted based on checking the at least one biomarker. The at least one sensor can include a temperature sensor, an oxygen sensor, a carbon dioxide sensor, a chemical analyte sensor (e.g., glucose, lactate, etc.). In another instance, any of the at least one sensor can include a rechargeable battery (so as to not require any external power) and a wireless transmitter to connect with one or more external devices including a display and a processor / controller (e.g., a computer, a tablet, a smartphone, a dedicated external device, or the like). The at least one sensor can wirelessly transmit collected data and / or warnings to the external device if data at a given time is outside pre-set thresholds. The external device can, for example, emit an audio, visual, and / ortactile alert to the user. The external device can optionally be in communication with a temperature control mechanism, one or more flow regulators, or the like to control temperature and / or perfusion of the system. During the hypothermic preservation the entire dynamic storage system can be transported from a first location to a second location (e.g., via wheels and / or handles). The transportation can include flying and / or driving the entire dynamic storage system without having to power the system and / or use specialty vehicles (e.g., for size and / or weight).V. Experimental

[0067] The following experiment describes storage of livers with a dynamic organ storage system (DOSS) - maintaining liver temperature at 10°C for 10 hours while delivering oxygenated perfusate and allowing real-time assessment of metabolic functions within a portable cooler. The DOSS was compared to conventional static cold storage (SCS) and to improve upon limitations of hypothermic oxygenated machine perfusion (MP) (HOPE). The experiment demonstrates that livers stored using the DOSS exhibited superior liver and biliary function compared to those preserved on conventional static cold storage (SCS). As shown, DOSS can enable improved lactate clearance, oxygen consumption, fibrinogen synthesis, bile production, cholangiocyte bicarbonate and sodium secretion, responsiveness to vasoactive agents, and cellular viability of livers. Technically, the experiment shows that DOSS maintained liver temperature at 10°C for 10 hours while delivering oxygenated perfusate and allowing real-time assessment of metabolic functions within a portable cooler. Moreover, DOSS enables continuous delivery of oxygenated perfusate to a liver graft at 10°C and realtime effluent sampling within a portable cooler, suggesting that DOSS can enhance DCD allograft function and assessment during organ transport, which could have direct clinical applications and contribute to the expansion of the donor pool. Additionally, this study aimed to minimize the need for external electrical power, improve organ quality, and reduce the size, cost, and complexity of operation of an organ preservation system. Integrating the DOSS circuit into a portable cooler facilitates its ease of transport and can be used in smaller aircraft compared to traditional devices. Thus, the DOSS has the potential to offer numerous benefits over current HOPE systems, particularly in terms of cost and logistical feasibility.

[0068] Materials and Methods

[0069] Study Design

[0070] The harvested livers in a porcine DCD model were preserved using either DOSS or SCS for a duration of 10 hours (n = 5 each). After the preservation period, NMP was performed for 4 hours to assess the physiological functions of the liver and bile duct. Liver and biliary tissues were collected at three time points: after procurement, following 10 hours of preservation, and after 4 hours of NMP, for histological evaluation. FIG. 23, element (a) shows an illustrated schematic of the experiment.

[0071] Liver Procurement in a Porcine DCD Model

[0072] The study protocol was approved by the Institutional Animal Care and UseCommittee at Vanderbilt University Medical Center. Male Yorkshire x Landrace swine, weighing between 45 and 54 kg, were utilized in this study. Following the induction of general anesthesia, the donor porcine received a bolus of 30,000 units of heparin and whole blood was collected using the acute normovolemic hemodilution technique. After hypoxemic cardiac arrest, a midline laparotomy was performed, and the liver (1 .380 ± 0.182 kg) was flushed with 3 L of cold University of Wisconsin (UW) solution (CoStorSol, Preservation Solutions, Inc., Elkhorn, Wl) through the abdominal aorta. The liver was subsequently procured in a standard manner and placed on the back table.

[0073] Back Table Procedure

[0074] FIG. 23, element (b) shows a liver graft placed in the liver chamber with cannulations. The livers were flushed with 1 L of UW solution via the portal vein (PV). In the DOSS group, the hepatic artery (HA) and PV were cannulated with 18G cannula (Covidien, Medtronic, Ireland) for perfusion during storage. Additionally, a small sample tube was inserted into the inferior vena cava (IVC) to collect real-time effluent samples for metabolic assessment during DOSS. After 10 hours of DOSS, the cannulas and sample tube were removed, and the HA, PV, IVC, and bile duct were cannulated with 12Fr, 28Fr, 36Fr, and 14Fr cannulas (Medtronic, Minneapolis, MN), respectively, to prepare for NMP. In the SCS group, the vessels were cannulated for NMP after procurement and the livers were stored in a cooler on ice.

[0075] Dynamic Organ Storage System

[0076] The DOSS (see FIG. 23, elements c and d) has been developed to deliver oxygenated perfusate at 10°C in a portable cooler (YETI Coolers, LLC., Austin, TX) with a minimal electrical power requirement of 12.96 Watt-hours. Eutectic gel packs (Traferox Technologies, Inc., Mississauga, ON) were used to maintain the liver temperature at 10°C. Liver temperature was continuously monitored by temperature probe (Pendo TECH) placed at the edge of the left lobe. The liver was perfused with STEEN solution, which has been utilized in both acellular and cellular MP experiments. Prior to DOSS, the perfusate was oxygenated with 2 L / min of 02 using an oxygenator (Capiox FX15, Terumo Cardiovascular, Ann Arbor, Ml), achieving a final partial pressure of oxygen (pO2) of over 650 mmHg. In the initial three experiments, whole porcine blood was added to STEEN solution at a 1 :4 dilution as an oxygen carrier.However, due to the decreased oxygen demand of liver grafts at 10°C and more clinical applicability of acellular solution, acellular STEEN solution was used in the final two experiments. The target perfusate flow rates for the HA and PV were 50 mL / hr and 150 mL / hr, respectively, achieved using infusion pumps (VET / IV 2.2 Infusion Pump, Heska, Loveland, CO). The perfusate drained from the liver without recirculation, collecting in a waste bag attached to the bottom of the liver chamber.

[0077] Perfusate samples from the perfusate bag and liver effluent samples from the IVC were assessed every 2 hours using a blood gas analyzer (Element POC, Heska, Loveland, CO). Liver oxygen consumption during DOSS was calculated as previously described. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) in the effluent were measured at the initiation of DOSS and at 10 hours of DOSS. FMN was measured from liver effluent samples, which were separated from blood components by centrifugation, using a Synergy HTX microplate reader (Agilent BioTek Instruments, Santa Clara, CA).

[0078] Normothermic Machine Perfusion (NMP)

[0079] After 10 hours of storage, NMP was continued for 4 hours as previously reported. Briefly, NMP system comprised a centrifugal pump, oxygenator, organ chamber, perfusate reservoir, dialysis filter, heat exchanger, flow and saturation probes, and pressure transducers. The circuit was primed with porcine whole blood. Perfusionwas initiated by progressively increasing the pump speed, with target flow rates set at 1 .0-1 .2 L / min for the PV and 0.2-0.5 L / min for the HA, respectively.

[0080] Parameters of Hepatocellular Functions during NMP

[0081] During NMP, perfusate samples from the IVC were collected hourly and analyzed by blood gas analyzer. Comprehensive metabolic panels (Antech Diagnostics, Seville, OH) were measured at the initiation of NMP and 4 hours of NMP. Fibrinogen levels were measured and evaluated as a marker of synthetic liver function. Perfusate samples from the PV were also collected to calculate oxygen consumption. After 4 hours of NMP, 4 mg of phenylephrine was administered into the HA cannula to assess graft vasoresponsiveness to selective a1 -adrenergic receptor activation.

[0082] Parameters of Cholangiocyte Functions during NMP

[0083] During NMP, bile samples were collected hourly under mineral oil for analysis of bile volume and biomarkers using a blood gas reader. Furthermore, the bile / perfusate glucose and sodium ratio were calculated to account for variations in perfusate concentration.

[0084] Histological Assessment

[0085] All liver and biliary biopsies were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 5 pm thickness. Hematoxylin and Eosin (H&E) and Trichrome staining were performed on liver tissues to evaluate composite acute injury scores. Apoptosis in liver tissues was evaluated through Terminal Deoxynucleotidyl Transferase-mediated Deoxyuridine Triphosphate Nick-end Labeling (TUNEL) and immunohistochemistry (IHC) for cleaved caspase-3. Biliary tissue sections were stained with H&E and analyzed for histological scores indicative of IC. Immunofluorescence (IF) staining with CD31 , CK7, and SOX9 was performed to quantify the perivascular plexus, cholangiocytes, and progenitor cells, respectively.

[0086] Histological composite injury scores for both liver and bile duct tissues were assessed by a blinded pathologist. Quantitative analysis of TUNEL, cleaved caspase-3, CD31 , CK7, and SOX9 positive cells was automatically performed using General Analysis 3 in NIS-Elements. The positive counts were normalized per high-powered field (hpf), defined as 0.252 mm2of tissue.

[0087] Metabolomic Analysis

[0088] Metabolomics analysis was carried out for liver and biliary tissues collected following 4 hours of NMP using MetaboAnalyst version 5.0 (detailed description in Supplementary Methods). Differentially expressed metabolites were identified by volcano plot with a fold-difference threshold of 1 .5 and p < 0.05. The key metabolic pathways were identified based on pathway enrichment analysis with p < 0.05 and based on pathway topological importance with an impact score threshold of 0.1 .

[0089] Statistical Analysis

[0090] Continuous variables are presented as mean ± standard deviation. Two- tailed Student's f-test was used to compare liver and biliary biomarkers at 4 hours of NMP and other continuous variables, including histological injury scores, between two groups. Linear mixed effect model was used to compare the response to phenylephrine administration over time between groups. All statistical analyses were performed using EZR, with p-values less than 0.05 considered statistically significant.

[0091] Results

[0092] Assessment of Liver Graft Condition during DOSS

[0093] Liver temperature remained stable for 10 hours during DOSS, with an initial temperature of 9.5 ± 1.2 °C and a temperature of 10.4 ± 1.2 °C at 10 hours (see FIG 24, element (a)). The pO2 in the cellular and acellular perfusate was 729.3 ± 26.8 mmHg and 709.3 ± 29.3 mmHg at the initiation of DOSS, and 394.5 ± 0.8 mmHg and 548.1 ± 62.5 mmHg at 10 hours of DOSS, respectively (see FIG. 24, element (b). During the 10 hours of storage, oxygen consumption remained stable, measured at 0.039 ± 0.009 mL / min / kg at 2 hours and 0.036 ± 0.005 mL / min / kg at 10 hours (see FIG. 24, element (c)). Lactate concentration in the effluent was 1 .6 ± 0.7 mmol / L at 2 hours of DOSS and 2.3 ± 1.7 mmol / L at 10 hours of DOSS (see FIG. 24, element (d)). Levels of AST, ALT, and LDH numerically decreased from the initiation of DOSS (AST: 1164 ± 743 U / L, ALT: 50 ± 30 U / L, LDH: 1152 ± 745 U / L) to (AST: 461 ± 461 U / L, ALT: 27 ± 13 U / L, LDH: 543 ± 369 U / L) after 10 hours of DOSS, while FMN concentration increased from 150 ± 55 ng / mL to 192 ± 61 ng / mL (see FIG. 24, elements (e)-(h)).

[0094] Hemodynamics of Liver Grafts during NMP and after PhenylephrineAdministration

[0095] Both DOSS and SCS livers reached targeted physiological levels of HA and PV flow after 1 hour of NMP (see FIG. 25, element (a)). There was no significant difference in HA and PV vascular resistance at 4 hours of NMP. However, after phenylephrine administration, DOSS livers demonstrated lower HA vascular resistance over time when compared to SCS (p = 0.018) (see FIG. 25, element (b)).

[0096] Parameters of Hepatocellular functions during NMP

[0097] Lactate concentration in the NMP perfusate decreased in the DOSS group over 4 hours of NMP (see FIG. 26, element (a)). In contrast, lactate concentration increased in the SCS group from 2 hours to 4 hours of NMP. At 4 hours of NMP, the DOSS group showed significantly lower lactate concentration compared to the SCS group (DOSS: 0.78 ± 0.57 mmol / L, SCS: 2.19 ± 0.83 mmol / L, p = 0.023). There was no significant difference in the perfusate pH, bicarbonate, and glucose between the two groups (see FIG. 26, elements (b)-(d)). Oxygen consumption was significantly higher in the DOSS group compared to the SCS group at 4 hours of NMP (DOSS: 17.37 ± 5.99 mL / min / kg, SCS: 6.29 ± 4.43 mL / min / kg, p = 0.018) (see FIG. 26, element (e)). There were no significant differences in the levels of AST or ALT (see FIG. 26, elements (f) and (g)). Fibrinogen concentration increased from 0 to 4 hours of perfusion in all livers in the DOSS group, whereas it increased in only one liver in the SCS group. The relative increase in fibrinogen concentration from 0 to 4 hours of NMP was significantly greater in DOSS livers compared to SCS livers (DOSS: 31 ± 8%, SCS: -35 ± 31 %, p = 0.005) (see FIG. 26, elements (h)-(i)).

[0098] Parameters of Cholangiocyte Functions During NMP

[0099] All DOSS and SCS livers produced bile during 4 hours of NMP. However, one liver in the SCS group exhibited haemobilia and was excluded from the analysis of biliary biomarkers to mitigate any potential influence of blood contamination on the interpretation of biliary functions. DOSS livers produced significantly more bile than SCS livers at 4 hours of NMP (DOSS: 11 .5 ± 3.4 mL / hr, SCS: 2.9 ± 3.4 mL / hr, p = 0.013) (see FIG. 27, element (a)). Bile pH was similar between groups after 4 hours of NMP (see FIG. 27, element (b)). Bile bicarbonate was significantly higher in DOSS livers compared to SCS livers (DOSS: 18.6 ± 3.5 mmol / L, SCS: 10.2 ± 4.0 mmol / L, p = 0.035) (see FIG. 27, element (c)). There was no significant difference in the bile glucoseconcentration and bile / perfusate glucose ratio between the two groups (see FIG. 27, elements (d) and (e)). The bile / perfusate sodium ratio was significantly higher in DOSS livers compared to SOS livers at 4 hours of NMP (DOSS: 1 .05 ± 0.02, SCS: 0.97 ± 0.01 , p = 0.002) (see FIG. 27, element (f)).

[0100] Histological A ssessment

[0101] Qualitative images of liver tissues on H&E, Trichrome, cleaved caspase-3, and TUNEL are shown in FIG. 28, element (a). Histologic liver injury scoring demonstrated no statistically significant difference between two groups after procurement, storage, or NMP (see FIG. 28, element (b)). Apoptotic assessment in liver tissues showed that there was no significant difference between two groups in the number of TUNEL and cleaved caspase-3 positive cells after procurement. However, after 4 hours of NMP, there were significantly fewer TUNEL (DOSS: 84 + 74 cells / hpf, SCS: 234 + 46 cells / hpf, p = 0.009) and cleaved caspase-3 (DOSS: 127 + 56 cells / hpf, SCS: 263 + 95 cells / hpf, p = 0.039) positive cells in DOSS livers compared to SCS livers (see FIG. 28, elements (c) and (d)).

[0102] Qualitative images of biliary tissues on H&E and IF are shown in FIG. 29, element (a). Biliary injury scoring did not show a statistical significance after procurement, storage, or NMP (see FIG. 29, element (b)). Moreover, there was no significant difference in the number of CD31 , CK7, an SOX9 positive cells after NMP (see FIG. 29, elements (c) and (d)).

[0103] From the above description, those skilled in the art will perceive improvements, changes, and modifications. Such improvements, changes and modifications are within the skill of one in the art and are intended to be covered by the appended claims.

Claims

The following is claimed:1 . An organ preservation system comprising: an insulating container; and a securable lid for enclosing an internal volume of the insulating container, wherein the internal volume of the insulating container houses: an organ container configured to house an organ at least partially submerged in a volume of a perfusate, having a lid configured to enclose the organ container, wherein the lid comprises at least one inlet port and at least one outlet port; at least one pressure bag, storing another volume of the perfusate, positioned above the organ container and tilted at an angle relative to the lid of the organ container; at least one infusion line enters the organ container via the at least one inlet port and connects the at least one pressure bag to the organ to gravity flow the perfusate from the at least one pressure bag into the organ; a sampling line connected to the organ and configured to allow a user to monitor a condition of the organ, wherein the sampling line exits the organ container via the at least one outlet port; and at least one cooling mechanism configured to maintain the organ at a hypothermic temperature for a time without requiring power.

2. The organ preservation system of claim 1 , further comprising: an effluent vessel housed within the internal volume of the insulating container and positioned below the organ container and configured to receive effluents from the organ through a drainage tube connected to the organ and extending from the organ container to the effluent vessel through the at least one outlet port.

3. The organ preservation system of claim 1 , wherein the at least one infusion line and the sampling line each comprise a flow regulator configured to regulate a rate theother volume of the perfusate flows to the organ and to regulate a rate of sampling of perfusate from the organ, respectively.

4. The organ preservation system of claim 1 , wherein the organ container is secured within the insulating container via at least one removable attachment mechanism.

5. The organ preservation system of claim 4, wherein the removable attachment mechanism comprises at least one of a hook, a snap, a button, and / or Velcro.

6. The organ preservation system of claim 1 , wherein the angle relative to the lid of the organ container is between 5 and 90 degrees to facilitate the gravity flow of the other volume of the perfusate in the at least one pressure bag to the organ.

7. The organ preservation system of claim 1 , wherein the at least one cooling mechanism is positioned around the organ container to uniformly control the temperature of the organ for the time.

8. The organ preservation system of claim 1 , wherein the system is mobile and at least partially re-usable.

9. The organ preservation system of claim 1 , wherein the hypothermic temperature is between 0 and 14 degrees Celsius, and the time is 24 hours or less.

10. The organ preservation system of claim 1 , wherein the perfusate is preoxygenated prior to use of the system.11 . The organ preservation system of claim 1 , wherein the organ is a liver, a lung, or a heart.

12. The organ preservation system of claim 1 , wherein when the organ is the liver, the at least one infusion line is a first infusion line connected to a hepatic artery of the liver and a second infusion line connected to the portal vein of the liver, wherein the first and second infusion lines flow the perfusate into the liver.

13. The organ preservation system of claim 12, wherein the sampling line is connected to an inferior vena cava of the liver.

14. The organ preservation system of claim 1 , wherein the sampling line further comprises one or more sensors configured to record and / or detect a change in one or more biomarkers indicative of a change in the condition of the organ.

15. The organ preservation system of claim 14, wherein the system further comprises: an external device comprising: a display, and a controller configured to analyze recordings from the one or more sensors in the sampling line and produce read outs of at least one of the biomarkers of the organ, oxygen consumption of the organ, and effluent composition.

16. The organ preservation system of claim 14, wherein the external device is configured to display organ health data and / or biomarkers recorded by the one or more sensors and / or analyzed by the controller.

17. A method for hypothermic organ preservation, the method comprising: placing an organ in an organ container so that the organ is at least partially submerged within a volume of perfusate; connecting at least one infusion line and a sampling line to the organ;-SI-enclosing the organ container with a lid comprising at least one inlet port that accepts the at least one infusion line and at least one outlet port that accepts the sampling line; placing the organ container in an insulating container having an internal volume, a securable lid for enclosing the internal volume, and at least one cooling mechanism positioned in the internal volume; connecting the at least one infusion line to at least one pressure bag; positioning the at least one pressure bag, storing another volume of the perfusate, above the organ container in the insulating container and tilted at an angle relative to the lid of the organ container so that the other volume of the perfusate gravity flows to the organ; cooling, by the at least one cooling mechanisms in the internal volume of the insulating container, the internal volume of the insulated container to maintain the organ at a hypothermic temperature for a time, without requiring power; perfusing, by the at least one pressure bag, the organ through the at least one infusion line cannulated into at least one vein and / or artery of the organ for the time, without requiring power; and allowing a user to monitor a condition of the organ using at least one sensor within at least the sampling line, wherein the at least one sensor is accessible with the securable lid closed.

18. The method of claim 16, further comprising checking, by the sampling line, at least one biomarker of the organ during the cooling and the perfusing.

19. The method of claim 17, further comprising adjusting a flow regulator coupled to the at least one infusion line to adjust the flow of the other volume of the perfusate into the organ.

20. The method of claim 16, further comprising moving the organ preservation system from a first location to a second location.

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