Heart profound hypothermia cryopreservation system for organ transplantation

Abstract

The invention belongs to the technical field of medical instruments, and provides a heart profound hypothermia cryopreservation system for organ transplantation. According to the invention, the quasi-homogeneous interface is completely attached to the inner wall of the container, so that the direct contact between the contained substance and the container body is avoided; meanwhile, the organic gel layer infiltrated with the dimethyl silicone oil is used as a quasi-homogeneous interface, so that pores in the organic gel layer can be fully filled with the dimethyl silicone oil, and the direct contact between the contained substances and the container body is further isolated; the contained substances are isolated from the container body, so that heterogeneous nucleation induced by liquid-gas and liquid-solid interfaces is effectively reduced, and the freezing risk is reduced; due to the construction of a quasi-homogeneous interface between the containing substance and the containing container body, heterogeneous nucleation sites are reduced, and the freezing risk is reduced; by means of the two characteristics, the deep hypothermia cryopreservation system is not prone to freezing of contained substances at the low temperature such as-3 DEG C; and the storage temperature is lower, so that the storage time of the contained substances is prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to a deep-temperature freezing preservation system for a heart used for organ transplantation. Background Art

[0002] Organ transplantation is one of the most effective treatments for patients with end-stage organ failure. However, the severe shortage of donor organs has always restricted the widespread implementation of transplant surgery. Although the exploration of xenotransplantation and artificial organs has made continuous progress in recent years, the most pressing bottleneck in clinical transplantation at this stage is still how to prolong the storage time of donor organs and improve their functional retention rate. The cold preservation method currently widely used in clinical practice is static cold preservation at 4°C, but the above static cold preservation can only maintain a transplantable window of 4 to 6 hours for high metabolic organs such as the heart, which is far from meeting the needs of off-site donor allocation and the global transplant network.

[0003] To extend the preservation time of organs, the academic community has proposed new strategies including mechanical perfusion preservation and supercooling. These methods have improved the potential of organ preservation to a certain extent, but they also have obvious limitations. For example: (1) Mechanical perfusion equipment is complex and expensive, and there are problems with cryoprotectant blockage and unstable perfusion pressure under low temperature conditions; (2) Supercooling requires the use of large amounts of permeable or non-permeable cryoprotectants (such as DMSO and glycerol), which have high toxicity risks and are difficult to completely elute.

[0004] Therefore, a new type of low-temperature preservation device is urgently needed. Summary of the Invention

[0005] In light of this, the present invention aims to provide a deep-temperature heart preservation system for organ transplantation. This system can prevent frozen contents at even lower temperatures, such as -3°C, thereby extending the preservation period. Furthermore, it offers a simple structure, low cost, and the absence of cryoprotectants.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a deep-temperature cryopreservation system for heart organ transplantation, comprising a holding system and a temperature control system;

[0008] The containing system includes a containing container and a sealing cover matched with the containing container;

[0009] The inner wall of the container is completely covered with a quasi-homogeneous interface, and the shape of the cavity enclosed by the quasi-homogeneous interface matches the shape of the substance to be contained;

[0010] The quasi-homogeneous interface is an organic gel layer infiltrated with dimethyl silicone oil; the material of the organic gel layer is polydimethylsiloxane;

[0011] The temperature control system includes a medium, a medium container for holding the medium, and a temperature control circulation device for controlling the temperature of the medium;

[0012] The medium is alcohol or an alcohol-water mixture.

[0013] Preferably, the thickness of the organic gel layer is greater than or equal to 5 μm.

[0014] Preferably, the viscosity of the dimethyl silicone oil is 10 to 20 mPa·s.

[0015] Preferably, the container is made of quartz glass.

[0016] Preferably, the medium is alcohol.

[0017] Preferably, the method for preparing the container comprises the following steps:

[0018] pre-treating the inner wall of the container to obtain a pre-treated container;

[0019] Applying a polydimethylsiloxane precursor to the inner wall of the pretreatment container, placing a mold inside the pretreatment container, and curing; the shape of the mold matches the shape of the material to be contained;

[0020] After curing, the mold is removed to obtain a container with an inner wall fully covered with an organic gel layer;

[0021] The container with the organic gel layer fully attached to the inner wall is immersed in dimethyl silicone oil to obtain the container.

[0022] Preferably, the pretreatment includes rinsing and plasma cleaning in sequence; the rinsing includes rinsing with anhydrous ethanol and rinsing with water in sequence; the power of the plasma cleaning is 18 to 24 W, and the time is 10 to 15 minutes.

[0023] Preferably, the curing temperature is 80-85°C and the curing time is 2-2.5 hours;

[0024] The immersion time is 20 to 24 hours.

[0025] The present invention also provides a method for using the heart deep-cryopreservation system for organ transplantation described in the above technical solution, comprising the following steps:

[0026] Place the substance in the cavity of the container, inject UW liquid until it completely covers the substance, perform oil sealing, and cover with a sealing cover;

[0027] Use temperature control circulation equipment to cool the medium in the medium container to make the medium reach the storage temperature;

[0028] Completely immerse the system in a medium that has reached the storage temperature for storage.

[0029] Preferably, after the storage, the method further comprises rewarming for use; the rewarming for use comprises the following steps:

[0030] Set the temperature-raising program of the temperature-controlled circulation equipment to raise the temperature;

[0031] After reaching the set rewarming temperature, take out the contents for use.

[0032] The present invention provides a deep-temperature freezing preservation system for a heart used for organ transplantation.

[0033] The present invention has a quasi-homogeneous interface attached to the inner wall of the container, which can prevent the contained material, such as an organ, from directly contacting the container body. At the same time, the organic gel layer soaked with dimethyl silicone oil is used as the quasi-homogeneous interface, in which the dimethyl silicone oil can fully fill the pores in the organic gel layer, further isolating the contained material from direct contact with the container body. The isolation of the contained material from the container body effectively reduces the heterogeneous nucleation induced by the liquid-gas and liquid-solid interfaces, reducing the risk of freezing. The setting of the quasi-homogeneous interface between the contained material and the container body, due to the construction of the quasi-homogeneous interface, reduces the heterogeneous nucleation sites and reduces the risk of freezing. The above two features make the deep-low temperature freezing preservation system for heart organ transplantation not easy to freeze even at a lower temperature such as -3°C. The lower storage temperature prolongs the storage time of the contained material. The low-temperature freezing preservation system of the present invention can be safely stored for more than 12 hours in a -3°C environment and can maintain the good function of the contained material, such as an organ. The cryopreservation system of the present invention avoids the toxic side effects of cryoprotectants (such as DMSO and glycerol), achieving stable deep cryopreservation without cryoprotectants or mechanical perfusion. This system overcomes key barriers in existing technologies, including interface control, physical support, and temperature control accuracy, laying a new foundation for remote deployment and xenotransplantation of high-risk organs.

[0034] Compared with existing organ preservation technologies, the heart deep cryopreservation system for organ transplantation of the present invention exhibits significant advantages in the following three aspects:

[0035] (1) Low operating difficulty and high repeatability

[0036] The heart deep hypothermic cryopreservation system for organ transplantation of the present application has simple structure, cancels the dependence on complex mechanical perfusion system, realizes automatic adhesion and sealed preservation of the organ through the cooperative design of the containing cavity and the sealing cover. Meanwhile, the matched temperature control system is convenient to operate, the cooling process and the warming process can be standardized and controlled, the organ damage caused by operation difference is avoided, and the repeatability of the preservation process is significantly improved.

[0037] (2) No need to use cryoprotective agents

[0038] The heart deep hypothermic cryopreservation system for organ transplantation of the present application maintains the supercooling state of the liquid around the containing substance such as organ by constructing a quasi-homogeneous interface and reducing the heterogeneous nucleation induced by liquid-gas and liquid-solid interfaces, thereby avoiding the structural damage caused by icing. Therefore, there is no need to add cryoprotective agents such as DMSO or glycerol, the risk of organ toxicity is reduced, and the subsequent processing process is simplified.

[0039] (3) Good function after resuscitation

[0040] The heart preserved by the heart deep hypothermic cryopreservation system for organ transplantation of the present application still maintains a good heart function state after transplantation, and the tissue structure is complete. The excellent resuscitation effect is due to the uniform and slow temperature change in the preservation process, and no severe icing damage, which maximizes the metabolic and structural integrity of myocardial cells.

[0041] In summary, the heart deep hypothermic cryopreservation system for organ transplantation of the present application significantly improves the safety, practicability and transformation prospect of organ cryopreservation through the cooperative optimization of structure and physical environment. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 The structural schematic diagram of the heart deep hypothermic cryopreservation system for organ transplantation provided by the present application is shown in the figure;

[0043] Figure 2 The B-ultrasound diagram of the heart preserved by the heart deep hypothermic cryopreservation system after in-situ transplantation of Case 1 is shown in the figure;

[0044] Figure 3 The pulsed Doppler imaging diagram of the mitral valve inflow (upper) and the aortic outflow (lower) of the heart preserved by the heart deep hypothermic cryopreservation system after in-situ transplantation of Case 1 is shown in the figure;

[0045] Figure 4 The general appearance diagram of the heart preserved by the heart deep hypothermic cryopreservation system after in-situ transplantation of Case 1 for 72 hours is shown in the figure;

[0046] Figure 5 The HE staining result diagram of the heart is shown in the figure. DETAILED DESCRIPTION

[0047] The present invention provides a deep-temperature cryopreservation system for heart organ transplantation, comprising a holding system and a temperature control system;

[0048] The containing system includes a containing container and a sealing cover matched with the containing container;

[0049] The inner wall of the container is completely covered with a quasi-homogeneous interface, and the shape of the cavity enclosed by the quasi-homogeneous interface matches the shape of the substance to be contained;

[0050] The quasi-homogeneous interface is an organic gel layer infiltrated with dimethyl silicone oil; the material of the organic gel layer is polydimethylsiloxane;

[0051] The temperature control system includes a medium, a medium container for holding the medium, and a temperature control circulation device for controlling the temperature of the medium;

[0052] The medium is alcohol or an alcohol-water mixture.

[0053] Figure 1 This is a schematic diagram of the structure of the deep-low temperature cryopreservation system for heart organ transplantation provided by the present invention. Figure 1 The heart deep-low temperature cryopreservation system for organ transplantation provided by the present invention is described in detail.

[0054] The heart deep-low temperature freezing preservation system for organ transplantation provided by the present invention comprises a containing system, which comprises a containing container and a sealing cover matched with the containing container.

[0055] In the present invention, the container is preferably made of quartz glass. The inner diameter of the container is preferably 10 to 20 cm, more preferably 10 cm, 12 cm, 14 cm, 16 cm, 18 cm, or 20 cm. The wall thickness of the container is preferably 2 to 5 mm, more preferably 2 mm, 3 mm, 4 mm, or 5 mm.

[0056] In the present invention, the inner wall of the container is completely coated with a quasi-homogeneous interface, and the shape of the cavity enclosed by the quasi-homogeneous interface matches the shape of the substance to be contained. In the present invention, the cavity is used to hold the substance. The present invention does not specifically limit the shape of the cavity, and those skilled in the art can design it based on the shape of the substance to be contained. In one embodiment of the present invention, the substance to be contained is preferably an organ.

[0057] In the present invention, the quasi-homogeneous interface is an organic gel layer impregnated with dimethyl silicone oil. In the present invention, the material of the organic gel layer is polydimethylsiloxane. In the present invention, the thickness of the organic gel layer is preferably greater than or equal to 5 μm. In the present invention, the thickness of the organic gel layer is set to be greater than or equal to 5 μm, which can completely avoid the contact between the contained material and the container body, so that the contained material is in contact with the quasi-homogeneous interface; the non-direct contact between the contained material and the container body effectively reduces the heterogeneous nucleation induced by the liquid-gas and liquid-solid interfaces, and reduces the risk of freezing; and the construction of the quasi-homogeneous interface reduces the heterogeneous nucleation sites and reduces the risk of freezing; the above two characteristics make it difficult for the contained material to freeze in the deep-low temperature freezing preservation system even at a lower temperature such as -3°C; and the lower storage temperature extends the storage time of the contained material.

[0058] In the present invention, the viscosity of the dimethyl silicone oil is preferably 10-20 mPa·s, and more preferably 10 mPa·s, 11 mPa·s, 12 mPa·s, 13 mPa·s, 14 mPa·s, 15 mPa·s, 16 mPa·s, 17 mPa·s, 18 mPa·s, 19 mPa·s, or 20 mPa·s. In the present invention, the infiltration of the dimethyl silicone oil can further fill the pores in the organic gel layer, further preventing direct contact between the contained material and the container body, and further reducing the risk of freezing.

[0059] In the present invention, the method for preparing the container preferably comprises the following steps:

[0060] pre-treating the inner wall of the container to obtain a pre-treated container;

[0061] Applying a polydimethylsiloxane precursor to the inner wall of the pretreatment container, and placing a mold inside the pretreatment container for curing; the shape of the mold matches the shape of the material to be contained;

[0062] After curing, the mold is removed to obtain a container with an inner wall fully covered with an organic gel layer;

[0063] The container with the organic gel layer fully attached to the inner wall is immersed in dimethyl silicone oil to obtain the container.

[0064] The present invention pre-treats the inner wall of the holding container to obtain a pre-treated holding container. In the present invention, the pre-treatment preferably includes rinsing and plasma cleaning in sequence. In the present invention, the rinsing preferably includes rinsing with anhydrous ethanol and rinsing with water in sequence; after the water rinsing, the present invention preferably further includes blow-drying and subsequent plasma cleaning. In the present invention, the power of the plasma cleaning is preferably 18 to 24 W, specifically preferably 18 W, 19 W, 20 W, 21 W, 22 W, 23 W or 24 W, and the time is preferably 10 to 15 minutes, specifically preferably 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes; the plasma cleaning is preferably carried out in a plasma cleaning apparatus. In the present invention, the pre-treatment can enhance the surface wettability of the inner wall of the holding container and improve the adhesion between the inner wall of the holding container and PDMS.

[0065] After obtaining a pretreatment container, the present invention applies a polydimethylsiloxane precursor to the inner wall of the pretreatment container and places a mold inside the pretreatment container for curing; the shape of the mold matches the shape of the material to be contained. In the present invention, the method for preparing the polydimethylsiloxane precursor preferably includes the following steps: mixing the corresponding elastomer matrix and curing agent in a mass ratio of 10:1, stirring, and degassing in sequence to obtain the polydimethylsiloxane precursor. In the present invention, the stirring speed is preferably 800-1200 rpm, more preferably 1000 rpm, and the duration is preferably 8-12 minutes, more preferably 10 minutes. The stirring is preferably performed in a centrifugal mixer to uniformly mix the elastomer matrix and curing agent. In the present invention, the degassing time is preferably 30 minutes or longer, preferably performed in a vacuum chamber; the degassing removes bubbles from the resulting mixture. In the present invention, the application preferably includes pouring the polydimethylsiloxane precursor onto the inner wall of the pretreatment container to ensure uniform spreading. In the present invention, the mold is preferably made of polytetrafluoroethylene (PTFE). The present invention does not specifically limit the shape of the mold, and those skilled in the art can set it according to the shape of the material to be contained. In the present invention, the curing temperature is preferably 80-85°C, specifically preferably 80°C, 81°C, 82°C, 83°C, 84°C, or 85°C; and the curing time is preferably 2-2.5 hours, specifically preferably 2 hours or 2.5 hours.

[0066] After curing, the present invention removes the mold to obtain a container with an inner wall completely covered with an organic gel layer. The present invention does not limit the removal operation, and those skilled in the art can configure it according to actual needs.

[0067] After obtaining a container having an inner wall fully coated with an organic gel layer, the present invention immerses the container having the inner wall fully coated with an organic gel layer in dimethyl silicone oil to obtain the container. In the present invention, the immersion time is preferably 20 to 24 hours, more preferably 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 25 hours; the immersion is preferably carried out at room temperature and pressure.

[0068] The heart cryopreservation system for organ transplantation provided by the present invention includes a temperature control system comprising a medium, a medium container for holding the medium, and a temperature control circulation device for controlling the temperature of the medium. In the present invention, the medium is an alcohol or an alcohol-water mixture, more preferably an alcohol, preferably ethanol, and even more preferably anhydrous ethanol. In one embodiment of the present invention, the temperature control system is preferably a water bath refrigerator, and the water bath refrigerator is filled with the medium ethanol.

[0069] In the present invention, the containing system is completely immersed in the medium of the temperature control system.

[0070] The present invention also provides a method for using the heart deep-cryopreservation system for organ transplantation described in the above technical solution, comprising the following steps:

[0071] Place the substance in the cavity of the container, inject UW liquid until it completely covers the substance, perform oil sealing, and cover with a sealing cover;

[0072] Use temperature control circulation equipment to cool the medium in the medium container to make the medium reach the storage temperature;

[0073] Completely immerse the system in a medium that has reached the storage temperature for storage.

[0074] The present invention places the contained material in the cavity of the containing container, injects UW liquid until the contained material is completely covered, performs oil sealing, and covers the container with a sealing cover.

[0075] The present invention does not specifically limit the method for obtaining the contained material. In one embodiment of the present invention, when the contained material is a heart, the method for obtaining the contained material is preferably as follows: the heart is removed under general anesthesia and aseptic conditions, and the heart is rapidly cold-arrested by retrograde perfusion of 500 mL each of Thomas solution and UW solution via the ascending aorta. The heart is then immersed in UW solution for later use. In the present invention, the temperature of the UW solution is preferably 4°C.

[0076] In the present invention, the oil seal preferably comprises: introducing oil to a sealing liquid surface, pouring liquid nitrogen to cool and solidify the oil to form a seal. In the present invention, the oil is preferably olive oil, and the thickness of the oil introduced is preferably greater than or equal to 0.5 mm.

[0077] The present invention utilizes a temperature control circulation device to cool the medium in the medium container to a storage temperature. In a specific embodiment of the present invention, when the contained substance is an organ, the storage temperature is preferably -3°C.

[0078] After the seal is sealed and the medium reaches the storage temperature, the present invention completely immerses the container system in the medium at the same temperature for storage. In the present invention, "complete immersion" means that the container system is completely immersed in the medium. In the present invention, during the storage process, the temperature change of the medium is preferably controlled to within ±0.2°C.

[0079] After the storage, the present invention preferably further comprises rewarming for use; the rewarming for use preferably comprises the following steps:

[0080] Set the temperature-raising program of the temperature-controlled circulation equipment to raise the temperature;

[0081] After reaching the set rewarming temperature, take out the contents for use.

[0082] In the present invention, the heating rate of the heating program is preferably less than or equal to 0.5°C / min.

[0083] In the present invention, the rewarming temperature is preferably set according to actual conditions. In a specific embodiment of the present invention, when the contained substance is preferably an organ, the rewarming temperature is preferably 4°C.

[0084] The present invention does not specifically limit the manner of use, and those skilled in the art can perform the method according to actual circumstances.

[0085] The heart deep-cryopreservation system for organ transplantation provided by the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0086] Example 1

[0087] 1. Raw Materials Preparation

[0088] Polydimethylsiloxane (PDMS) precursor (Sylgard 184, Dow Corning): elastomer matrix: curing agent mass ratio of 10:1; quartz glass container (thickness 3 mm, inner diameter 16 cm); polytetrafluoroethylene (PTFE) heart mold; dimethyl silicone oil, Sigma-Aldrich, viscosity 10 mPa·s; olive oil, medical grade, Macklin; anhydrous ethanol, purity >99.7%, Sigma-Aldrich.

[0089] 2. Preparation of quartz glass container

[0090] The inner wall of the quartz glass container was rinsed with anhydrous ethanol and ultrapure water in sequence and dried; the container was treated in a plasma cleaner at 18 W power for 10 minutes to enhance the surface wettability and PDMS adhesion ability of the inner wall of the quartz glass container, thereby obtaining a pretreated container.

[0091] The PDMS elastomer matrix and the curing agent were weighed and mixed in a mass ratio of 10:1, and stirred in a centrifugal mixer at 1000 rpm for 10 minutes to obtain a uniform mixture. The mixture was placed in a vacuum chamber for degassing for more than 30 minutes to remove bubbles in the system to obtain an organogel layer precursor.

[0092] Slowly pour the organogel layer precursor onto the inner wall of the pre-treated container, ensuring even spreading. Place the PTFE heart mold vertically in the center to form a cavity for the organ.

[0093] The mold was heated and cured at 80° C. for 2 hours, and the mold was carefully removed after cooling to obtain a container with an organic gel layer fully attached to the inner wall.

[0094] The container with the organic gel layer fully attached to the inner wall was completely immersed in dimethyl silicone oil for 24 hours to form a uniform wetting interface on the surface and improve the quasi-homogeneous state, thereby obtaining a container.

[0095] 3. Organ preservation operation methods

[0096] (1) Heart acquisition and processing

[0097] The heart was removed under general anesthesia and aseptic conditions. 500 mL of Thomas solution and 500 mL of UW solution (4°C) were quickly perfused retrogradely through the ascending aorta to cold arrest the heart, and then the heart was immersed in UW solution (4°C) for later use.

[0098] (2) Organ placement and packaging

[0099] Place the perfused heart in the cavity of the container, slowly inject cooled UW solution until the heart is completely covered, introduce olive oil to a thickness of 0.5 mm, pour a small amount of liquid nitrogen to cool and solidify the olive oil, form an airtight seal, and cover with a sealing lid.

[0100] (3) Treatment of temperature control system

[0101] The temperature of the medium anhydrous ethanol in the medium container is cooled by using a temperature control circulation device to make the medium reach a storage temperature of -3°C.

[0102] (4) Place the system in anhydrous ethanol at -3°C and keep it at a constant temperature for 12 hours. During the storage process, the temperature control accuracy is maintained at ±0.2°C. After 12 hours of storage, the sample is collected for pathological examination.

[0103] 4. Rewarming and use

[0104] After the end of the preservation, the temperature program of the temperature control circulation device was set to 0.5°C / min for temperature increase; after reaching the set rewarming temperature of 4°C, the contained substance was taken out and connected to an extracorporeal circulation system or subjected to transplantation operation.

[0105] Two cases of heart preservation for 12 hours were completed using the heart deep hypothermic freezing preservation system of Example 1, which were recorded as Case 1 and Case 2, respectively.

[0106] After rewarming of the case 1 and case 2 pig hearts preserved for 12 hours, orthotopic heart transplantation was performed, and the case 1 and case 2 pig hearts were successfully resuscitated after orthotopic heart transplantation.

[0107] After 72 hours of orthotopic heart transplantation, the heart was stopped by thoracotomy perfusion, and pathological examination was performed after sampling.

[0108] Comparative Example 1: Ordinary freezing preservation

[0109] The heart was taken under general anesthesia and aseptic conditions. The heart was rapidly cooled and stopped by retrograde perfusion of 500 mL of Thomas and UW solution (4°C) through the ascending aorta, and then the heart was immersed in UW solution (4°C) for standby.

[0110] The above heart was simply placed in the UW solution and preserved at 4°C for 12 hours.

[0111] The myocardial tissue of the pig heart preserved by ordinary freezing for 12 hours was severely damaged and did not have the conditions for orthotopic heart transplantation, and was directly subjected to HE staining characterization.

[0112] The process of pathological examination was as follows:

[0113] At the end of the experiment, the chest cavity was reopened along the original thoracotomy incision. After clamping the ascending aorta and the superior and inferior vena cava, a perfusion needle was inserted into the aortic root, and 500 mL of Thomas cardioplegia solution was injected to induce cardiac arrest. At the same time, the right atrial appendage was cut for decompression and drainage. After perfusion, the heart was completely removed, and all collected tissue samples were stored in 10% neutral buffered formalin (ZLI-9381, Zhongshan Jinqiao). The fixed myocardial tissue was subjected to gradient dehydration, transparentization and paraffin embedding: immersed in 50%, 70%, 80%, 95% and anhydrous ethanol for 1 hour each for dehydration, xylene I and II for transparentization for 30 minutes each, and immersed in paraffin at 60°C for 1 hour. After embedding and cooling, it was cut into 4 μm thin slices using a paraffin microtome (Leica), and the slices were spread in a 48°C water bath. After mounting, the slices were dried at room temperature. Paraffin sections were dried at 65°C for 2 hours to remove residual wax, dewaxed in xylene I and II for 15-20 minutes each, rehydrated in anhydrous ethanol for 10 minutes, and then in 95%, 85%, 75%, and 50% ethanol for 5 minutes each. Rinse with double-distilled water for 5 minutes. Stain with hematoxylin for 5 minutes, rinse with running water, differentiate with 1% hydrochloric acid and ethanol for 1 second, and then stain with eosin for 5 minutes after bluing. Dehydrated in 75%, 85%, and 95% ethanol for 5 minutes each, and anhydrous ethanol I and II for 10 minutes each. Clear in xylene I and II for 15 minutes each, and mount with neutral gum.

[0114] Figure 2 This is an ultrasound image of Case 1, a heart preserved in a heart cryopreservation system, obtained after orthotopic transplantation. Figure 2 In the figure, Pre-op stands for before orthotopic transplantation surgery, Post-op stands for after orthotopic transplantation surgery, End-diastole stands for end-diastole, and End-systole stands for end-systole. Figure 2 The results showed that the left ventricular (LV) and right ventricular (RV) chamber dimensions and systolic function remained stable.

[0115] Figure 3 Pulsed Doppler imaging of the mitral valve inflow tract (top) and aortic outflow tract (bottom) of Case 1 heart preserved in the heart cryopreservation system after orthotopic transplantation. Figure 3 In the figure, Mitral inflow pulsed-wave Doppler image represents mitral valve ultrasound Doppler blood flow; Aortic inflow pulsed-wave Doppler image represents aortic valve ultrasound Doppler blood flow. Figure 3 It can be seen that the left ventricular filling and contractile function are good.

[0116] The quantitative analysis results of key cardiac function parameters are shown in Table 1.

[0117] Table 1 Quantitative analysis results of key cardiac function parameters

[0118] Before surgery Postoperative -0h Postoperative - 48 hours Postoperative - 72 hours Left ventricular ejection fraction / % 54 51 60 54 Short axis shortening / % 16 21 33 32 Stroke volume / ml 40 23 24 25 <![CDATA[心输出量 / ml·min -1 ]]> 3000 2783 3096 3025 Area change fraction / % 48 36 32 41 ratio of early to late mitral flow velocity 1.47 1.68 - 1.65 <![CDATA[最大流速 / m·s -1 ]]> 0.93 0.86 - 0.83

[0119] As can be seen from Table 1: After the operation, the left ventricular ejection fraction was stable at more than 50%, the cardiac output was stable, and the other major indices remained stable.

[0120] In conclusion, after 12 hours of deep cryopreservation, the orthotopic transplanted heart successfully maintained hemodynamic stability.

[0121] Figure 4 This is the gross appearance of Case 1, a heart preserved in a heart cryopreservation system, 72 hours after orthotopic transplantation. The left image is an overall view, and the right image is an anatomical view, showing the LV (left ventricle); RV (right ventricle); IVS (interventricular septum); PA (pulmonary artery); AO (aorta); LA (left atrium); RA (right atrium); LVOT (left ventricular outflow tract); and LVIT (left ventricular inflow tract).

[0122] Figure 5 The HE staining results of the heart are shown in the figure on the left. Specifically, the HE staining results of the heart preserved by ordinary cryopreservation for 12 hours are shown in the figure on the middle. The HE staining results of Case 1 heart preserved by the deep-low temperature cryopreservation system in Example 1 and transplanted in situ for 72 hours are shown in the figure on the right. The HE staining results of Case 2 heart preserved by the deep-low temperature cryopreservation system in Example 1 and transplanted in situ for 72 hours are shown in the figure on the right. Hypothermic preservation is ordinary cryopreservation; Region 1 represents region 1; Region 2 represents region 2; Figure 5 It can be seen that in the myocardial tissue after 12 hours of conventional cryopreservation, large areas of coagulative necrosis (black arrows), interstitial edema (blue arrows), and perinuclear vacuolation (yellow arrows) were visible; in Case 1 and Case 2, hearts preserved in the deep-temperature cryopreservation system for 12 hours in Example 1 and transplanted in situ for 72 hours, the heart structure was intact and the cardiac chamber morphology was symmetrical, and the myocardial structure was well preserved, with only mild perinuclear vacuolation and edema. This indicates that compared with the hearts preserved for 12 hours of conventional cryopreservation, deep-temperature cryopreservation only caused mild myocardial damage.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A deep-cryogenic heart preservation system for organ transplantation, comprising a storage system and a temperature control system; The containing system includes a containing container and a sealing cover matched with the containing container; The inner wall of the container is completely covered with a quasi-homogeneous interface, and the shape of the cavity enclosed by the quasi-homogeneous interface matches the shape of the substance to be contained; The quasi-homogeneous interface is an organic gel layer infiltrated with dimethyl silicone oil; the material of the organic gel layer is polydimethylsiloxane; The temperature control system includes a medium, a medium container for holding the medium, and a temperature control circulation device for controlling the temperature of the medium; The medium is alcohol or an alcohol-water mixture.

2. The heart deep cryopreservation system for organ transplantation according to claim 1, characterized in that: The thickness of the organic gel layer is greater than or equal to 5 μm.

3. The heart deep cryopreservation system for organ transplantation according to claim 1, characterized in that: The viscosity of the dimethyl silicone oil is 10 to 20 mPa·s.

4. The heart deep cryopreservation system for organ transplantation according to claim 1, characterized in that: The material of the containing container is quartz glass.

5. The heart deep cryopreservation system for organ transplantation according to claim 1, characterized in that: The medium is alcohol.

6. The heart deep cryopreservation system for organ transplantation according to claim 1, characterized in that: The preparation method of the holding container comprises the following steps: pre-treating the inner wall of the container to obtain a pre-treated container; Applying a polydimethylsiloxane precursor to the inner wall of the pretreatment container, placing a mold inside the pretreatment container, and curing; the shape of the mold matches the shape of the material to be contained; After curing, the mold is removed to obtain a container with an inner wall fully covered with an organic gel layer; The container with the organic gel layer fully attached to the inner wall is immersed in dimethyl silicone oil to obtain the container.

7. The heart deep cryopreservation system for organ transplantation according to claim 6, characterized in that: The pretreatment includes rinsing and plasma cleaning in sequence; the rinsing includes rinsing with anhydrous ethanol and rinsing with water in sequence; the power of the plasma cleaning is 18 to 24 W, and the time is 10 to 15 minutes.

8. The heart deep cryopreservation system for organ transplantation according to claim 6, characterized in that: The curing temperature is 80-85°C and the curing time is 2-2.5 hours; The immersion time is 20 to 24 hours.

9. The method for using the heart deep cryopreservation system for organ transplantation according to any one of claims 1 to 8, characterized in that: The following steps are involved: Place the substance in the cavity of the container, inject UW liquid until it completely covers the substance, perform oil sealing, and cover with a sealing cover; Use temperature control circulation equipment to cool the medium in the medium container to make the medium reach the storage temperature; Completely immerse the system in a medium that has reached the storage temperature for storage.

10. The method of use according to claim 9, characterized in that: After the storage, the method further includes rewarming for use; the rewarming for use includes the following steps: Set the temperature-raising program of the temperature-controlled circulation equipment to raise the temperature; After reaching the set rewarming temperature, take out the contents for use.

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