A self-pressurized supercooled storage and transportation device for large-volume biological samples

Abstract

The present application relates to a kind of self-pressurized supercooled storage and transport devices of large volume biological sample, belong to human life article technical field, for biological sample preservation and transport.The device includes the following contents: self-pressurized storage and transport tank, with rigid outer layer and flexible inner layer, separate into freezing chamber and sample storage chamber.Through different surface coating, freezing chamber freezes in advance, pass through flexible film to sample storage chamber Pressure transmission, maintain its supercooling steady state;Active heating / cooling system and passive cold storage and transport system are divided into two layers.Upper layer includes self-pressurized storage and transport tank installation groove, cooling liquid circulating chamber and inflation chamber.Low-temperature storage device transport box can be separated alone, and conveniently transported.Inflation chamber is inflated by air pump, so that it is attached to self-pressurized storage and transport tank.The liquid in cooling liquid circulating chamber is used for the cooling / heating of self-pressurized storage and transport tank.The overall design can realize the long-time supercooling preservation and convenient transport of biological sample.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of human life supplies, and particularly relates to a self-pressurized supercooled storage and transportation device for large-volume biological samples. BACKGROUND

[0002] With the increasing demand for organ transplantation in clinical medicine, the supply of organs is far less than the number of patients waiting for transplantation, and the demand for preservation methods of transplantable donor organs is becoming more urgent. The current mainstream method of cryopreservation is to keep biological samples in a frozen state at-80 DEG C or-196 DEG C, which will face fatal ice crystal damage and protective agent damage for large-volume samples such as organs, and will also face serious thermal stress damage during rewarming. Supercooling preservation is a promising alternative method for large-volume samples (such as organs), which can extend the preservation time of biological samples in the system and avoid ice crystal damage to biological samples through technical means. Thus, the time and space limitations faced by biological sample transportation are greatly reduced, which has very important significance for short-term high-quality storage and transportation of biological samples. SUMMARY

[0003] The purpose of the present application is to provide a self-pressurized supercooled storage and transportation device for large-volume biological samples, which solves the technical problems of damage caused by ice crystals, protective agents and thermal stress, too small amount of preserved samples, strict time and space limitations during transportation and inconvenient operation during transportation in the existing cryopreservation technology.

[0004] The technical scheme of the present application is as follows:

[0005] A self-pressurized supercooled preservation device for large-volume biological samples, comprising a self-pressurized storage and transportation tank, an active heating / cooling system and a passive cold storage and transportation system;

[0006] The self-pressurized storage and transportation tank comprises a rigid tank body, a flexible membrane is arranged in the inner chamber of the rigid tank body, and the inner chamber of the rigid tank body is divided into a freezing chamber and a sample storage chamber by the flexible membrane, a sample storage liquid is stored in the sample storage chamber, a freezing liquid is stored in the freezing chamber, the freezing chamber is arranged around the sample storage chamber, and a sample adding port and a freezing liquid feeding port are arranged on the rigid tank body respectively for the sample storage chamber and the freezing chamber;

[0007] The active heating / cooling system and the non-rim cold storage and transportation system comprises a low-temperature storage device and a temperature control system, the low-temperature storage device is provided with a self-pressurized storage and transportation tank placing groove, a cooling liquid circulating chamber is arranged on the outer periphery of the self-pressurized storage and transportation tank placing groove, an inflation chamber is arranged on the outer periphery of the cooling liquid circulating chamber, and the self-pressurized storage and transportation tank is placed in the self-pressurized storage and transportation tank placing groove; the temperature control system is arranged below the low-temperature storage device and comprises a thermoelectric refrigerator and a circulating water pump and a gas pump connected with the thermoelectric refrigerator respectively, the circulating water pump and the gas pump are communicated with the cooling liquid circulating chamber and the inflation chamber through pipelines respectively, and a separation valve is arranged on each of the two pipelines.

[0008] The low-temperature storage device and the temperature control system are detachably connected through the separation valve, when the low-temperature storage device is separated from the low-temperature storage device, the self-pressurized storage and transportation tank is stored and transferred through the low-temperature storage device.

[0009] The active heating / cooling system and the non-rim cold storage and transportation system control the temperature of the self-pressurized storage and transportation tank through the thermoelectric refrigerator, the circulating water pump, the cooling liquid circulating chamber, the gas pump and the inflation chamber, the cooling liquid circulating chamber is extruded by the inflation chamber to tightly adhere to the self-pressurized storage and transportation tank, and then the temperature of the cooling liquid circulating chamber is rapidly synchronized and kept stable.

[0010] Further, the surface of the flexible membrane in contact with the freezing chamber is coated with a coating for increasing the crystallization probability of the freezing liquid, and the surface of the flexible membrane in contact with the sample storage chamber is coated with an inert coating for reducing the crystallization probability of the sample storage liquid.

[0011] Further, the sample storage liquid is a low-temperature preservation liquid for a large-volume biological sample; the freezing liquid is added with a nucleating agent for increasing the heterogeneous nucleation probability of the liquid and improving the ice crystal generation efficiency.

[0012] Further, when the freezing liquid crystallizes, the volume of the freezing chamber increases and the pressure rises, the pressure is transmitted to the sample storage chamber through the flexible membrane, the pressure of the sample storage chamber rises to inhibit the nucleation and crystallization of the low-temperature preservation liquid, and the biological sample is in a high-pressure supercooled stable state.

[0013] Further, the sample adding port and the freezing liquid feeding port are respectively provided with a storage chamber sealing cover and a freezing chamber sealing cover, and the storage chamber sealing cover and the freezing chamber sealing cover are respectively provided with a drainage protrusion and a sealing ring.

[0014] Further, the large-volume biological sample includes high-throughput preservation of small-volume biological samples and single large-volume biological samples; and the low-temperature preservation liquid is a solution added with a single or multiple low-temperature protective agents and adjusted for different biological samples.

[0015] Further, the temperature control system is further provided with a storage battery for providing power for the self-pressurized supercooled storage and transportation device in an emergency.

[0016] Further, the low-temperature storage device is also provided with a phase-change cold storage plate for reducing temperature fluctuation during operation, thereby avoiding temperature fluctuation of the self-pressurized storage tank.

[0017] Further, the low-temperature storage device is also provided with a low-temperature storage device transfer box top cover made of high cold storage material and having sealing property, for avoiding temperature fluctuation caused by interaction with external gas after being covered.

[0018] Further, the side of the cooling liquid circulating chamber facing the inflation chamber is made of flexible anti-stretching material.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] 1. The device is based on the thermodynamic and kinetic principles of ice crystal formation, and can effectively reduce the freezing damage to biological samples under traditional low-temperature storage conditions through supercooling preservation.

[0021] 2. The deformable flexible film of the present application can be made of high-strength deformation materials such as stainless steel film, which can realize self-pressurization effect and maintain at low temperature, so that stable supercooling effect can be achieved through high pressure without the need to introduce external substances.

[0022] 3. The sample storage chamber and the freezing chamber for providing pressure for rapid crystallization of the sample are filled with liquid through respective filling ports, which can avoid the failure of supercooling preservation caused by mixing of liquids during operation, and significantly reduce the operation difficulty during sample addition.

[0023] 4. Different surface treatments and solution formulations in the sample storage chamber and the freezing chamber of the present application can further improve the ice crystal control efficiency, so that efficient icing is achieved in the freezing chamber, and the supercooling state in the sample storage chamber is effectively maintained during the cooling process.

[0024] 5. The present application can maintain a deep and temperature supercooling system, greatly extending the preservation time of the sample, thereby significantly reducing the time and space limitations in the transfer process of biological samples.

[0025] 6. The device of the present application provides a stable environmental temperature for the self-pressurized storage tank during transfer by using an active heating / cooling system and a passive cold storage storage system, so that the temperature inside the self-pressurized storage tank does not fluctuate, thereby ensuring the preservation quality of the sample.

[0026] 7. The device of the present application is simple, does not require complex pressurization equipment, is easy to operate, and is convenient for transfer of biological samples.

[0027] 8、The device provided by the device can not only realize supercooling preservation, but also realize precise control of temperature during sample transportation, so as to avoid temperature fluctuation in actual operation and affect the preservation quality of the sample. In addition, the integrated storage and transportation device can standardize the whole process from sample preservation to transportation to application, so as to optimize the preservation process to the greatest extent through precise control of each process, and make the sample preservation effect better. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a structure schematic view of a self-pressurizing supercooling storage and transportation device for large-volume biological samples according to the present application;

[0029] Figure 2 It is a structure schematic view of a self-pressurizing supercooling storage and transportation device for large-volume biological samples according to the present application before the self-pressurizing storage and transportation tank works.

[0030] Figure 3 It is a contrastive view of the outer layer of the flexible film in contact with the frozen liquid before and after adopting a-Alumina coating.

[0031] Figure 4 It is a schematic view of the activity of superoxide dismutase of rat kidney after low-temperature preservation.

[0032] Figure 5 It is a schematic view of the malondialdehyde metabolic content of rat kidney after low-temperature preservation.

[0033] Figure 6 It is a schematic view of the activity of sodium-potassium pump ATPase of rat kidney after low-temperature preservation.

[0034] Figure 7 It is a structure schematic view of an active heating / cooling system and a passive cold storage and transportation system.

[0035] 2, low-temperature storage device; 21, self-pressurizing storage and transportation tank placement groove; 22, cooling liquid circulating chamber; 23, inflation chamber; 24, low-temperature storage device top cover; 25, phase change cold storage plate; 3, self-pressurizing storage and transportation tank; 31, rigid tank body; 32, freezing chamber; 33, flexible film; 34, sample storage chamber; 35, frozen liquid filling port; 36, sample adding port; 37, sample storage chamber sealing cover; 38, freezing chamber sealing cover; 4, temperature control system; 41, thermoelectric refrigerator; 42, circulating water pump; 43, air pump; 44, battery; 5, separation valve. DETAILED DESCRIPTION

[0036] The following is a more detailed description of a self-pressurized subcooled storage and transportation device for large-volume biological samples, with reference to schematic diagrams, which illustrate preferred embodiments of the invention. It should be understood that those skilled in the art can modify the invention described herein while still achieving its advantageous effects. Therefore, the following description should be understood as being of general knowledge to those skilled in the art and is not intended to limit the invention.

[0037] like Figure 1 As shown, a self-pressurized subcooled storage and transportation device for large-volume biological samples includes an electrically heated / cooled system, an electrically unheated cold storage and transportation system, and a self-pressurized storage and transportation tank 3.

[0038] The self-pressurized storage and transportation tank 3 includes a rigid tank body 31. The outer layer of the rigid tank body 31 is rigid and can withstand a pressure of 100 MPa. It can be made of stainless steel and is used to confine the pressure to the interior. Its internal cavity is divided into a freezing chamber 32 and a sample storage chamber 34 by a flexible membrane 33. The freezing chamber 32 is filled with freezing fluid, and the sample storage chamber 34 is filled with sample storage fluid. The flexible membrane 33 can transfer heat and pressure, but cannot transfer substances. The flexible membrane 33 is flexible and has high strength, used to direct pressure to the sample storage fluid and achieve complete isolation between the freezing fluid and the biological sample. The outer layer of the flexible membrane 33 in contact with the freezing fluid has an a-Alumina coating, which can increase the probability of freezing fluid crystallization. The inner layer of the flexible membrane 33 in contact with the sample storage fluid is coated with an inert coating, which can reduce the probability of sample storage fluid crystallization. The inert coating is such as a BiofloatFlex coating.

[0039] The sample storage solution is a solution containing one or more cryoprotectants, including but not limited to dimethyl sulfoxide (DMSO), propylene glycol, glycerol, ethylene glycol, trehalose, sucrose, glucose, polyols and polymers (such as polyethylene glycol) or any combination thereof, and organ storage solutions such as HTK solution and UW solution.

[0040] Figure 3 This study presents a comparison of the effects of applying an a-Alumina coating to the outer layer of the flexible membrane 33 before and after it came into contact with the freezing liquid. When pure water was used as the freezing liquid, it was observed in the cooling and freezing experiment that the solution only began to freeze after being supercooled to below -6°C. Figure 3 a. After freezing begins, the internal pressure of the container rises sharply, quickly reaching the pressure corresponding to the temperature. However, the frozen solution, due to the breaking of its supercooled state, experiences a temperature rebound, and the rapidly rising pressure drops accordingly. It then continues to cool in a mixture of ice and water, failing to reach thermal equilibrium for a considerable period, thus the pressure fluctuates within a certain range. During this process, the rapid pressure increase in a short period can cause organ damage, and the supercooling of the freezing solution may cause the cryopreservation solution to freeze simultaneously, damaging the sample. Therefore... Figure 3b shows the effect of taking a-Alumina coating on the outer layer of flexible film 33 in contact with frozen liquid. It directly leads to the freezing of frozen liquid at 0℃, the pressure increases synchronously with the decrease of temperature, avoiding the impact of instantaneous pressure on the sample, avoiding the supercooling of the cryoprotective solution, and accelerating the thermal equilibrium of the container. There is no obvious repeated fluctuation of pressure.

[0041] Further, to increase the probability of frozen liquid crystallization, we add nucleating agents such as nanocellulose crystals and cholesterol crystals to increase the probability of heterogeneous nucleation of frozen liquid and improve the efficiency of ice crystal generation. The sample storage solution is a cryoprotective solution for large volume biological samples, and the composition of the cryoprotective solution includes but is not limited to dimethyl sulfoxide (DMSO), propylene glycol, glycerol, ethylene glycol, trehalose, sucrose, glucose, polyethylene glycol, or any combination of the foregoing, and organ storage solutions such as HTK solution and UW solution. Large volume biological samples include high-throughput preservation of small volume biological samples and single large volume biological samples, such as one or more biological molecules, cell components, cells, viruses, embryos, tissues, organs or entire organisms.

[0042] The freezing chamber 32 and the sample storage chamber 34 are respectively provided with a frozen liquid filling port 35 and a sample filling port 36 above the rigid tank body 31 for adding frozen liquid and sample storage solution. The frozen liquid filling port 35 is provided with a freezing chamber sealing cover 38, and the sample filling port 36 is provided with a sample storage chamber sealing cover 37. Both sealing covers include a drainage protrusion and a sealing ring.

[0043] The heating / cooling system with or without margin and the cold storage and transportation system include a low-temperature storage device 2 and a temperature control system 4. The low-temperature storage device 2 includes a self-pressurized storage and transportation tank placement groove 21 arranged in the middle for placing a self-pressurized storage and transportation tank storing large biological samples. The outer periphery of the self-pressurized storage and transportation tank placement groove is provided with a cooling liquid circulating chamber 22, and the lower half of the cooling liquid circulating chamber 22 is provided with an aeration chamber 23. The top of the low-temperature storage device 2 is also provided with a low-temperature storage device transfer box top cover 24 made of high cold storage material and having sealing property to avoid temperature fluctuation caused by interaction with external gas after covering.

[0044] The temperature control system 4 is arranged below the low-temperature storage device 2 and includes a thermoelectric refrigerator 41, a circulating water pump 42, an air pump 43 and a storage battery 44. The circulating water pump 42 and the air pump 43 are respectively communicated with the cooling liquid circulating chamber 22 and the aeration chamber 23 through pipelines (respectively provided with a cooling liquid inlet and an aeration chamber air inlet), and the storage battery 44 is used to provide power for the thermoelectric refrigerator 41 in emergency conditions.

[0045] Both pipes are provided with a separation valve 5, and the low-temperature storage device 2 and the temperature control system 4 are detachably connected through the separation valve 5.

[0046] After the self-pressurized storage tank 3 is filled, it is placed in the self-pressurized storage tank 3 accommodation groove of the low-temperature storage device 2 of the active heating / cooling system and the passive cold storage and transportation system, and then the air pump 43 is opened, the inside of the inflation chamber 23 is filled with gas, and the cooling liquid circulation chamber 22 is extruded, wherein the surfaces of the cooling liquid circulation chamber 22 and the inflation chamber 23 and the self-pressurized storage tank 3 are flexible and resistant to stretching. After the cooling liquid circulation chamber 22 is extruded, it is tightly attached to the self-pressurized storage tank 3, so that the temperature of the self-pressurized storage tank 3 is rapidly synchronized with the cooling liquid circulation chamber 22 and remains stable. The temperature of the cooling liquid circulation chamber 22 is controlled by the circulating water pump 42 and the thermoelectric refrigerator 41, which can not only refrigerate but also heat, and has an integrated function. The battery 44 can provide power to the entire device as an independent power source during equipment movement. The low-temperature storage device 2 transfer box can be independently separated from the active heating / cooling system and the passive cold storage and transportation system, the connection between the inflation chamber 23 and the cooling liquid circulation chamber 22 and the air pump 43 and the circulating water pump 42 in the lower layer is cut off through the separation valve 5, and the separation is carried out under the premise of maintaining the inflation chamber 23 full and continuing to extrude the cooling liquid circulation chamber 22.

[0047] The working principle of the present application is as follows:

[0048] Because the side of the flexible film 33 facing the freezing chamber 32 is coated with an alpha alumina coating, and the injected freezing liquid contains nucleating agents such as nano-cellulose crystals, and the side facing the sample storage chamber 34 is coated with a BiofloatFlex inert coating, the injected cryoprotective liquid is not prone to nucleation and crystallization. Therefore, during the cooling process, the freezing liquid in the freezing chamber 32 will freeze first. The volume increase trend caused by freezing combined with the high pressure caused by the constraint of the rigid outer wall is transmitted to the biological sample system in the sample storage chamber 34 through the flexible film 33 between the freezing chamber 32 and the sample storage chamber 34. As known from the water triple point, water solutions above -30℃ are more difficult to nucleate and crystallize under the same temperature and pressure, and the components in the cryoprotective agent inhibit the formation of ice crystals during the cooling process. Under the combined action of high pressure and cryoprotective agent, the biological sample system in the sample storage chamber 34 can be cooled to below 0℃ without forming ice crystals, thereby maintaining a stable state of deep supercooling. After the cooling process is completed, the self-pressurized storage tank 3 only needs to be maintained at a certain temperature to ensure the temperature of the supercooling state of the biological sample system. During the warming process, because the heat is conducted from the rigid outer wall to the inside, the liquid in the freezing chamber is thawed first, and after thawing, the system pressure disappears, and then the biological sample can be normally operated.

[0049] Calculation of the volume ratio of the sample storage chamber and the freezing chamber:

[0050] In the case of the total volume of the container is determined, the volume ratio between the freezing chamber and the sample storage chamber is approximately equal to the ratio of the phase fraction of the solid-liquid two-phase in the constant volume container, that is, the freezing chamber is solid phase and the sample storage chamber is liquid phase. The traditional water phase diagram is described according to the Gibbs free energy function G, and the formula is:

[0051] dG=-SdT+Vdp+μdN

[0052] Wherein, temperature T and pressure P are natural variables, S is entropy, V is volume, μ is chemical potential, and N is particle number. The lowest free energy phase in the Gibbs free energy function is projected on the natural variable axis to form a T-P water phase diagram. And in the constant volume condition, because the volume of the container is constant, the two-phase equilibrium of water and ice cannot be directly quantified by the traditional T-P water phase diagram. For the freezing condition under the constant volume condition, another thermodynamic calculation method needs to be used for analysis, that is, the Helmholtz free energy function F is used, and the formula is:

[0053] dF=-SdT-PdV+μdN

[0054] Wherein, temperature T and volume V are natural variables, S is entropy, P is pressure, μ is chemical potential, and N is particle number. In the same way, the free energy is projected on the natural variable to construct a T-V water phase diagram, which is used to describe the two-phase equilibrium zone under the constant volume condition, and a two-dimensional solid-liquid mixed steady-state zone can be obtained instead of the one-dimensional solid-liquid phase coexistence line described by the Gibbs free energy function G. The phase fraction ratio of the two phases in the constant volume container can be obtained by the lever rule.

[0055] The specific process of the biological sample transportation and storage of the application is as follows:

[0056] First, the freezing liquid mixed with nanocellulose crystals or other ice nucleating agents is injected from the freezing liquid inlet 35 into the freezing chamber 32, and the injection rate should not be too fast to prevent the injection of freezing liquid mixed with air bubbles. Then, the freezing chamber sealing cover 38 is sealed, and the drainage protrusion on the freezing chamber sealing cover 38 extrudes the excess freezing liquid from the freezing liquid inlet 35, and cooperates with the sealing ring to ensure that the freezing liquid inlet 35 does not retain air. Then, the biological sample is mixed with the cryoprotective solution and fully loaded, and then the biological sample system is filled into the sample storage chamber 34 through the sample inlet 36, and the filling process should be slow enough to avoid air bubbles mixing into the sample storage chamber. In the same way, the sample storage chamber 34 is sealed by the sample storage chamber sealing cover 37 to ensure that the chamber is free of air and only filled with cryopreservation solution and biological samples. Then, the self-pressurized storage tank 3 loaded with the sample is transferred to the low-temperature storage device 2 in the heating / cooling system with a rim and the cold storage system without a rim. At this time, the low-temperature storage device 2 is connected with the temperature control system 4, and the self-pressurized storage tank 3 is cooled by the temperature control system 4. Different cooling rates are set according to different samples, and after cooling to the required temperature, the low-temperature storage device 2 with the self-pressurized storage tank 3 is separated from the temperature control system 4 by the separation valve 5, and the next transfer and storage operation is performed. When the transfer and storage are completed, the low-temperature storage device 2 with the self-pressurized storage tank 3 is transferred to other locations of the heating / cooling system with a rim and the cold storage system without a rim and connected with the temperature control system 4 through the separation valve 5, and the rewarming operation is performed. When the temperature rises to the set temperature, the biological sample can be taken out for subsequent elution and other operations, and the transfer and storage process of the biological sample is completed.

[0057] The specific sample implementation case results are shown in Table 1. Figures 4 to 6 The biochemical indicators of the rat kidney sample after isochoric supercooling preservation are shown in Table 1. The specific indicators are sodium-potassium pump ATPase (ATP), superoxide dismutase (SOD), and malondialdehyde (MDA). The higher the values of ATP and SOD and the lower the value of MDA, the less damage to the kidney sample during isochoric supercooling preservation, and the better the preservation effect. The specific operation is as follows.

[0058] After the rats were anesthetized, the abdominal cavity was opened, and the aorta, inferior vena cava and left kidney were selected during dissection. After the aorta, inferior vena cava and left kidney were isolated, the blood flow of the top aorta was blocked, and heparinized saline was injected into the left kidney through the infusion needle. When the color of the kidney turned to brown, the membrane and fat around the kidney were stripped, and the blood vessels were disconnected, and the kidney was taken out, which retained the arterial and venous access. In the experimental group, two commonly used organ preservation solutions (HTK solution and UW solution) were respectively injected into the kidney at 4°C, and then the kidney was transferred into the self-pressurized storage tank 3, immersed in the sample storage room, and filled with the corresponding organ preservation solution through the sample inlet. Then the sample storage room was evacuated and sealed by the lid. The freezing chamber was filled with freezing liquid, and then the air in the freezing chamber was evacuated and sealed by the lid. Then the self-pressurized storage tank 3 was placed in the environment of 4°C and-4°C respectively, and placed for 48H. Then the actual box was detected by ATP, SOD and MDA, and the biochemical indexes of the preserved kidney were analyzed. The results are shown in Figures 4 to 6 . Wherein Figure 4 is the SOD activity of the rat kidney preserved at 4°C static cold storage and-4°C isochoric supercooling for 48H, and the change of SOD of the rat kidney preserved at-4°C for 48H and 72H. Similarly Figure 5 is the MDA content of the rat kidney, Figure 6 is the ATP enzyme activity of the rat kidney.

[0059] The above are only preferred embodiments of the present application and do not have any limiting effect on the present application. Any person skilled in the art can make any form of equivalent replacement, modification or change of the technical solutions and technical contents disclosed in the present application without departing from the scope of the technical solutions of the present application, which still belongs to the protection scope of the present application.

Claims

1. A self-pressurized subcooled storage and transportation device for large-volume biological samples, characterized in that, This includes self-pressurized storage and transportation tanks, active heating / cooling systems, and uninsulated cold storage and transportation systems; The self-pressurized storage and transportation tank includes a rigid tank body. The internal cavity of the rigid tank body is provided with a flexible membrane, and the internal cavity of the rigid tank body is divided into a freezing chamber and a sample storage chamber by the flexible membrane. The sample storage chamber stores sample storage solution, and the freezing chamber stores freezing solution. The freezing chamber is arranged around the sample storage chamber. The sample storage chamber and the freezing chamber are respectively provided with sample feeding port and freezing solution feeding port on the rigid tank body. The active heating / cooling system and the passive cold storage and transportation system include a low-temperature storage device and a temperature control system. The low-temperature storage device is equipped with a self-pressurizing storage tank placement tank, a coolant circulation chamber around the outside of the self-pressurizing storage tank placement tank, and an air filling chamber around the outside of the coolant circulation chamber. The self-pressurizing storage tank is placed inside the self-pressurizing storage tank placement tank. The temperature control system is located below the low-temperature storage device and includes a thermoelectric chiller and a circulating water pump and an air pump connected to the thermoelectric chiller respectively. The circulating water pump and the air pump are connected to the coolant circulation chamber and the air filling chamber respectively through pipelines, and both pipelines are equipped with separation valves. The cryogenic storage device and the temperature control system are detachably connected via a separation valve. When the cryogenic storage device is separated from the temperature control system, the self-pressurized storage and transportation tank is stored in the cryogenic storage device and then transferred. The active heating / cooling system and the passive cold storage and transportation system control the temperature of the self-pressurized storage tank through a thermoelectric chiller and a circulating water pump in conjunction with the coolant circulation chamber. The system also uses an air pump in conjunction with an air filling chamber to compress the coolant circulation chamber, making it fit tightly against the self-pressurized storage tank, thereby rapidly synchronizing the temperature with the coolant circulation chamber and maintaining it stably. The flexible membrane in contact with the freezing chamber is coated with a coating to increase the probability of freezing liquid crystallization, and the flexible membrane in contact with the sample storage chamber is coated with an inert coating to reduce the probability of sample storage liquid crystallization. The sample storage solution is a cryopreservation solution for large-volume biological samples; the freezing solution contains nucleating agents to increase the probability of heterogeneous nucleation and improve the efficiency of ice crystal formation. When the freezing solution crystallizes, the volume of the freezing chamber increases and the pressure rises. The freezing chamber transmits the pressure to the sample storage chamber through the flexible membrane. The pressure rise in the sample storage chamber inhibits the nucleation and crystallization of the cryopreservation solution, and the biological sample is in a high-pressure supercooled steady state.

2. The self-pressurized subcooled storage and transportation device for large-volume biological samples according to claim 1, characterized in that, The sample inlet and the freezing liquid inlet are respectively equipped with a storage chamber sealing cover and a freezing chamber sealing cover. Both the storage chamber sealing cover and the freezing chamber sealing cover are equipped with drainage protrusions and sealing rings.

3. The self-pressurized subcooled storage and transportation device for large-volume biological samples according to claim 1, characterized in that, Large-volume biological samples include high-throughput preservation of small-volume biological samples and single large-volume biological samples; the cryopreservation solution is a solution formulated for different biological samples and containing one or more cryoprotectants.

4. The self-pressurized subcooled storage and transportation device for large-volume biological samples according to claim 1, characterized in that, The temperature control system is also equipped with a battery for providing power to the self-pressurized subcooled storage and transportation device in emergency conditions.

5. The self-pressurized subcooled storage and transportation device for large-volume biological samples according to claim 1, characterized in that, The cryogenic storage device is also equipped with a phase change cold storage plate to reduce temperature fluctuations during operation, thereby avoiding temperature fluctuations in the self-pressurized storage tank.

6. The self-pressurized subcooled storage and transportation device for large-volume biological samples according to claim 1, characterized in that, The cryogenic storage device is also equipped with a top cover for a cryogenic storage device transfer box. The top cover is made of a high-cold-storage material and has a sealing function to prevent temperature fluctuations caused by interaction with the outside gas after the cover is closed.

7. The self-pressurized subcooled storage and transportation device for large-volume biological samples according to claim 1, characterized in that, The side of the coolant circulation chamber facing the air chamber is made of a flexible, tensile-resistant material.

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