A method for preserving ex vivo organs by supercooling

Abstract

This invention discloses a method for preserving supercooled ex vivo organs. The method involves placing the ex vivo organ in a preservation solution and then cooling it for preservation. The key feature is that the ex vivo organ is placed within a magnetic field range and its temperature is lowered to a supercooled range of 0°C to -5°C for preservation. This invention can better reduce the organ preservation temperature, thereby extending the preservation time and improving the preservation effect and organ viability. It is particularly suitable for clinical organ preservation and research sample preservation.

Description

Technical Field

[0001] This invention relates to the field of ex vivo organ preservation technology, and specifically to a method for preserving supercooled ex vivo organs. Background Technology

[0002] In the medical field, organ transplantation surgery requires transplanted organs to have strong viability in order to better ensure the success of the surgery. Therefore, organs need to be preserved in a special organ preservation device from the time they are removed from the body until the time of surgery in order to prolong their survival time and ensure their good viability.

[0003] Currently, the mainstream clinical protocol for ex vivo organ preservation remains static cryopreservation (SCS), with a preservation temperature of 0–4°C and preservative solutions primarily consisting of UW or HTK solutions. For the liver, the clinically accepted safe cold ischemia time is approximately 12 hours; for the heart and lungs, it is even shorter, typically requiring transplantation within 6 hours. In recent years, mechanical perfusion techniques (such as hypothermic oxygenation perfusion and hypothermic mechanical perfusion) have gradually entered clinical research, which can improve oxygen supply and metabolite clearance to some extent. However, these techniques are complex, costly, and still cannot exceed the preservation time limit above freezing. Therefore, the existing static cryopreservation (SCS) method, limited by temperatures above freezing, results in insufficient metabolic inhibition, and local ice crystal formation still occurs in the 0–4°C environment, leading to mitochondrial damage and cell death. This is one of the fundamental reasons why the preservation time is difficult to extend.

[0004] To lower the preservation temperature below freezing (-2°C to -8°C), some studies have attempted to use cryoprotectants (CPAs) for vitrification. However, high concentrations of cryoprotectants have significant chemical toxicity to organs, and devitrification (ice crystal reformation) easily occurs during rewarming. Currently, they have only found limited application in small tissues or simple cell systems and have not yet become a feasible solution for the preservation of large organs. Therefore, existing cryoprotectant (CPAs) preservation methods are chemically toxic and complex to operate, making them unsuitable for clinical transplantation of large organs such as the liver and heart.

[0005] Recent literature reports that electrostatic or magnetic fields can affect the nucleation temperature and growth morphology of ice crystals in aqueous solutions. Therefore, the applicant believes that if this technology could be applied to the field of organ preservation, it could better inhibit the growth of ice crystals during sub-zero preservation, thereby improving the preservation effect and activity of organs. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is: how to provide a supercooled ex vivo organ preservation method that can better reduce the organ preservation temperature, improve the preservation effect and organ activity, and make it suitable for clinical organ transportation and scientific research sample preservation.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for preserving supercooled ex vivo organs involves placing the ex vivo organ in a preservation solution and then preserving it after cooling. The method is characterized by placing the ex vivo organ within a magnetic field range and preserving it at a temperature within the supercooled range of 0°C to -5°C.

[0008] Magnetic fields can influence and lower the nucleation temperature of ice crystals in aqueous solutions. Existing experiments have shown that magnetic fields, under certain strengths, can delay water freezing and increase supercooling, thus indirectly affecting the freezing process. This phenomenon is already being practically applied in refrigerator preservation technology, using magnetic fields to inhibit ice crystal growth, thereby increasing supercooling, lowering refrigerator preservation temperatures, and extending the shelf life of food. Therefore, the applicant is considering applying this principle to the preservation of ex vivo organs. Through experimental verification, the applicant has successfully lowered the organ preservation temperature to the supercooled range of 0℃ to -5℃ without freezing, using conventional preservation solutions. This effectively lowers the organ preservation temperature, extends the preservation time, and improves survival rates.

[0009] Furthermore, this method includes the following steps: (1) Organ pretreatment: Obtain the human organ to be preserved and use pre-cooled perfusion fluid (temperature usually 15-20℃) to rinse the residual blood inside the organ in order to reduce the initial temperature of the organ and reduce the impact of residual blood on freezing stability. (2) Preservation solution immersion: The pretreated organ is placed in the preservation solution so that the whole organ is completely immersed in the preservation solution to establish a stable liquid phase heat transfer environment; Furthermore, the preservation solution is pre-cooled to the same temperature as the pre-treated organ (usually 15-20°C) before organ placement. Because the organ was flushed with pre-cooled perfusion solution beforehand, the preservation solution is pre-cooled to the same temperature as the organ and then cooled synchronously to avoid organ hypothermia stress and improve organ preservation results.

[0010] Furthermore, the main components of the cryopreservation solution include hydroxyethyl starch, lactobionic acid, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, raffinose, adenosine, allopurinol, glutathione, polyethylene glycol, and glucose. Lactobionic acid and raffinose maintain osmotic pressure balance and reduce cell edema; hydroxyethyl starch and polyethylene glycol provide colloidal osmotic protection and enhance supercooling stability; potassium dihydrogen phosphate and magnesium sulfate heptahydrate maintain the stability of the solution's ionic environment; adenosine, as an energy metabolism precursor, participates in cellular metabolic regulation; allopurinol and glutathione alleviate oxidative stress damage; glucose enhances cryoprotection; insulin promotes glucose utilization; and dexamethasone reduces inflammatory damage. This cryopreservation solution is based on University of Wisconsin (UW) cryopreservation solution (a commonly used organ preservation solution), with the addition of polyethylene glycol, glucose, insulin, and dexamethasone, forming a supercooled cryopreservation solution with strong osmotic protection, antioxidant, and supercooling stability properties, making it suitable for organ cryopreservation environments.

[0011] (3) Gradient cooling under magnetic field: The organ and preservation fluid are placed within the magnetic field range, and the temperature of the preservation system (i.e., the organ and preservation fluid) is gradually reduced to the supercooled range by gradient cooling to achieve preservation.

[0012] Furthermore, during cooling, the temperature is first reduced to 4 °C at a rate of 1-5 °C / min, then to 0 °C at a rate of 0.5-1 °C / min, and finally to the supercooled range of 0 °C to -5 °C at a rate of 0.1-0.5 °C / min. This initial rapid cooling pre-cooling reduces organ metabolic levels and achieves preliminary pre-cooling, followed by a slow cooling to near 0 °C to minimize tissue stress caused by the temperature gradient. Near the freezing point, the cooling rate is reduced, allowing the system to slowly transition from 0 °C to sub-zero temperatures, thereby inhibiting the triggering of heterogeneous nucleation. Therefore, this method better avoids thermal shock and premature nucleation caused by rapid cooling, improves supercooled stability, and enhances preservation effectiveness.

[0013] As an optimized option, the magnetic field is a static magnetic field. This allows the use of permanent magnets to obtain a static magnetic field, which is more cost-effective.

[0014] Specifically, in this case, the method is implemented using an organ preservation device based on a static magnetic field. The organ preservation device based on a static magnetic field includes a box body with an opening at the top and a lid. The box body includes an outer shell on the outside and an inner liner on the inside. There are also multiple pairs of permanent magnets arranged symmetrically between the outer shell and the inner liner. A semiconductor cooling chip is installed on the lid, with the cold end of the semiconductor cooling chip facing downward. A battery is also installed on the box body or the lid and connected to the semiconductor cooling chip for power supply.

[0015] In this way, when the device is in use, the inner liner contains the organ preservation solution for storing the organs. After the lid is closed, the inner liner is cooled by a semiconductor cooling chip to provide a preservation temperature environment. Then, a permanent magnet creates a magnetic field inside the inner liner, which inhibits the nucleation and growth of ice crystals in the aqueous solution, thus achieving the effect of non-crystalline, low-damage preservation of ex vivo organs in a sub-zero environment.

[0016] Furthermore, the outer shell is made of high-strength ABS engineering plastic. It is low in cost, high in strength, and easy to manufacture.

[0017] Furthermore, an intermediate layer consisting of heat-insulating material or a hollow structure is provided between the outer shell and the inner liner. This forms a sealed storage cavity that isolates the interior from heat exchange, effectively reducing the impact of ambient temperature fluctuations on the temperature control accuracy within the cavity.

[0018] Furthermore, the insulation material is polyurethane foam, which is easy to prepare.

[0019] Furthermore, the inner liner is cylindrical, and the permanent magnet is a vertical strip that is fitted into the inner liner in a ring shape.

[0020] This allows for a more effective magnetic field to be generated inside the inner liner. In implementation, a customized cylindrical Halbach permanent magnet array structure can be used, specifically high-energy-product rare-earth neodymium iron boron permanent magnets (N52 grade), arranged circumferentially according to the Halbach magnetic circuit structure, and nested entirely within the inner liner to form an integrated cylindrical structure. A cylindrical, uniform magnetic field cavity with an axially penetrating center is formed within the array, achieving a uniform static magnetic field strength of 2mT-5mT within the cavity, with a uniform area of ​​<5% magnetic field non-uniformity accounting for ≥90%. This permanent magnet solution achieves continuous magnetic field output with zero power consumption without external power supply, generates no Joule heat during operation, and has no thermal conflict with the semiconductor cooling system, fundamentally solving the defects of traditional electromagnetic coil solutions, such as severe heat generation and conflict with cooling requirements. Simultaneously, the integrated array structure reduces weight by more than 80% compared to electromagnetic coil solutions with the same field strength, significantly reducing the overall weight of the device and adapting to portable transportation needs.

[0021] Furthermore, the inner liner is made of stainless steel or high borosilicate glass, and the length of the permanent magnet is equal to or exceeds the length of the inner liner.

[0022] This ensures that the container is completely covered by a uniform static magnetic field throughout the entire process, better guaranteeing the uniformity of the internal magnetic field. The stainless steel (or high borosilicate glass) material used in the container has excellent low-temperature impact resistance and corrosion resistance. It exhibits no structural deformation or release of harmful substances in sub-zero environments of -10℃ to 0℃, is easy to sterilize, and fully meets the aseptic and biosafety requirements for clinical ex vivo organ preservation.

[0023] Furthermore, a heat-conducting plate is attached to the lower surface of the cold end of the semiconductor cooling chip. The heat-conducting plate has a circular heat-conducting protrusion extending downward around it. The height of the upper port of the inner liner is lower than the height of the upper port of the box body, and when the box cover is closed, the lower end face of the circular heat-conducting protrusion is in contact with the surface of the upper port of the inner liner.

[0024] This allows the thermoelectric cooler to conduct heat downwards more effectively.

[0025] Furthermore, the heat-conducting sheet and the circular heat-conducting boss are made of copper, resulting in higher thermal conductivity.

[0026] Furthermore, a wind-cooled heat sink is provided on the outside of the cold end of the semiconductor cooling chip, and a ventilation window is provided on the upper surface of the cover corresponding to the wind-cooled heat sink. The wind-cooled heat sink is connected to the battery for power supply.

[0027] This makes it easier for the thermoelectric cooler to transfer heat outwards. In implementation, the air-cooled heat sink is an existing mature device, specifically a finned high-density aluminum alloy heat sink, matched with a low-noise DC axial fan. The bottom surface of the heat sink is tightly attached to the hot end face of the thermoelectric cooler through high thermal conductivity nano-thermal conductive silicone grease, which continuously removes the heat generated by the hot end of the thermoelectric cooler during operation, ensuring stable cooling efficiency.

[0028] Furthermore, one side of the lid and one side of the upper part of the box are hinged by stainless steel hinges, and the other side is equipped with a push-type latch to achieve fastening. A sealing ring is also provided on the lower surface of the lid corresponding to the upper part of the box opening.

[0029] This allows for better rapid opening and closing of the cavity and negative pressure sealing, ensuring the stability of the low-temperature environment within the cavity. During implementation, the sealing ring is preferably made of food-grade silicone rubber.

[0030] Furthermore, a control unit is provided on one side of the enclosure, and a temperature sensor is provided on the inner liner and connected to the control unit. The control unit is connected to the semiconductor cooling chip and the air-cooled heat sink.

[0031] This allows for better temperature control. In implementation, a multi-point distributed platinum resistance temperature sensor (PT1000) can be used. Furthermore, the temperature sensors can be deployed inside the inner liner, on the outer wall of the inner liner, and at the cold and hot ends of the thermoelectric cooler. This allows for real-time acquisition of end-to-end temperature data and feedback to the PID controller, which serves as the control unit. The controller, through closed-loop regulation of the thermoelectric cooler's input power, achieves continuous adjustment within the -10℃ to 0℃ temperature range inside the cavity, with a temperature control accuracy of ±0.2℃. The maximum temperature difference in the sample area is controlled within 0.3℃, completely avoiding ice crystal formation caused by localized overcooling and tissue metabolic damage caused by localized overheating. In implementation, the control unit is equipped with an interactive LCD touchscreen display on the outer side of the enclosure for convenient temperature control and on / off operation.

[0032] Furthermore, a telescopic pull rod is installed on the other side of the box, and casters are installed at the bottom of the box.

[0033] This design facilitates the transport and delivery of the enclosure, adapting to the mobility needs of clinical transport scenarios. Further, the telescopic pull rod, made of high-strength aviation-grade aluminum alloy, is nested within the vertical limiting groove on the back of the outer enclosure. It features a multi-position telescopic locking structure, allowing for adjustment of the telescopic length according to operator needs. When not in use, it is completely retracted into the back of the enclosure, occupying no extra space. Four sets of silent swivel casters with self-locking brakes are fixedly installed at the four corners of the enclosure's bottom. These casters, made of wear-resistant rubber, allow for 360° rotation in a flat surface. Combined with the brake locking structure, they enable movement and fixed-point positioning of the device during transport, addressing the pain point of traditional equipment's inability to adapt to rapid clinical transport.

[0034] Alternatively, the magnetic field can be a dynamic magnetic field. In this way, a rotating magnet can be used to provide a dynamic magnetic field for the preservation system.

[0035] This is because organs and biological tissues contain a certain amount of ferromagnetic components (such as hemoglobin, ferritin, metal ion-rich areas, iron-deposited microregions, biomineralized magnetic microstructures, and other magnetically sensitive biological components), which possess certain magnetic response characteristics and can exhibit dynamic response behavior under the influence of an external dynamic magnetic field. Under the influence of a dynamic magnetic field, the magnetic response particles or magnetic components within the organ and tissue can be induced to generate microscale movements, creating continuous disturbances at the liquid-solid interface, thereby weakening the stable attachment of crystal nuclei and delaying ice crystal formation. Furthermore, the applicant has verified through specific experiments that the preservation effect of a dynamic magnetic field on organs is superior to that of a static magnetic field.

[0036] The supercooled ex vivo organ preservation method using a dynamic magnetic field can be implemented using a single-arm dynamic magnetic field organ preservation device. This device includes a base, with a cylindrical organ preservation container (made of a non-ferromagnetic material, such as high borosilicate glass) mounted on the upper end of the base. The upper end of the organ preservation container is open and fitted with an openable, sealed lid. A cryogenic fluid jacket surrounds the outer periphery of the inner cavity of the organ preservation container. A cryogenic fluid inlet is connected to one side of the lower end of the cryogenic fluid jacket, and a cryogenic fluid outlet is connected to the other side of the lower end of the cryogenic fluid jacket. The cryogenic outlet (in practice, cryogenic inlet and cryogenic outlet are connected to cryogenic pipes and a liquid pump connected to the cryogenic tank, and the cryogenic pipes are cooled and heat exchanged through the evaporator of an air conditioning system). A rotary motor is set directly opposite the upper axis of the organ preservation container. The rotary motor is fixedly mounted on a motor mounting bracket fixed relative to the base. The output shaft of the rotary motor is set vertically downward and fixedly connected to a rotating rod. One end of the rotating rod is fixedly connected downward to a strip-shaped permanent magnet arranged vertically at intervals on the outside of the organ preservation container. The other end of the rotating rod is fixedly connected downward to a counterweight.

[0037] This method controls the temperature of the organ preservation container by regulating the temperature of the cryosol and its input and output, thereby enabling the preservation of the ex vivo organ under the influence of a dynamic magnetic field. It has the advantages of simple device structure and ease of implementation. During preservation, a dynamic magnetic field is applied. The dynamic disturbance of the magnetic field weakens the stable adhesion state of the liquid-solid interface at potential heterogeneous nucleation sites within the organ tissue, inhibits the formation of locally ordered crystal nuclei, and reduces the probability of initial crystal nucleus stabilization. Simultaneously, the periodic microscale interface disturbances formed under the continuous action of the rotating magnetic field further disrupt the local ordered arrangement of water molecules in the supercooled state, increasing the overall ice crystal nucleation free energy barrier of the system, thus delaying the initial ice crystal formation time and maintaining the organ in a sub-zero ice-free supercooled state.

[0038] Furthermore, the coolant jacket has multiple baffles arranged in a spiral direction. The upper end of the baffles is left a distance from the top of the coolant jacket to form an annular mixing zone at the top of the coolant jacket. Spiral-arranged coolant upward channels and coolant downward channels are formed between adjacent baffles. The upper ends of the coolant upward channels and coolant downward channels are connected to the mixing zone, the lower end of the coolant upward channels is connected to the coolant inlet, and the lower end of the coolant downward channels is connected to the coolant outlet.

[0039] In this way, multiple refrigerant inlets simultaneously feed refrigerant along the spiral upward refrigerant channel to the mixing zone, and then the refrigerant flows downward through the downward refrigerant channel back to the refrigerant outlet. Therefore, this sandwich structure enables rapid flow and fluid exchange of refrigerant within the sandwich, greatly improving heat exchange efficiency.

[0040] In the case of using a dynamic magnetic field, the supercooled ex vivo organ preservation method can also be implemented using a unipolar dynamic magnetic field organ preservation device. The unipolar dynamic magnetic field organ preservation device includes a base, with a cylindrical organ preservation container (made of non-ferromagnetic material, such as high borosilicate glass) at the top of the base. The organ preservation container has an opening at the top and is fitted with an openable sealed container lid. A cryo-fluid jacket is arranged around the outer periphery of the organ preservation container. A cryo-fluid inlet is connected to one side of the lower end of the cryo-fluid jacket, and a cryo-fluid outlet is connected to the other side of the lower end of the cryo-fluid jacket (in practice, the cryo-fluid inlet and outlet are connected to a cryo-fluid pipe and a liquid pump, which are connected to a cryo-fluid tank. The cryo-fluid pipe passes through an evaporator of an air conditioning system for cooling and heat exchange). Multiple pairs of permanent magnets are also installed at intervals around the cryo-fluid jacket in an axisymmetric arrangement. The lower ends of the permanent magnets are fixed on a circular rotating seat. The rotating seat is rotatably mounted on the outer periphery of the base by means of bearings. A rotary motor is also installed on the rotating seat, and the main shaft of the rotary motor is connected to the rotating seat for transmission.

[0041] In this way, by controlling the temperature of the cryosol and its input and output, the temperature of the organ preservation container is regulated, thereby enabling the preservation of the ex vivo organ under the action of a dynamic magnetic field. This method has the advantages of simple device structure and ease of implementation. Furthermore, compared to the aforementioned single-arm dynamic magnetic field organ preservation device, this device exhibits better operational stability. Moreover, compared to the aforementioned organ preservation devices based on static magnetic fields, this device utilizes a rotating magnetic field to construct a continuously changing, time-varying perturbation environment, avoiding the saturation problem caused by a single steady-state magnetic field. This significantly improves the dynamic control capability over potential freezing interfaces within the complex internal structure of the organ, thereby enhancing the stability of supercooled preservation and improving the sustained anti-icing effect.

[0042] Furthermore, the coolant jacket has multiple baffles arranged in a spiral direction. The upper end of the baffles is left a distance from the top of the coolant jacket to form an annular mixing zone at the top of the coolant jacket. Spiral-arranged coolant upward channels and coolant downward channels are formed between adjacent baffles. The upper ends of the coolant upward channels and coolant downward channels are connected to the mixing zone, the lower end of the coolant upward channels is connected to the coolant inlet, and the lower end of the coolant downward channels is connected to the coolant outlet.

[0043] In this way, multiple refrigerant inlets simultaneously feed refrigerant along the spiral upward refrigerant channel to the mixing zone, and then the refrigerant flows downward through the downward refrigerant channel back to the refrigerant outlet. Therefore, this sandwich structure enables rapid flow and fluid exchange of refrigerant within the sandwich, greatly improving heat exchange efficiency.

[0044] Furthermore, the permanent magnet is a vertically elongated strip arranged in a ring with intervals between it and the organ preservation container. This further facilitates the formation of a uniform magnetic field inside.

[0045] Furthermore, a gear ring is horizontally fixed on the lower surface of the rotating seat, and a gear is mounted on the main shaft of the rotary motor that meshes with the gear ring to achieve a transmission connection. The structure is simple, and the transmission is stable and reliable.

[0046] In the case of using a dynamic magnetic field, the supercooled ex vivo organ preservation method can also be implemented using a composite dynamic magnetic field organ preservation device. The composite dynamic magnetic field organ preservation device includes a base, with a cylindrical organ preservation container (made of non-ferromagnetic material, such as high borosilicate glass) at the top of the base. The organ preservation container has an opening at the top and is fitted with an openable, sealed container lid. A cryogenic liquid jacket is arranged around the outer periphery of the organ preservation container. A cryogenic liquid inlet is connected to one side of the lower end of the cryogenic liquid jacket, and a cryogenic liquid outlet is connected to the other side of the lower end of the cryogenic liquid jacket (in practice, the cryogenic liquid inlet and outlet are connected to a cryogenic liquid pipe and a liquid pump connected to a cryogenic liquid tank, and the cryogenic liquid pipe passes through an evaporator of an air conditioning system for cooling and heat exchange). A pair of vertically arranged, axially symmetrical, electromagnetic coils are also installed at intervals on the left and right sides outside the cryogenic liquid jacket. A pair of horizontally arranged, axially symmetrical, electromagnetic coils are also installed at intervals on the upper and lower sides outside or inside the vertical electromagnetic coils. The vertical and horizontal electromagnetic coils are each connected to a control unit.

[0047] This method controls the temperature of the organ preservation container by regulating the temperature of the cryosol and its input and output, thereby preserving the ex vivo organ under the influence of a dynamic magnetic field. It boasts advantages such as simple device structure and ease of implementation. Simultaneously, the device relies on a control unit to control the current of two symmetrically arranged electromagnetic coils. By supplying multiphase alternating current (usually three-phase) to the spatially symmetrically distributed coil windings, the phase difference of each phase current and the spatial geometric arrangement can synthesize a continuously rotating magnetic field in space (this technique of controlling the symmetrical coil current to form a rotating magnetic field is a mature existing technology and will not be detailed here). Furthermore, this device superimposes two rotating magnetic fields in the horizontal and vertical directions within its internal space. Compared to organ preservation devices with a single-pole dynamic magnetic field, this allows the dynamic magnetic field to create a three-dimensional disturbance response effect on the ferromagnetic components inside the ex vivo organ, better ensuring the uniformity of the disturbance response effect at various locations within the organ preservation container, and better improving the dynamic control capability of potential freezing interfaces in the complex internal structure of the organ, thus significantly improving the live preservation effect of the ex vivo organ.

[0048] Furthermore, the coolant jacket has multiple baffles arranged in a spiral direction. The upper end of the baffles is left a distance from the top of the coolant jacket to form an annular mixing zone at the top of the coolant jacket. Spiral-arranged coolant upward channels and coolant downward channels are formed between adjacent baffles. The upper ends of the coolant upward channels and coolant downward channels are connected to the mixing zone, the lower end of the coolant upward channels is connected to the coolant inlet, and the lower end of the coolant downward channels is connected to the coolant outlet.

[0049] In this way, multiple refrigerant inlets simultaneously feed refrigerant along the spiral upward refrigerant channel to the mixing zone, and then the refrigerant flows downward through the downward refrigerant channel back to the refrigerant outlet. Therefore, this sandwich structure enables rapid flow and fluid exchange of refrigerant within the sandwich, greatly improving heat exchange efficiency.

[0050] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention does not require the addition of exogenous magnetic particles or other magnetic response media to the preservation system. It can directly utilize the natural magnetic response components inside the organ to achieve cryo-freezing regulation, avoiding the biotoxicity, tissue residue and clinical safety risks that may be caused by exogenous additives. (2) By applying a static magnetic field or constructing a continuous dynamic magnetic disturbance environment, the present invention can effectively suppress heterogeneous nucleation during organ preservation, improve the supercooling stability of the organ system, delay ice crystal formation, and maintain a sub-zero ice-free preservation state. (3) This invention utilizes the continuous dynamic action of a rotating magnetic field to replace the traditional unidirectional steady-state action of a static magnetic field, thereby enhancing the ability to regulate the multi-interface freezing area inside complex organs and improving the overall ice suppression uniformity and stability. (4) The present invention is simple to operate, non-contact, easy to implement, has good clinical adaptability and engineering application potential, can significantly extend the effective preservation time of human organs outside the body, and improve the success rate of organ transplantation.

[0051] In summary, this invention can better reduce organ preservation temperature, improve preservation effect and organ viability, and is especially suitable for clinical organ preservation and scientific research sample preservation. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the organ preservation device based on a static magnetic field used in Embodiment 1 of the present invention.

[0053] Figure 2 This is a schematic diagram of the single-arm dynamic magnetic field organ preservation device used in Embodiment 2 of the present invention.

[0054] Figure 3 for Figure 2A schematic diagram showing the internal cryogenic fluid upward and downward channels of the individual organ preservation container.

[0055] Figure 4 This is a schematic diagram of the unipolar dynamic magnetic field organ preservation device used in Embodiment 3 of the present invention.

[0056] Figure 5 This is a schematic diagram of the composite dynamic magnetic field organ preservation device used in Embodiment 4 of the present invention.

[0057] Figure 6 This is a schematic diagram of the temperature-time change curves of different systems during the cooling process in the experiment to verify the principle of this invention. Detailed Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments.

[0059] Example 1: A method for preserving supercooled ex vivo organs, wherein the ex vivo organ is placed in a preservation solution and then cooled for preservation. The method is characterized by placing the ex vivo organ within a magnetic field range and preserving it in a supercooled range of 0°C to -5°C.

[0060] Magnetic fields can influence the nucleation behavior of ice crystals in aqueous solutions, lowering the nucleation temperature and increasing supercooling under certain conditions. Previous studies have shown that an external magnetic field may alter the hydrogen bond structure and interfacial energy state of water molecules, thereby increasing the nucleation free energy barrier and inhibiting crystal formation. Although the specific mechanism is not yet fully understood, experimental results consistently show that an appropriate magnetic field strength can effectively delay the freezing process of water and aqueous solutions, increase supercooling, and regulate ice crystal formation. This phenomenon has already been practically applied in refrigerator preservation technology, using magnetic fields to inhibit ice crystal growth, thereby increasing supercooling, lowering refrigerator preservation temperatures, and extending the shelf life of food. Therefore, the applicant considered applying this principle to the preservation of ex vivo organs. Experiments conducted by the applicant have verified that, using conventional preservation solutions, the organ preservation temperature can be successfully lowered to the supercooled range of 0°C to -5°C without freezing. This effectively lowers the organ preservation temperature, extends the preservation time, and improves survival rates.

[0061] When implemented, this method includes the following steps: (1) Organ pretreatment: Obtain the human organ to be preserved and use pre-cooled perfusion fluid (temperature usually 15-20℃) to rinse the residual blood inside the organ in order to reduce the initial temperature of the organ and reduce the impact of residual blood on freezing stability. (2) Preservation solution immersion: The pretreated organ is placed in the preservation solution so that the whole organ is completely immersed in the preservation solution to establish a stable liquid phase heat transfer environment; The preservation solution is pre-cooled to the same temperature as the pre-treated organ (usually 15-20℃) before organ placement. Because the organ was flushed with pre-cooled perfusion solution beforehand, the preservation solution is pre-cooled to the same temperature as the organ and then cooled synchronously to avoid organ hypothermia stress and improve the organ preservation effect.

[0062] The cryopreservation solution primarily comprises hydroxyethyl starch, lactobionic acid, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, raffinose, adenosine, allopurinol, glutathione, polyethylene glycol, and glucose. Lactobionic acid and raffinose maintain osmotic balance and reduce cell edema; hydroxyethyl starch and polyethylene glycol provide colloidal osmotic protection and enhance supercooling stability; potassium dihydrogen phosphate and magnesium sulfate heptahydrate maintain ionic stability; adenosine, as an energy metabolism precursor, participates in cellular metabolic regulation; allopurinol and glutathione mitigate oxidative stress damage; glucose enhances cryoprotection; insulin promotes glucose utilization; and dexamethasone reduces inflammatory damage. This cryopreservation solution is based on University of Wisconsin (UW) cryopreservation solution (a commonly used organ preservation solution), with the addition of polyethylene glycol, glucose, insulin, and dexamethasone. It possesses strong osmotic protection, antioxidant, and supercooling stability properties, making it suitable for cryopreservation of organs.

[0063] Of course, when implementing this method, the preservation solution can also be the existing UW solution or HTK preservation solution (full name: histidine-tryptophan-ketoglutarate solution, which is an existing conventional organ preservation solution), etc.

[0064] (3) Gradient cooling under magnetic field: The organ and preservation fluid are placed within the magnetic field range, and the temperature of the preservation system (i.e., the organ and preservation fluid) is gradually reduced to the supercooled range by gradient cooling to achieve preservation.

[0065] The cooling process involves first lowering the temperature to 4 °C at a rate of 1-5 °C / min, then to 0 °C at a rate of 0.5-1 °C / min, and finally to the supercooled range of 0 °C to -5 °C at a rate of 0.1-0.5 °C / min. This initial rapid cooling reduces organ metabolic levels and achieves preliminary pre-cooling. The subsequent slow cooling to near 0 °C minimizes tissue stress caused by the temperature gradient. Approaching the freezing point, the cooling rate is reduced, allowing the system to slowly transition from 0 °C to sub-zero temperatures, thus inhibiting heterogeneous nucleation. Therefore, this method better avoids thermal shock and premature nucleation caused by rapid cooling, improves supercooled stability, and enhances preservation effectiveness.

[0066] In this embodiment 1, the magnetic field is a static magnetic field. This allows the use of permanent magnets to obtain a static magnetic field, which is more cost-effective.

[0067] Specifically, in this embodiment, the method employs an organ preservation device based on a static magnetic field. (See [link to embodiment]). Figure 1 The organ preservation device based on static magnetic field includes a box body with an opening at the top and a box cover 6. The box body includes an outer shell 1 on the outside and an inner liner 2 on the inside. The device is characterized in that there are multiple pairs of permanent magnets 3 arranged symmetrically between the outer shell 1 and the inner liner 2. A semiconductor cooling chip 4 is installed on the box cover with the cold end of the semiconductor cooling chip 4 facing downward. A battery 5 is also installed at the bottom of the box body and connected to the semiconductor cooling chip for power supply.

[0068] In this way, when the device is in use, the inner liner contains the organ preservation solution for storing the organs. After the lid is closed, the inner liner is cooled by a semiconductor cooling chip to provide a preservation temperature environment. Then, a permanent magnet creates a magnetic field inside the inner liner, which inhibits the nucleation and growth of ice crystals in the aqueous solution, thus achieving the effect of non-crystalline, low-damage preservation of ex vivo organs in a sub-zero environment.

[0069] The outer shell 1 is made of high-strength ABS engineering plastic. It is low in cost, high in strength, and easy to manufacture.

[0070] The outer shell 1 and the inner liner 2 are further separated by an intermediate layer 7 formed by heat insulation material or a hollow structure. This creates a sealed storage cavity that isolates the environment from heat exchange, effectively reducing the impact of ambient temperature fluctuations on the temperature control accuracy within the cavity.

[0071] The thermal insulation material is polyurethane foam, which is easy to prepare.

[0072] The inner liner 2 is cylindrical, and the permanent magnet 3 is a vertical strip that fits into the inner liner in a ring shape.

[0073] This allows for a more effective magnetic field to be generated inside the inner liner. In implementation, a customized cylindrical Halbach permanent magnet array structure can be used, specifically high-energy-product rare-earth neodymium iron boron permanent magnets (N52 grade), arranged circumferentially according to the Halbach magnetic circuit structure, and nested entirely within the inner liner to form an integrated cylindrical structure. A cylindrical, uniform magnetic field cavity with an axially penetrating radius is formed at the center of the array, achieving a uniform static magnetic field strength of 2mT-5mT within the cavity, with a uniform area of ​​<5% magnetic field non-uniformity accounting for ≥90%. This permanent magnet solution achieves continuous magnetic field output with zero power consumption without external power supply, generates no Joule heat during operation, and has no thermal conflict with the semiconductor cooling system. Simultaneously, the integrated array structure reduces weight by more than 80% compared to electromagnetic coil solutions with the same field strength, significantly reducing the overall weight of the device and meeting portable transportation requirements.

[0074] The inner liner 2 is made of food-grade 304 stainless steel, and the length of the permanent magnet is equal to or exceeds the length of the inner liner.

[0075] This design offers advantages such as high strength, good thermal conductivity, and safe material properties. Alternatively, a high borosilicate glass liner can be used. This type of container utilizes high borosilicate glass, which possesses excellent low-temperature impact resistance and biocompatibility. It exhibits no structural deformation or release of harmful substances in sub-zero environments (-10℃ to 0℃), fully meeting the sterile and biosafety requirements for clinical ex vivo organ preservation.

[0076] A heat-conducting plate 8 is attached to the lower surface of the cold end of the semiconductor cooling chip 4. The heat-conducting plate 8 has a circular heat-conducting protrusion 9 extending downward around it. The height of the upper port of the inner liner is lower than the height of the upper port of the box body. When the box cover is closed, the lower end face of the circular heat-conducting protrusion is in contact with the upper port surface of the inner liner 2.

[0077] This allows the thermoelectric cooler to conduct heat downwards more effectively.

[0078] The heat-conducting sheet 8 and the circular heat-conducting boss 9 are made of copper, resulting in higher thermal conductivity.

[0079] The semiconductor cooling chip 4 has a fan-cooled heat sink 10 on its cold end, and the upper surface of the cover has a ventilation window 11 corresponding to the fan-cooled heat sink. The fan-cooled heat sink is connected to the battery 5 for power supply.

[0080] This makes it easier for the thermoelectric cooler to transfer heat outwards. In implementation, the air-cooled heat sink is an existing mature device, specifically a finned high-density aluminum alloy heat sink, matched with a low-noise DC axial fan. The bottom surface of the heat sink is tightly attached to the hot end face of the thermoelectric cooler through high thermal conductivity nano-thermal conductive silicone grease, which continuously removes the heat generated by the hot end of the thermoelectric cooler during operation, ensuring stable cooling efficiency.

[0081] The lid 6 is hinged to the upper part of the box body on one side by a stainless steel hinge 12, and a push-type latch 13 is provided on the other side to achieve fastening. A sealing ring 14 is also provided on the lower surface of the lid corresponding to the box opening at the upper end of the box body.

[0082] This allows for better rapid opening and closing of the cavity and negative pressure sealing, ensuring the stability of the low-temperature environment within the cavity. During implementation, the sealing ring is preferably made of food-grade silicone rubber.

[0083] The box body has a control unit 15 on one side, and a temperature sensor (not shown in the figure) is installed on the inner liner and connected to the control unit. The control unit 15 is connected to the semiconductor cooling chip and the air-cooled heat sink.

[0084] This approach better facilitates temperature control. In implementation, a multi-point distributed platinum resistance temperature sensor (PT1000) can be used. These sensors can be strategically placed inside the inner chamber, on the outer wall of the inner chamber, and at the cold and hot ends of the thermoelectric cooler. This allows for real-time acquisition of end-to-end temperature data, which is then fed back to the PID controller acting as the control unit. The controller, through closed-loop regulation of the thermoelectric cooler's input power, achieves continuous adjustment within the -10℃ to 0℃ temperature range inside the chamber, with a temperature control accuracy of ±0.2℃. The maximum temperature difference within the sample area is controlled within 0.3℃, completely avoiding ice crystal formation caused by localized overcooling and tissue metabolic damage caused by localized overheating. In implementation, the control unit is equipped with an interactive LCD touchscreen display located on the outer side of the chamber for convenient temperature control and on / off operation.

[0085] The other side of the box is equipped with an external telescopic pull rod 16 (not shown in the figure), and the bottom of the box is equipped with a caster device 17.

[0086] This design facilitates the transport and delivery of the enclosure, adapting to the mobility needs of clinical transport scenarios. During implementation, the telescopic pull rod, made of high-strength aviation-grade aluminum alloy, is nested within the vertical limiting groove on the back of the outer enclosure. It features a multi-position telescopic locking structure, allowing for adjustment of the telescopic length according to operator needs. When not in use, it is completely retracted into the back of the enclosure, occupying no extra space. Four sets of silent swivel casters with self-locking brakes are fixedly installed at the four corners of the bottom of the enclosure. These casters, made of wear-resistant rubber, allow for 360° rotation in a flat surface. Combined with the brake locking structure, they enable movement and fixed-point positioning of the device during transport, addressing the pain point of traditional equipment's inability to adapt to rapid clinical transport.

[0087] Example 2: The difference between the supercooled ex vivo organ preservation method in Example 2 and Example 1 is that the magnetic field in this example is a dynamic magnetic field (the other method steps are the same as in Example 1). This is because under the action of a rotating or alternating magnetic field, the system will further generate microscale disturbances or shearing effects on the ferromagnetic components, making it difficult for crystal nuclei to grow stably, thereby delaying the freezing process and prolonging the nucleation time.

[0088] Specifically, the supercooled ex vivo organ preservation method in this embodiment is implemented using a single-arm dynamic magnetic field organ preservation device, which is described in [reference needed]. Figure 2 and Figure 3The system includes a base 21, with a cylindrical organ preservation container 22 (made of non-ferromagnetic material, such as borosilicate glass) mounted on the upper end of the base 21. The organ preservation container 22 has an openable, sealed container lid 23. A cryogenic fluid jacket surrounds the outer periphery of the inner cavity of the organ preservation container. A cryogenic fluid inlet 24 is connected to one side of the lower end of the cryogenic fluid jacket, and a cryogenic fluid outlet 25 is connected to the other side of the lower end of the cryogenic fluid jacket. A rotary motor 30 is positioned directly opposite the axis at the upper end of the organ preservation container. The rotary motor is fixedly mounted on a motor mounting bracket 31 fixed relative to the base. The output shaft of the rotary motor is vertically downward and fixedly connected to a rotating rod 32. One end of the rotating rod is fixedly connected downward to a strip-shaped permanent magnet 33 arranged vertically at intervals on the outside of the organ preservation container, and the other end of the rotating rod 32 is fixedly connected downward to a counterweight 34.

[0089] In addition, during implementation, the refrigerant inlet and outlet need to be connected to a refrigeration system to achieve refrigerant cooling control. In this embodiment, the refrigeration system is a compression refrigeration cycle system directly installed inside the base. The compression refrigeration cycle system includes an evaporator 26, a throttle valve 27, a condenser 28, and a compressor 29. In other embodiments, the refrigeration system can also be an external refrigeration system.

[0090] This method controls the temperature of the organ preservation container by regulating the temperature of the cryosol and its input and output, thereby enabling the preservation of the ex vivo organ under the influence of a dynamic magnetic field. It has the advantages of simple device structure and ease of implementation. During preservation, a dynamic magnetic field is applied. The dynamic disturbance of the magnetic field weakens the stable adhesion state of the liquid-solid interface at potential heterogeneous nucleation sites within the organ tissue, inhibits the formation of locally ordered crystal nuclei, and reduces the probability of initial crystal nucleus stabilization. Simultaneously, the periodic microscale interface disturbances formed under the continuous action of the rotating magnetic field further disrupt the local ordered arrangement of water molecules in the supercooled state, increasing the overall ice crystal nucleation free energy barrier of the system, thus delaying the initial ice crystal formation time and maintaining the organ in a sub-zero ice-free supercooled state.

[0091] The coolant jacket contains multiple baffles 35 arranged in a spiral direction. The upper end of each baffle is left a distance from the top of the coolant jacket to form an annular mixing zone 36 at the top of the coolant jacket. A spirally arranged coolant upward channel 37 and coolant downward channel 38 are formed between adjacent baffles. The upper ends of the coolant upward channel and the coolant downward channel are connected to the mixing zone. The lower end of the coolant upward channel is connected to the coolant inlet, and the lower end of the coolant downward channel is connected to the coolant outlet.

[0092] In this way, multiple refrigerant inlets simultaneously feed refrigerant along the spiral upward refrigerant channel to the mixing zone, and then the refrigerant flows downward through the downward refrigerant channel back to the refrigerant outlet. Therefore, this sandwich structure enables rapid flow and fluid exchange of refrigerant within the sandwich, greatly improving heat exchange efficiency.

[0093] Example 3: The supercooled ex vivo organ preservation method in Example 3 differs from that in Example 2 only in the apparatus used. Specifically, in Example 3, the supercooled ex vivo organ preservation method employs a unipolar dynamic magnetic field organ preservation device, see [link to example]. Figure 4 The unipolar dynamic magnetic field organ preservation device includes a base 41, with a cylindrical organ preservation container 42 (made of non-ferromagnetic material, such as high borosilicate glass) at the upper end of the base 41. The organ preservation container has an opening at the upper end and is fitted with an openable sealed container lid. A cryofluid jacket 43 is arranged around the outer periphery of the organ preservation container. A cryofluid inlet 44 is connected to one side of the lower end of the cryofluid jacket, and a cryofluid outlet 45 is connected to the other side of the lower end of the cryofluid jacket. Multiple pairs of permanent magnets 46 are also installed at intervals outside the cryofluid jacket in an axisymmetric arrangement. The lower ends of the permanent magnets are fixed on a circular rotating seat 47. The rotating seat is rotatably mounted on the outer periphery of the base by means of bearings. A rotary motor 48 is also installed on the rotating seat, and the main shaft of the rotary motor is connected to the rotating seat 47 for transmission.

[0094] In addition, during implementation, the refrigerant inlet and outlet need to be connected to a refrigeration system to control the refrigerant temperature. In this embodiment, the refrigeration system is a compression refrigeration cycle system directly installed inside the base, and the structure of the compression refrigeration cycle system is the same as in Embodiment 2. In other embodiments, the refrigeration system can also be an external refrigeration system.

[0095] In this way, by controlling the temperature of the cryosol and its input and output, the temperature of the organ preservation container is regulated, thereby enabling the preservation of the ex vivo organ under the action of a dynamic magnetic field. This method has the advantages of simple device structure and ease of implementation. Furthermore, compared to the aforementioned single-arm dynamic magnetic field organ preservation device, this device exhibits better operational stability. Moreover, compared to the aforementioned organ preservation devices based on static magnetic fields, this device utilizes a rotating magnetic field to construct a continuously changing, time-varying perturbation environment, avoiding the saturation problem caused by a single steady-state magnetic field. This significantly improves the dynamic control capability over potential freezing interfaces within the complex internal structure of the organ, thereby enhancing the stability of supercooled preservation and improving the sustained anti-icing effect.

[0096] The refrigerant jacket contains multiple partitions arranged in a spiral direction. The upper end of each partition is left a distance from the top of the refrigerant jacket to form an annular mixing zone at the top of the refrigerant jacket. Spiral-arranged refrigerant upward and downward channels are formed between adjacent partitions. The upper ends of the refrigerant upward and downward channels are connected to the mixing zone, the lower end of the refrigerant upward channel is connected to the refrigerant inlet, and the lower end of the refrigerant downward channel is connected to the refrigerant outlet. (The internal structure of the refrigerant jacket in this embodiment is the same as that in Embodiment 2, and can be connected with reference to its attached drawings. The diagrams are not repeated here.)

[0097] In this way, multiple refrigerant inlets simultaneously feed refrigerant along the spiral upward refrigerant channel to the mixing zone, and then the refrigerant flows downward through the downward refrigerant channel back to the refrigerant outlet. Therefore, this sandwich structure enables rapid flow and fluid exchange of refrigerant within the sandwich, greatly improving heat exchange efficiency.

[0098] The permanent magnet 46 is a vertically elongated strip arranged in a ring around the organ preservation container, which is more conducive to forming a uniform magnetic field inside.

[0099] A gear ring 49 is horizontally fixed on the lower surface of the rotating seat, and a gear 50 is mounted on the main shaft of the rotary motor, which meshes with the gear ring 49 to achieve a transmission connection. The structure is simple and the transmission is stable and reliable.

[0100] Example 4: The supercooled ex vivo organ preservation method in Example 4 differs from that in Example 2 only in the device used. Specifically, in Example 4, the supercooled ex vivo organ preservation method employs a composite dynamic magnetic field organ preservation device. See [link to example]. Figure 5 The composite dynamic magnetic field organ preservation device includes a base 51, with a cylindrical organ preservation container 52 (made of non-ferromagnetic material, such as high borosilicate glass) at the upper end of the base. The organ preservation container has an opening at the upper end and is fitted with an openable sealed container lid. A cryo-fluid jacket 53 is arranged around the outer periphery of the organ preservation container. A cryo-fluid inlet 54 is connected to one side of the lower end of the cryo-fluid jacket, and a cryo-fluid outlet 55 is connected to the other side of the lower end of the cryo-fluid jacket. A pair of vertically arranged, axially symmetrical vertical electromagnetic coils 56 are also installed at intervals on the left and right sides of the cryo-fluid jacket. A pair of horizontally arranged, axially symmetrical horizontal electromagnetic coils 57 are also installed at intervals on the upper and lower sides of the vertical electromagnetic coils, either outside or inside. The vertical electromagnetic coils 56 and the horizontal electromagnetic coils 57 are each connected to a control unit (not shown in the figure).

[0101] In addition, during implementation, the refrigerant inlet and outlet need to be connected to a refrigeration system to control the refrigerant temperature. In this embodiment, the refrigeration system is a compression refrigeration cycle system directly installed inside the base, and the structure of the compression refrigeration cycle system is the same as in Embodiment 2. In other embodiments, the refrigeration system can also be an external refrigeration system.

[0102] This method controls the temperature of the organ preservation container by regulating the temperature of the cryosol and its input and output, thereby preserving the ex vivo organ under the influence of a dynamic magnetic field. It boasts advantages such as simple device structure and ease of implementation. Simultaneously, the device relies on a control unit to control the current of two symmetrically arranged electromagnetic coils. By supplying multiphase alternating current (usually three-phase) to the spatially symmetrically distributed coil windings, the phase difference of each phase current and the spatial geometric arrangement can synthesize a continuously rotating magnetic field in space (this technique of controlling the symmetrical coil current to form a rotating magnetic field is a mature existing technology and will not be detailed here). Furthermore, this device superimposes two rotating magnetic fields in the horizontal and vertical directions within its internal space. Compared to organ preservation devices with a single-pole dynamic magnetic field, this allows the dynamic magnetic field to create a three-dimensional disturbance response effect on the ferromagnetic components inside the ex vivo organ, better ensuring the uniformity of the disturbance response effect at various locations within the organ preservation container, and better improving the dynamic control capability of potential freezing interfaces in the complex internal structure of the organ, thus significantly improving the live preservation effect of the ex vivo organ.

[0103] The refrigerant jacket contains multiple baffles arranged in a spiral direction. The upper ends of the baffles are spaced a distance from the top of the refrigerant jacket to form an annular mixing zone. Spiral-arranged upward and downward refrigerant channels are formed between adjacent baffles. The upper ends of the upward and downward refrigerant channels connect to the mixing zone, the lower end of the upward refrigerant channel connects to the refrigerant inlet, and the lower end of the downward refrigerant channel connects to the refrigerant outlet. (The internal structure of the refrigerant jacket in this embodiment is the same as in Embodiment 2, and can be referred to in the attached drawings for connection; the diagrams and descriptions are not repeated here.) In this way, multiple refrigerant inlets simultaneously feed refrigerant along the spiral upward refrigerant channel to the mixing zone, and then the refrigerant flows downward through the downward refrigerant channel back to the refrigerant outlet. Therefore, this sandwich structure enables rapid flow and fluid exchange of refrigerant within the sandwich, greatly improving heat exchange efficiency.

[0104] To better explore and verify the core principles of the method of this invention, the applicant conducted comparative experiments. Samples under four environmental conditions were compared and tested in a low-temperature constant-temperature environment: deionized water, magnetic fluid, magnetic fluid under a static magnetic field, and magnetic fluid under a dynamic magnetic field. During the experiment, each group of samples was placed in a container of the same volume, with an initial temperature of approximately 10–12 °C. They were then placed in a low-temperature constant-temperature device for cooling, with the ambient temperature controlled at approximately -15 °C. Temperature changes were recorded in real time using thermocouples. For the magnetic field group, a stable magnetic field (or rotating magnetic field) was applied during the cooling process, with the magnetic field strength controlled within the range of 0–20 mT.

[0105] In biological tissues, organs such as the liver and heart are rich in endogenous ferritin and hemoglobin, with concentrations typically ranging from approximately 500–2000 mg / kg and 100–500 mg / kg, respectively. Related studies have shown that ferritin is paramagnetic at room temperature, and its magnetic susceptibility is linearly related to the iron concentration within the tissue. Therefore, under the influence of an external magnetic field, biological tissues as a whole exhibit a typical weak magnetic response system.

[0106] To experimentally simulate this type of weak magnetic response environment, a magnetic nanoparticle system can be introduced, and a correspondence between the two can be established based on the principle of magnetic susceptibility equivalence. According to the fundamental theory of magnetic media, the magnetic response intensity of the system can be expressed as the product of magnetic susceptibility and volume fraction:

[0107] Where M represents the magnetization of the system, φ represents the volume fraction of the magnetic component in the system, χ represents the magnetic susceptibility of the material, and H represents the applied magnetic field strength. Therefore, by maintaining equivalent magnetization, the magnetic response in biological tissue can be equivalent to a low-concentration dispersion state in a magnetic nanoparticle system.

[0108] Considering the magnetic susceptibility of ferritin (approximately 10) -5 -10 -4 It is significantly lower than Fe3O4 nanoparticles (approximately 10). -1 –1), the difference in their magnetic response capabilities is approximately 10. 4 -10 5 Based on this difference, and combined with the actual iron content range of the tissue, the equivalent concentration of Fe3O4 nanoparticles can be calculated to be in the ppm range. Specifically, the equivalent concentration in liver tissue is approximately 1–10 ppm, and in heart tissue it is approximately 0.1–5 ppm.

[0109] The above equivalence relationship is established based on the consistency of macroscopic magnetic response, mainly reflecting the equivalence at the level of magnetic field intensity. Therefore, a 1 ppm concentration of magnetohydrodynamic fluid was used to replace organ tissue in experiments with static and rotating magnetic fields.

[0110] Experimental results are as follows Figure 6 As shown in the image, based on the analysis of the supercooling ΔT (defined as the difference between the equilibrium freezing temperature 0 ℃ and the nucleation temperature T_n) in the image, we can obtain the following results: the supercooling of the deionized water system is 12.818 ℃, the supercooling of the magnetohydrodynamic system is 5.618 ℃, the supercooling of the magnetohydrodynamic system under static magnetic field is 8.521 ℃, and the supercooling of the magnetohydrodynamic system under dynamic magnetic field is 9.412 ℃.

[0111] Under the same cooling conditions, although the deionized water system can achieve a relatively large degree of supercooling, its latent heat is rapidly released once nucleation occurs, limiting its ability to maintain stable supercooling. In contrast, the supercooling of the magnetohydrodynamic (MHD) system without an applied magnetic field is significantly reduced to 5.618 °C, a decrease of approximately 56.2% compared to deionized water. This indicates that the introduction of magnetic particles provides a large number of heterogeneous nucleation sites, lowers the nucleation energy barrier, and promotes earlier nucleation. Under the influence of an applied magnetic field, the supercooling capability of the MHD system is significantly restored and enhanced: under a static magnetic field, the supercooling increases to 8.521 °C, an increase of approximately 2.903 °C compared to the condition without a magnetic field, representing an increase of approximately 51.7%; under a dynamic magnetic field, the supercooling further increases to 9.412 °C, an increase of approximately 3.794 °C compared to the condition without a magnetic field, representing an increase of approximately 67.5%, while still showing an increase of approximately 0.891 °C compared to the static magnetic field. These results indicate that a dynamic magnetic field has a stronger inhibitory effect on nucleation than a static magnetic field. Based on the nucleation time, it can be seen that the nucleation time is earliest in the non-magnetic magnetic fluid system, while the nucleation time is significantly delayed after the magnetic field is applied. The delay effect is most significant under the dynamic magnetic field condition, indicating that the magnetic field not only increases the supercooling, but also enhances the system's ability to maintain metastable state.

[0112] The above results indicate that the introduction of magnetic particles promotes heterogeneous nucleation and reduces supercooling. An external magnetic field, by controlling the spatial arrangement and dynamic behavior of the magnetic particles (such as the formation and reconstruction of chain-like structures), weakens effective nucleation sites and increases the nucleation energy barrier, thereby partially offsetting or even reversing the nucleation-promoting effect of the magnetic particles. In particular, the dynamic magnetic field, by continuously perturbing the magnetic structure, makes it more difficult for the system to form stable nucleation cores, thus exhibiting a superior supercooling enhancement effect.

[0113] Previous studies have shown that static magnetic fields can, to some extent, increase the supercooling of biological tissue systems and delay ice crystal formation. The results of this experiment are consistent with this pattern and further demonstrate that magnetic fields have a universal regulatory effect on systems containing magnetically responsive components. Given that biological tissues (such as the liver) contain endogenous magnetically responsive substances such as ferritin, their microenvironment may also undergo similar regulation under the influence of an external magnetic field, thereby affecting ice crystal nucleation behavior.

[0114] Therefore, it can be reasonably proven that the technical mechanism of magnetic field-controlled nucleation of the present invention is not only applicable to magnetofluid systems, but can also be extended to the cryopreservation process of biological tissues containing endogenous magnetic response components, so as to achieve the technical effects of increasing supercooling, delaying freezing and reducing ice crystal damage.

Claims

1. A method for preserving supercooled ex vivo organs, comprising placing the ex vivo organ in a preservation solution, cooling it, and then preserving it, characterized in that... The isolated organ was placed within a magnetic field and preserved by cooling it to a supercooled range of 0°C to -5°C.

2. The method for preserving supercooled ex vivo organs as described in claim 1, characterized in that, Includes the following steps: (1) Organ pretreatment: Obtain human organs to be preserved and use pre-cooled perfusion fluid to rinse the residual blood inside the organs in order to reduce the initial temperature of the organs and reduce the impact of residual blood on freezing stability. (2) Preservation solution immersion: The pretreated organ is placed in the preservation solution so that the whole organ is completely immersed in the preservation solution to establish a stable liquid phase heat transfer environment; (3) Gradient cooling under magnetic field: The organ and preservation fluid are placed in the magnetic field range, and the temperature of the preservation system is gradually reduced to the supercooled range by gradient cooling to achieve preservation.

3. The method for preserving supercooled ex vivo organs as described in claim 2, characterized in that, In step (2), the preservation solution is pre-cooled to the same temperature as the pre-treated organ before organ placement.

4. The method for preserving supercooled ex vivo organs as described in claim 2, characterized in that, In step (2), the cryopreservation solution mainly comprises hydroxyethyl starch, lactobionic acid, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, raffinose, adenosine, allopurinol, glutathione, polyethylene glycol, and glucose. Lactobionic acid and raffinose are used to maintain osmotic pressure balance and reduce cell edema. Hydroxyethyl starch and polyethylene glycol are used to provide colloidal osmotic protection and enhance supercooling stability. Potassium dihydrogen phosphate and magnesium sulfate heptahydrate are used to maintain the stability of the solution's ionic environment. Adenosine, as an energy metabolism precursor, participates in cell metabolism regulation. Allopurinol and glutathione are used to reduce oxidative stress damage. Glucose is used to enhance the cryoprotective effect. Insulin is used to promote glucose utilization. Dexamethasone is used to reduce inflammatory damage.

5. The method for preserving supercooled ex vivo organs as described in claim 2, characterized in that, In step (3), the temperature is first reduced to 4 ℃ at a cooling rate of 1-5℃ / min, then reduced to 0 ℃ at a cooling rate of 0.5-1℃ / min, and then reduced to the supercooled range of 0 ℃ to -5 ℃ at a cooling rate of 0.1-0.5℃ / min.

6. The method for preserving supercooled ex vivo organs as described in claim 1, characterized in that, The magnetic field is a static magnetic field; The method is implemented using an organ preservation device based on a static magnetic field. The organ preservation device based on a static magnetic field includes a box body with an opening at the top and a box cover. The box body includes an outer shell on the outside and an inner liner on the inside. There are also multiple pairs of permanent magnets arranged symmetrically between the outer shell and the inner liner. A semiconductor cooling chip is installed on the box cover with the cold end of the semiconductor cooling chip facing downward. A battery is also installed on the box body or the box cover and connected to the semiconductor cooling chip for power supply. An intermediate layer, either made of heat-insulating material or a hollow structure, is also provided between the outer shell and the inner liner; The inner liner is cylindrical, and the permanent magnet is a vertical strip that fits into the inner liner in a ring shape. The inner liner is made of stainless steel, and the length of the permanent magnet is equal to or exceeds the length of the inner liner. A heat-conducting plate is attached to the lower surface of the cold end of the semiconductor cooling chip. The heat-conducting plate has a circular heat-conducting protrusion extending downward around it. The height of the upper port of the inner liner is lower than the height of the upper port of the box body. When the box cover is closed, the lower end face of the circular heat-conducting protrusion is in contact with the surface of the upper port of the inner liner. A wind-cooled heat sink is provided on the outside of the cold end of the semiconductor cooling chip, and a ventilation window is provided on the upper surface of the cover corresponding to the wind-cooled heat sink. The wind-cooled heat sink is connected to the battery for power supply. A control unit is provided on one side of the box, and a temperature sensor is provided on the inner liner and connected to the control unit. The control unit is connected to the semiconductor cooling chip and the air-cooled heat sink. A telescopic pull rod is also installed on the other side of the box, and casters are installed at the bottom of the box.

7. The method for preserving supercooled ex vivo organs as described in claim 1, characterized in that, The magnetic field is a dynamic magnetic field.

8. The method for preserving supercooled ex vivo organs as described in claim 7, characterized in that, The method employs a single-arm dynamic magnetic field organ preservation device, which includes a base. A cylindrical organ preservation container is mounted on the upper end of the base. The upper end of the organ preservation container is open and fitted with an openable, sealed container lid. A cryofluid jacket surrounds the outer periphery of the inner cavity of the organ preservation container. A cryofluid inlet is connected to one side of the lower end of the cryofluid jacket, and a cryofluid outlet is connected to the other side of the lower end of the cryofluid jacket. A rotary motor is positioned directly opposite the axis at the upper end of the organ preservation container. The rotary motor is fixedly mounted on a motor mounting bracket fixed relative to the base. The output shaft of the rotary motor is vertically downward and fixedly connected to a rotating rod. One end of the rotating rod is fixedly connected downward to a strip-shaped permanent magnet arranged vertically at intervals on the outside of the organ preservation container, and the other end of the rotating rod is fixedly connected downward to a counterweight. The coolant jacket has multiple baffles arranged in a spiral direction. The upper end of the baffles is left a distance from the top of the coolant jacket to form an annular mixing zone at the top of the coolant jacket. Spiral-arranged coolant upward and downward channels are formed between adjacent baffles. The upper ends of the coolant upward and downward channels are connected to the mixing zone, the lower end of the coolant upward channel is connected to the coolant inlet, and the lower end of the coolant downward channel is connected to the coolant outlet.

9. The method for preserving supercooled ex vivo organs as described in claim 7, characterized in that, The method employs a unipolar dynamic magnetic field organ preservation device, which includes a base with a cylindrical organ preservation container mounted on the upper end of the base. The organ preservation container has an opening at the top and is fitted with an openable, sealed container lid. A cryofluid jacket surrounds the organ preservation container, with a cryofluid inlet connected to one side of the lower end of the jacket and a cryofluid outlet connected to the other side. Multiple pairs of permanent magnets are also installed symmetrically around the cryofluid jacket, with their lower ends fixed to a circular rotating seat. The rotating seat is rotatably mounted on the outer periphery of the base by bearings, and a rotary motor is mounted on the rotating seat. The main shaft of the rotary motor is connected to the rotating seat via a transmission. The coolant jacket has multiple baffles arranged in a spiral direction. The upper end of the baffles is left a distance from the top of the coolant jacket to form an annular mixing zone at the top of the coolant jacket. Spiral-arranged coolant upward and downward channels are formed between adjacent baffles. The upper ends of the coolant upward and downward channels are connected to the mixing zone, the lower end of the coolant upward channel is connected to the coolant inlet, and the lower end of the coolant downward channel is connected to the coolant outlet. The permanent magnet is a vertically elongated strip and is arranged in a ring with the organ preservation container spaced apart. A gear ring is horizontally fixed on the lower surface of the rotating seat, and a gear is installed on the main shaft of the rotary motor to mesh with the gear ring to achieve transmission connection.

10. The method for preserving supercooled ex vivo organs as described in claim 7, characterized in that, The method employs a composite dynamic magnetic field organ preservation device, which includes a base with a cylindrical organ preservation container mounted on the upper end of the base. The organ preservation container has an open top and is fitted with an openable, sealed container lid. A cryo-fluid jacket surrounds the organ preservation container. A cryo-fluid inlet is connected to one side of the lower end of the cryo-fluid jacket, and a cryo-fluid outlet is connected to the other side of the lower end of the cryo-fluid jacket. A pair of vertically arranged, axially symmetrical electromagnetic coils are also installed at intervals on the left and right sides of the cryo-fluid jacket. A pair of horizontally arranged, axially symmetrical electromagnetic coils are also installed at intervals on the upper and lower sides of the vertical electromagnetic coils, either outside or inside the vertical electromagnetic coils. The vertical and horizontal electromagnetic coils are each connected to a control unit. The coolant jacket has multiple baffles arranged in a spiral direction. The upper end of the baffles is left a distance from the top of the coolant jacket to form an annular mixing zone at the top of the coolant jacket. Spiral-arranged coolant upward and downward channels are formed between adjacent baffles. The upper ends of the coolant upward and downward channels are connected to the mixing zone, the lower end of the coolant upward channel is connected to the coolant inlet, and the lower end of the coolant downward channel is connected to the coolant outlet.

Images