Organ supercooling storage container as well as preparation method and use method thereof

Abstract

The invention provides an organ supercooling storage container and a preparation method and a use method thereof.The organ supercooling storage container comprises a container body and a sealing layer, one side of the container body is open, and an oil gel layer is arranged on the inner side wall of the container body; the oil gel layer comprises a first polymer frame structure and first liquid oil, the first polymer frame structure is filled with the first liquid oil, the sealing layer is made of an ice dredging material, and the sealing layer is used for sealing an opening in one side of the container body and makes contact with the content in the container body. The sealing layer and the oil gel layer jointly improve the preservation system to form a quasi-homogeneous preservation system, heterogeneous nucleation sites of the preservation system in the preservation container can be eliminated, the stable duration of supercooling preservation is prolonged, and the preservation container can be used for long-term preservation of isolated organs in the environment with the temperature lower than zero DEG C without using a cryoprotectant.

Description

Technical Field

[0001] The present invention belongs to the technical field of organ transplantation, and particularly relates to an organ supercooling preservation container, a preparation method thereof, and a usage method thereof. Background Art

[0002] Organ transplantation is the most effective radical treatment for end-stage organ failure. Worldwide, there is a serious mismatch in the temporal and spatial distribution of donors and recipients, which has become a bottleneck factor restricting the development of organ transplantation. Safe and effective long-term storage technology has become an urgent common demand in the field of organ transplantation. Currently, the main preservation methods clinically used, such as 4°C static or mechanical perfusion, limit the ex vivo preservation time of most important tissues and organs to 24 hours, resulting in more than 70% of donated organs being discarded during preservation.

[0003] In recent years, the gradually developed supercooling technology reduces the preservation temperature below the freezing point and extends the preservation time, which is a highly potential organ preservation means in the future. However, during long-term preservation below the freezing point, uncontrollable ice crystal growth will cause fatal damage to tissues and organs. To maintain a stable supercooled state, traditional methods use a large amount of cryoprotectants to lower the freezing point of the preservation solution. However: firstly, cryoprotectants (such as ethylene glycol, dimethyl sulfoxide, 2,3-butanediol) are exogenous materials for organisms and may cause biosafety problems; secondly, the introduction of cryoprotectants increases the viscosity of the preservation solution. To ensure uniform distribution inside tissues and organs, a mechanically perfusion process that is costly and technically complex is often required during the preservation process, and too high perfusion pressure is likely to cause endothelial cell damage. Therefore, the harm brought by cryoprotectants in the actual organ preservation process offsets the gain in reduced metabolism brought by sub-zero low temperature, limiting the application of supercooling preservation. Summary of the Invention

[0004] In view of this, the present invention provides an organ supercooling preservation container, a preparation method thereof, and a usage method thereof, which can eliminate the heterogeneous nucleation sites in the preservation system inside the preservation container, increase the stable duration of supercooling preservation, and can be used for long-term preservation of ex vivo organs in a sub-zero environment without using cryoprotectants.

[0005] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides an organ supercooling preservation container, comprising: a container body, one side of the container body is open, and an oleogel layer is provided on the inner side wall, the oleogel layer includes a first polymer framework structure and a first liquid oil filled in the first polymer framework structure; and, a sealing layer, the sealing layer is composed of an ice-repellent material, the sealing layer is used to seal the open side of the container body and is in contact with the contents inside the container body.

[0006] Preferably, the first polymer framework structure includes a first polymer, and the first polymer includes at least one of polydimethylsiloxane, polyvinylidene fluoride, polyethylene, polyacrylamide, and polyvinyl chloride; and / or, The first liquid oil includes at least one of silicone oil, perfluoropolyether, and vegetable oil.

[0007] Preferably, the ice-repellent material includes a liquid ice-repellent material, and the liquid ice-repellent material includes at least one of paraffin oil, silicone oil, and liquid alkane.

[0008] Preferably, the ice-repellent material includes a solid ice-repellent material, and the solid ice-repellent material includes at least one of paraffin and organogel; wherein, the organogel includes a second polymer framework structure and a second liquid oil filled in the second polymer framework structure.

[0009] Preferably, a sealing assembly is further included for sealing the container body.

[0010] In a second aspect, the present invention provides a method for preparing the organ supercooling preservation container, the organ supercooling preservation container includes a container body with an organogel layer provided on the inner sidewall, and the method for preparing the container body with an organogel layer provided on the inner sidewall includes the following steps: S11. Mix the first polymer stock solution with the first curing agent, and evacuate to obtain a first mixed solution; S12. Add the first mixed solution into the container body, and then put a first mold adapted to the container body into the container body to obtain a container body to be formed; S13. Dry and mold the container body to be formed, demold, and then add the first liquid oil and soak to obtain a container body with an organogel layer provided on the inner sidewall.

[0011] Preferably, in step S11, the mass ratio of the first polymer stock solution to the first curing agent is (5-20):1; and / or, In step S13, the temperature for drying and molding is 60-100 °C, and the time for drying and molding is 15-180 min.

[0012] Preferably, the organ supercooling preservation container further includes a sealing layer, the sealing layer is composed of organogel, and the organogel includes a second polymer framework structure and a second liquid oil filled in the second polymer framework structure; the method for preparing the sealing layer includes the following steps: S21. Mix the second polymer stock solution with the second curing agent, and evacuate to obtain a second mixed solution; S22. Add the second mixture into a second mold adapted to seal the open side of the container body to obtain a to-be-formed sealing layer. S23. Dry and form the to-be-formed sealing layer, demold it, and soak it in a second liquid oil to obtain a sealing layer.

[0013] Thirdly, the present invention provides a method for using the organ supercooling preservation container as described above, including the following steps: A1. Place the excised organ into the container body with an oil gel layer provided on the inner side wall, and add a preservation solution to submerge the excised organ with the preservation solution. A2. Add an ice-melting prevention material into the container body to cover the preservation solution with the ice-melting prevention material to form a sealing layer. A3. Seal the container body through a sealing component. A4. Place the sealed container body into a temperature device for supercooling preservation.

[0014] Preferably, in step A1, the preservation solution is UW preservation solution; and / or, in step A2, the ice-melting prevention material includes a liquid ice-melting prevention material, and the liquid ice-melting prevention material includes at least one of paraffin oil, silicone oil, and liquid alkane; and / or, Step A4 includes: placing the sealed container body into a temperature device at 4°C, and then cooling it at a rate of 0.1 - 2°C / min to the supercooling preservation temperature for supercooling preservation; wherein, the supercooling preservation temperature is less than or equal to -4°C.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The stability of the organ supercooling preservation of the present invention is strong. When used to preserve a mouse heart, it can be stably supercooled at -4°C for 36 h. Compared with the commonly used 4°C static cold preservation in clinics, the preservation time is longer and the organ damage is significantly reduced.

[0016] (2) The operation of the organ supercooling preservation of the present invention is simple and the cost is low. It does not require steps with complex operations such as mechanical perfusion and expensive equipment. The equipment used is widely used scientific research equipment, and the materials used are inexpensive, with low economic cost and excellent promotion prospects.

[0017] (3) The present invention has strong universality. Except that the materials used for the oil gel layer and the sealing layer have a wide selection space, the preservation container and the using method can be applied to the preservation of various organs (including heart, liver, kidney, lung, etc.) of small animals such as mice and rats, large animals such as pigs, and even humans.

[0018] (4) The present invention has high biosafety. The materials used for the oil gel layer and the sealing layer are biomaterials or bio-friendly materials, and cryoprotectants with cytotoxicity can be not used.

[0019] (5) The present invention has strong durability. After the storage container is used, the oil gel layer can be restored to a complete state only by soaking it in the first liquid oil again, and the liquid ice-repellent material used for the sealing layer can also be taken out and recycled after rewarming. Description of the Drawings

[0020] Figure 1 is a schematic structural diagram of an embodiment of the storage container provided by the present invention; Figure 2 is a flowchart of an embodiment of the preparation method of the storage container provided by the present invention; Figure 3 is a flowchart of an embodiment of the usage method of the storage container provided by the present invention; Figure 4 is a graph of the interfacial ice formation temperature of the oil gel used in Example 1 of the present invention; Figure 5 is a graph of the storage stability of the oil gel used in Example 1 of the present invention; Figure 6 is the ice formation probability under different supercooled storage conditions provided by Example 2 and Comparative Example 1 of the present invention; Figure 7 is the survival rate at different storage times provided by Example 2 and Comparative Example 2 of the present invention; Figure 8 is the heart resuscitation rate at different storage times provided by Example 2 and Comparative Example 2 of the present invention.

[0021] Description of the reference numerals: container body 1; oil gel layer 2; sealing layer 3; preservation liquid 4; excised organ 5. Specific Embodiments

[0022] The following further describes the present invention in detail with reference to specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0023] In a first aspect, please refer to Figure 1 , the present invention provides an organ supercooled storage container, including: A container body 1, one side of the container body 1 is open, and an oil gel layer 2 is provided on the inner side wall. The oil gel layer 2 includes a first polymer framework structure and a first liquid oil filled in the first polymer framework structure; and, A sealing layer 3, the sealing layer 3 is composed of an ice-repellent material, the sealing layer 3 is used to seal the open side of the container body 1 and is in contact with the contents in the container body 1.

[0024] In the technical solution of the present invention, the inner side wall of the container body 1 is provided with the oleogel layer 2, so that the inner wall surface of the container body 1 forms an ice-repellent interface with a high nucleation barrier, and the ice nucleation temperature of this interface is close to the homogeneous nucleation temperature. The sealing layer 3 forms an ice-repellent interface with a high nucleation barrier on the upper surface of the preservation liquid 4, so that all the interfaces of the preservation liquid 4 in the container body 1 adopt the ice-repellent interface with a high nucleation barrier. The oleogel layer 2 and the sealing layer 3 jointly improve the preservation system to form a quasi-homogeneous preservation system, which can eliminate the heterogeneous nucleation sites in the preservation system in the preservation container, improve the stable duration of supercooled preservation, and can be used for the long-term preservation of the excised organ 5 in an environment below zero without using cryoprotectants, improve the biological safety, and the preservation effect is also better than the most commonly used static cold preservation method in current clinical practice.

[0025] It should be noted that the supercooling degree is the system temperature at which the system freezes under a constant cooling rate, and it is an evaluation index for the stability of supercooled preservation. The lower the supercooling degree, the more stable the supercooled preservation. The main nucleation methods in supercooled preservation are heterogeneous nucleation and homogeneous nucleation. Heterogeneous nucleation refers to the nucleation of the liquid phase on the external interface, and homogeneous nucleation refers to the nucleation of the liquid phase inside the bulk phase. The heterogeneous nucleation temperature is significantly higher than the homogeneous nucleation temperature. In the traditional preservation container, the oleogel layer 2 is not provided on the inner side wall of the container body 1, and the inner side wall is usually an ice-prone wall surface (such as metal, plexiglass, plastic, glass, etc.), and the upper part of the preservation liquid 4 is a gas-liquid interface.

[0026] It can be understood that the first liquid oil penetrates into the first polymer framework structure to form strong molecular interactions to form an oleogel, and the material of the oleogel layer 2 on the inner side wall of the container body 1 is an oleogel. When the preservation container preserves the excised organ 5, the content includes the preservation liquid 4 and the excised organ 5, and the preservation liquid 4 submerges the excised organ 5. The contact between the sealing layer 3 and the content in the container body 1 can be that the sealing layer 3 covers the upper surface of the preservation liquid 4; when the sealing layer 3 completely covers the upper surface of the preservation liquid 4, the supercooled preservation effect is better.

[0027] Further, the first polymer framework structure includes a first polymer, and the first polymer includes at least one of polydimethylsiloxane, polyvinylidene fluoride, polyethylene, polyacrylamide, and polyvinyl chloride.

[0028] Further, the first liquid oil includes at least one of silicone oil, perfluoropolyether, and vegetable oil. It should be noted that the silicone oil can include at least one of methyl silicone oil, phenyl silicone oil, ethyl silicone oil, hydrogen-containing silicone oil, and vinyl silicone oil, and the vegetable oil can include at least one of soybean oil, rapeseed oil, peanut oil, and olive oil. Different viscosities can also be used for the first liquid oil. For example, dimethyl silicone oil can be selected with a viscosity of 3 cST to 100000 cST.

[0029] Further, the ice-repellent material includes a liquid ice-repellent material, and the liquid ice-repellent material includes at least one of paraffin oil, silicone oil, and liquid paraffin.

[0030] Further, the ice-repellent material includes a solid ice-repellent material, and the solid ice-repellent material includes at least one of paraffin and organogel; wherein, the organogel includes a second polymer framework structure and a second liquid oil filled in the second polymer framework structure.

[0031] Further, the second polymer framework structure includes a second polymer, and the first polymer includes at least one of polydimethylsiloxane, polyvinylidene fluoride, polyethylene, polyacrylamide, and polyvinyl chloride.

[0032] Further, the second liquid oil includes at least one of silicone oil, perfluoropolyether, and vegetable oil.

[0033] It should be noted that when the sealing layer 3 composed of organogel is used, the second polymer and the second liquid oil used in the sealing layer 3 can be the same as or different from the first polymer and the first liquid oil of the organogel layer 2 on the inner side wall of the container body 1. When the sealing layer 3 is solid, the ice-repellent material can be made into a plug adapted to the container body 1. In addition, the materials used for the sealing layer 3 and the organogel layer 2 can be selected from biomaterials or bio-friendly materials, which can improve the biological safety.

[0034] Further, a sealing assembly is further included for sealing the container body 1. The sealing assembly is used to further seal an open side of the container body 1 to improve the supercooling preservation effect. It should be noted that there are various sealing assemblies. For example, the container body 1 can be sealed with a necked-in flat-weld flange and a screw, or sealed by means of a threaded plug. As long as the open side of the container body 1 can be effectively sealed (effectively sealing the sealing layer 3 inside the container body 1) and the heat transfer performance of the container body 1 is not greatly affected; please refer to Figure 2 , in a specific embodiment, the sealing assembly includes a first necked-in flat-weld flange and a second necked-in flat-weld flange respectively arranged at the upper end and the lower end of the container body 1. The first necked-in flat-weld flange and the second necked-in flat-weld flange are connected by a plurality of screws to fasten the first necked-in flat-weld flange and the second necked-in flat-weld flange to both ends of the container body 1. A gasket and a blind plate are sequentially covered on the first necked-in flat-weld flange, and the first necked-in flat-weld flange and the blind plate are fastened by a clamp to achieve the sealing of the container body 1.

[0035] In a second aspect, the present invention provides a preparation method of the organ supercooling preservation container described above. The organ supercooling preservation container includes a container body with an organogel layer provided on the inner side wall. The preparation method of the container body with an organogel layer provided on the inner side wall includes the following steps: S11. Mix the first polymer stock solution with the first curing agent and evacuate to obtain a first mixed solution. S12. Add the first mixed solution into the container body 1, and then place the first mold adapted to the container body 1 into the container body 1 to obtain a container body to be molded. S13. Dry and mold the container body to be molded, demold it, and then add the first liquid oil and soak it to obtain a container body with an oil gel layer provided on the inner side wall.

[0036] It should be noted that in step S11, the evacuation is carried out in a vacuum chamber to make the obtained first mixed solution clear and without obvious bubbles. In step S12, before adding the first mixed solution into the container body 1, the inner wall of the container body 1 can be cleaned with anhydrous ethanol and ultrapure water, dried, and then placed in a plasma cleaner for treatment or subjected to chemical silanization; the first mold can be a mold made of polytetrafluoroethylene customized according to the shape of the container body 1, and a commercial mold release agent can be sprayed on the surface; the specific operation of placing the first mold into the container body 1 can be to slowly place the first mold in the center of the container body 1 to avoid generating bubbles, so that the first mixed solution is evenly distributed on the wall surface of the container body 1, and the excess first mixed solution is removed by overflowing from the open side of the container body 1, and the first mixed solution is distributed on the entire inner side wall of the container body 1; in step S13, the container body to be molded can be horizontally placed in an oven for drying and molding, and the molded container can be placed in a refrigerator for freezing treatment before demolding to facilitate demolding; the soaking is to make the first liquid oil enter the network of the first polymer framework structure to form an oil gel layer 2 on the inner side wall of the container body 1.

[0037] Further, in step S11, the mass ratio of the first polymer stock solution to the first curing agent is (5 - 20):1.

[0038] Further, in step S13, the temperature for drying and molding is 60 - 100 °C, and the time for drying and molding is 15 - 180 min. It can be understood that the temperature and time for drying and molding are related and can be 60 °C / 3 h, 70 °C / 2.5 h, 80 °C / 2 h, or 100 °C / 15 min.

[0039] Further, the organ supercooling preservation container further includes a sealing layer, the sealing layer is composed of an oil gel, and the oil gel includes a second polymer framework structure and a second liquid oil filled in the second polymer framework structure; the preparation method of the sealing layer includes the following steps: S21. Mix the second polymer stock solution with the second curing agent and evacuate to obtain a second mixed solution. S22. Add the second mixture into a second mold adapted to seal the open side of the container body 1 to obtain a to-be-formed sealing layer. S23. Dry and form the to-be-formed sealing layer, demold it, and soak it in a second liquid oil to obtain the sealing layer 3.

[0040] It should be noted that the sealing layer 3 prepared by the above steps is an oil gel plug, and the specific operation can refer to the preparation steps of the container body with an oil gel layer provided on the inner side wall.

[0041] In a third aspect, please refer to Figure 3 , the present invention provides a method for using the organ supercooling preservation container, including the following steps: A1. Place the excised organ 5 into the container body 1 with an oil gel layer provided on the inner side wall, and add the preservation solution 4 to submerge the excised organ 5 with the preservation solution 4. A2. Add an ice-melting prevention material into the container body 1 to cover the preservation solution 4 with the ice-melting prevention material to form the sealing layer 3. A3. Seal the container body 1 through a sealing component. A4. Place the sealed container body 1 into a temperature device for supercooling preservation.

[0042] Using the above steps for supercooling preservation of the excised organ 5, no cryoprotectant is used, and there is no need for complex operations such as mechanical perfusion and expensive equipment steps. The operation is simple and the cost is low, having excellent promotion prospects.

[0043] It should be noted that the ice-melting prevention material can be a liquid ice-melting prevention material or a solid ice-melting prevention material. For the used preservation container, only by adding the first liquid oil to soak the container body 1, the oil gel layer 2 can be restored to a complete state; the preservation container has strong durability.

[0044] Further, in step A1, the preservation solution 4 is UW preservation solution 4.

[0045] Further, in step A2, the ice-melting prevention material includes a liquid ice-melting prevention material, and the liquid ice-melting prevention material includes at least one of paraffin oil, silicone oil, and liquid alkane. Using the above liquid ice-melting prevention material is convenient for operation, and after rewarming, the ice-melting prevention material can be taken out for recycling.

[0046] Further, step A4 includes: placing the sealed container body 1 in a temperature device at 4°C, and then cooling it at a rate of 0.1~2°C / min to the supercooled storage temperature for supercooled storage; wherein, the supercooled storage temperature is less than or equal to -4°C. It should be noted that when the sealed container body is placed in the temperature device from room temperature conditions, the temperature of the temperature device will rise. It can be preheated in the temperature device until the temperature stabilizes at 4°C and then cooled down.

[0047] Example 1 A method for preparing a storage container includes the following steps: S11. Mix the polydimethylsiloxane (PDMS) stock solution and the curing agent in a mass ratio of 10:1 (the PDMS stock solution and the curing agent are commercial products, model SYLGARD TM 184 Silicone Elastomer Kit), and stir thoroughly until completely mixed. Place it in a vacuum chamber for vacuum treatment. After pumping until the mixed liquid becomes clear and has no obvious bubbles, take it out and set aside; S12. Clean the inner wall of the container body (quartz glass tube) with absolute ethanol and ultrapure water, dry it, and place it in a plasma cleaner for 10 min; S13. Pour the prepared mixed liquid into the container body after plasma treatment, and let it stand for a period of time to remove the mixed bubbles; S14. Slowly place the customized polytetrafluoroethylene mold (sprayed with commercial mold release agent on the surface) centered according to the shape of the container body, avoiding the generation of bubbles, and at the same time making the mixed liquid evenly distributed on the wall surface of the container body. Remove the excess liquid by overflowing through the open end of the container body; S15. Place the container body horizontally in an 80°C oven for 2 h until the PDMS is formed; S16. Place the formed container body in a -4°C refrigerator for 1 h for easy demolding. After demolding, inject dimethyl silicone oil (10 mPa·s) to completely immerse the inner wall of the container and the surface of the PDMS, and soak for 24 h to make the dimethyl silicone oil enter the PDMS network, obtaining a container body with an oil gel layer on the inner side wall.

[0048] Example 2 An organ supercooled storage method includes the following steps: A1. Transfer the mouse heart without obvious damage into the container body prepared in Example 1, and inject 3 mL of UW preservation solution along the tube wall with a syringe. The mouse heart is immersed in the UW preservation solution; A2. Inject 10 mL of liquid paraffin oil along the tube wall with a syringe; A3. Use a flange device to seal both ends of the container body, cover the gasket and the blind plate in sequence, and tighten the clamp to ensure sealing; A4. Place the sealed container body in a 4°C low-temperature constant temperature water bath for insulation until the temperature of the water bath stabilizes at 4°C, and then cool it at a rate of 0.1°C / min to the supercooled storage temperature for supercooled storage.

[0049] Comparative Example 1 For the supercooled storage of an organ, the steps are the same as those in Example 2, except that the container body used is the container body (quartz glass tube) with the inner wall untreated in Example 1.

[0050] Comparative Example 2 For the static cold storage of an organ, the steps are the same as those in Example 2, except that step A4 is: Place the sealed container in a 4°C low-temperature constant temperature water bath for cold storage.

[0051] Performance Test and Results (1) Test on the ice nucleation temperature of the oil gel interface Place the oil gel and different materials (glass, aluminum, steel, polypropylene, PDMS, phenylmethyl silicone oil) flat on the refrigeration platform. Use a pipette to drop 10 μL of pure water droplets on each surface. The refrigeration platform cools down from 0°C at a rate of 0.1°C / s. Judge the occurrence of the nucleation process by observing the change in the transparency of the droplets. Record the wall supercooling degree at the first occurrence of nucleation as the ice nucleation temperature of this surface. Repeat several times. The whole test is carried out in a nitrogen atmosphere; among them, the oil gel is a square block structure (dimethyl silicone oil is filled in the PDMS network) prepared by the preparation method of the oil gel layer in Example 1. The results are shown in Figure 4 .

[0052] It can be seen from Figure 4 that the supercooling degree of the oil gel interface is lower, and using it as the interface or sealing layer of the inner wall of the container can reduce the freezing point of the preservation liquid.

[0053] (2) Test on the storage stability of the oil gel Prepare an oil gel with a square block structure (dimethyl silicone oil is filled in the PDMS network) by the preparation method of the oil gel layer in Example 1, with a thickness of 5 mm. Make a spindle-shaped tensile standard test sample through a mold. Immerse the sample after filling with oil in UW preservation liquid, and take samples on the 0th, 1st, 3rd, and 7th days of immersion for tensile test of elastic modulus. The results are shown in Figure 5 .

[0054] It can be seen from Figure 5 that after the oil gel is immersed in UW preservation liquid for 7 days, the elastic modulus basically does not change, and the stability of the oil gel is good.

[0055] (3) Test on the ice nucleation probability of organ supercooled storage The organ supercooling preservation was carried out using Example 2 and Comparative Example 1. By changing the supercooling preservation conditions (temperature and time), three groups were set for the supercooling preservation of Example 2 and Comparative Example 1 respectively. The first group: the supercooling preservation temperature was -4°C, and the supercooling preservation time was 36 h (-4°C / 36 h); the second group: the supercooling preservation temperature was -4°C, and the supercooling preservation time was 48 h (-4°C / 48 h); the third group: the supercooling preservation temperature was -5°C, and the supercooling preservation time was 72 h (-5°C / 72 h). After the supercooling preservation of each group, the ice formation probability of the mouse heart was detected respectively. The specific method was that during the supercooling preservation process, at key time points such as 12 h, 24 h, 36 h, etc., the preservation container was slowly taken out of the low-temperature constant temperature water bath, and it was observed whether ice formation occurred and recorded, so as to obtain the ice formation probability of each group. Among them, the ice formation probability = (the number of ice formation occurrences / the total number of experiments) × 100%, and the total number of experiments in each group was 40. The results are shown in Figure 6 , among which, the ice formation probability of Example 2 after preservation at -4°C / 36 h was 0%.

[0056] It can be seen from Figure 6 that the preservation container of the present invention can achieve stable supercooling preservation of excised organs below -4°C.

[0057] (4)Testing of the survival rate and heart re-beating rate of organ supercooling preservation The organ supercooling preservation was carried out using Example 2. The supercooling preservation temperature was -4°C, and four groups with supercooling preservation times of 18 h, 24 h, 36 h, and 42 h were set. After the supercooling preservation of each group, the survival rate and heart re-beating rate of the mouse heart were detected respectively. The specific method was that the preserved mouse heart was subjected to heterotopic heart transplantation surgery, and the postoperative survival and re-beating conditions were observed to obtain the survival rate and re-beating rate of each group. Among them, the survival rate = (the number of survivors / the total number of experiments) × 100%, and the re-beating rate = (the number of re-beating occurrences / the total number of experiments) × 100%. The total number of experiments in each group was 6. The survival rate results at different preservation times are shown in Figure 7 (-4°C), and the heart re-beating rate results at different preservation times are shown in Figure 8 (-4°C).

[0058] The organ static cold preservation was carried out using Comparative Example 2. The cold preservation temperature was 4°C, and four groups with cold preservation times of 18 h, 24 h, 36 h, and 42 h were set. After the cold preservation of each group, the survival rate and heart re-beating rate of the rat heart were detected respectively; the survival rate results at different preservation times are shown in Figure 7 (4°C), among which, the survival rates at preservation times of 36 h and 42 h were both 0%, and the heart re-beating rate results at different preservation times are shown in Figure 8 (4°C), among which, the heart re-beating rates at preservation times of 36 h and 42 h were both 0%.

[0059] From Figure 7 and Figure 8 It can be seen that, compared with the 4°C static cold preservation in clinical practice, the stable supercooled preservation at -4°C improves the 24-hour survival rate and the rate of heart restoration, can effectively extend the preservation time, reduce organ damage, and improve the quality of organ preservation.

[0060] In the present invention, the specific raw materials not described are all existing substances and can be directly purchased from the market.

[0061] The above are only the preferred embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An organ supercooled storage container, characterized in that, Comprising: A container body, one side of the container body is open, and an oil gel layer is provided on the inner side wall. The oil gel layer includes a first polymer framework structure and a first liquid oil filled in the first polymer framework structure; and, A sealing layer, the sealing layer is made of an ice-repellent material, the sealing layer is used to seal the open side of the container body and is in contact with the contents inside the container body.

2. The organ supercooled storage container according to claim 1, wherein The first polymer framework structure includes a first polymer, and the first polymer includes at least one of polydimethylsiloxane, polyvinylidene fluoride, polyethylene, polyacrylamide, and polyvinyl chloride; and / or, The first liquid oil includes at least one of silicone oil, perfluoropolyether, and vegetable oil.

3. The organ supercooling preservation container according to claim 1, wherein The ice-repellent material includes a liquid ice-repellent material, and the liquid ice-repellent material includes at least one of paraffin oil, silicone oil, and liquid alkane.

4. The organ supercooled preservation container according to claim 1, wherein, The ice-repellent material includes a solid ice-repellent material, and the solid ice-repellent material includes at least one of paraffin and oil gel; wherein, the oil gel includes a second polymer framework structure and a second liquid oil filled in the second polymer framework structure.

5. The organ supercooled storage container according to claim 1, wherein, It further includes a sealing assembly for sealing the container body.

6. The preparation method of the organ supercooling preservation container according to any one of claims 1-5, characterized in that, The organ supercooling preservation container includes a container body with an oil gel layer provided on the inner side wall. The preparation method of the container body with an oil gel layer provided on the inner side wall includes the following steps: S11. Mix the first polymer stock solution with the first curing agent and evacuate to obtain a first mixed solution; S12. Add the first mixed solution into the container body, and then put a first mold adapted to the container body into the container body to obtain a container body to be formed; S13. Dry and form the container body to be formed, demold, and then add the first liquid oil and soak to obtain a container body with an oil gel layer provided on the inner side wall.

7. The preparation method of the organ supercooling preservation container according to claim 6, wherein In step S11, the mass ratio of the first polymer stock solution to the first curing agent is (5~20):1; and / or, In step S13, the temperature for drying and forming is 60~100 °C, and the time for drying and forming is 15~180 min.

8. The preparation method of the organ supercooling preservation container according to claim 6, characterized in that, The organ supercooling preservation container further includes a sealing layer, the sealing layer is made of oil gel, and the oil gel includes a second polymer framework structure and a second liquid oil filled in the second polymer framework structure; the preparation method of the sealing layer includes the following steps: S21. Mix the second polymer stock solution with the second curing agent and evacuate to obtain a second mixed solution; S22. Add the second mixed solution into a second mold adapted to seal the open side of the container body to obtain a sealing layer to be formed; S23. Dry and form the sealing layer to be formed, demold, and soak it in the second liquid oil to obtain the sealing layer.

9. The method of using an organ supercooling preservation container according to any one of claims 1 - 5 or an organ supercooling preservation container prepared by the preparation method according to any one of claims 6 - 8, characterized in that, Including the following steps: A1. Put the excised organ into the container body with an oil gel layer provided on the inner side wall, and add a preservation solution to submerge the excised organ with the preservation solution; A2. Add an ice-repellent material into the container body to cover the preservation solution with the ice-repellent material to form a sealing layer; A3. Seal the container body through the sealing assembly; A4. Put the sealed container body into a temperature device for supercooling preservation.

10. The method for using the organ supercooling preservation container according to claim 9, characterized in that, In step A1, the preservation solution is UW preservation solution; and / or, In step A2, the ice-scavenging material includes a liquid ice-scavenging material, and the liquid ice-scavenging material includes at least one of paraffin oil, silicone oil, and liquid alkane; and / or, Step A4 includes: placing the sealed container body into a temperature device at 4°C, and then cooling it to the supercooled preservation temperature at a rate of 0.1~2°C / min for supercooled preservation; wherein, the supercooled preservation temperature is less than or equal to -4°C.

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