A method for long-term low-temperature preservation of autologous bone tissue

Abstract

The invention belongs to the technical field of bone tissue cryopreservation, and specifically is a method for long-term cryopreservation of autologous bone tissue, comprising the following steps: S1, obtaining a bone tissue sample, washing the bone tissue with physiological saline or phosphate buffer, and removing blood and other residues; S2, preparing a cryopreservation medium, pouring the cryopreservation medium into an inner barrel of a preservation device, wrapping the bone tissue sample with a gauze net bag and hanging it under the middle sealing cover of an outer barrel, and the outer barrel and the inner barrel can slide relative to each other, thereby sinking the bone tissue sample into the cryopreservation medium for preservation; S3, placing the preservation device in a freezing device to pre-freeze the bone tissue; S4, after the pre-freezing is completed, taking out the preservation device and putting it into a liquid nitrogen storage tank to preserve the bone tissue at low temperature; the invention separates the bone tissue and the cryopreservation medium before taking out, so as to reduce the subsequent cleaning workload, reduce the work intensity of medical staff, and improve work efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of bone tissue cryopreservation, and specifically is a method for long-term cryopreservation of autologous bone tissue. Background Art

[0002] Bone tissue can be cryogenically stored with the help of liquid nitrogen. Liquid nitrogen is a substance that is gaseous at room temperature and becomes liquid when the temperature drops to -196°C. At this temperature, the metabolic activities of cells and tissues are almost completely stopped, which effectively delays the aging and degradation process of biological tissues. By placing the container containing bone tissue in liquid nitrogen, we can achieve long-term preservation while maintaining its structural and functional integrity. When using liquid nitrogen to preserve bone tissue, the bone tissue is usually not directly in contact with liquid nitrogen. The temperature of liquid nitrogen is extremely low. If bone tissue comes into direct contact with liquid nitrogen, it may cause rapid temperature changes, which may cause structural damage inside the bone tissue, such as cell rupture and tissue destruction. Therefore, some protective measures are usually taken to ensure that the contact between liquid nitrogen and bone tissue is indirect to avoid potential damage caused by direct contact.

[0003] In the prior art, bone tissue is usually placed in a cryovial, sample bag or dedicated storage container, which is then immersed in liquid nitrogen for preservation. In order to further protect the bone tissue, freezing media are usually used. These chemicals can help protect tissue cells, prevent frostbite and the formation of ice crystals, thereby ensuring the safety and integrity of the bone tissue.

[0004] When removing bone tissue, the bone tissue needs to be removed from the freezing medium first. After the bone tissue is removed from the freezing medium, the freezing medium attached to the surface of the bone tissue needs to be cleaned. When cleaning, a solution containing a buffer solution is usually used for multiple cleanings to ensure that the freezing medium is effectively removed. The cleaning workload is large and the cleaning efficiency is low. To this end, the present invention provides a method for long-term low-temperature storage of autologous bone tissue. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, at least one technical problem raised in the background technology is solved.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a method for long-term low-temperature preservation of autologous bone tissue described in the present invention comprises the following steps:

[0007] S1. Obtain bone tissue samples and flush the bone tissue with saline or phosphate buffer to remove blood and other residues;

[0008] S2, preparing a cryopreservation medium, pouring the cryopreservation medium into the inner barrel of the preserver, wrapping the bone tissue sample with a gauze net bag and hanging it under the middle sealing cover of the outer barrel, so that the outer barrel and the inner barrel can slide relative to each other, thereby sinking the bone tissue sample into the cryopreservation medium for preservation;

[0009] S3, placing the preservation device in a freezing device to pre-freeze the bone tissue;

[0010] S4. After the prefreezing is completed, the preservation device is taken out and placed in a liquid nitrogen storage tank to preserve the bone tissue at low temperature;

[0011] S5. When taking out the bone tissue sample, separate the bone tissue sample from the freezing medium by lifting the outer barrel, so that the freezing medium attached to the surface of the bone tissue sample can drip freely, thereby reducing the amount of freezing medium attached;

[0012] S6. Open the sealing cover on the top of the outer barrel and take out the bone tissue sample.

[0013] Preferably, the preserver in step S2 comprises an outer barrel and an inner barrel; the outer barrel is provided with openings at the upper and lower ends respectively, the top of the inner barrel is open, and the inner barrel is sealingly slidably installed in the lower opening of the outer barrel, the sealing cover is threadedly connected to the upper opening of the outer barrel, a vertical rod is provided at the bottom of the sealing cover, a hanging ring is fixed at the bottom of the vertical rod, and the bone tissue sample is suspended below the hanging ring through a net bag.

[0014] Preferably, in step S4, the liquid nitrogen storage tank comprises a base and a tank body, the tank body is fixed to the top of the base, a cavity 2 for placing the retainer is provided in the middle of the tank body, a sealing ring 1 is movably installed in the cavity 2 for sealing, a through hole is provided in the middle of the sealing ring 1 for allowing the inner barrel to pass through, a stepped groove is provided on the upper part of the through hole, the bottom of the outer barrel is clamped in the stepped groove, there is a gap between the outer barrel and the inner wall of the cavity 2, a screw rod is rotatably installed in the gap, the sealing ring 1 is threadedly connected to the screw rod, a motor for driving the screw rod to rotate is fixed to the top of the tank body, a cavity 1 for storing liquid nitrogen is provided in the middle of the side wall of the tank body, a through hole is provided in the middle of the cavity 1 and the top of the cavity 2, a sealing ring 2 is sealingly and slidably installed inside the cavity 1, and a spring 4 is installed at the bottom of the sealing ring 2 to reset it.

[0015] Preferably, the vertical rod is rotatably installed in the middle position of the lower end surface of the sealing cover, a gear 1 is fixed on the vertical rod, an air hole is opened on the sealing cover, a sealing plug is threadedly connected to the top of the air hole, a bracket is fixed below the air hole, a rotating shaft is rotatably installed in the middle of the bracket, the top end of the rotating shaft extends into the air hole, an impeller is fixed on the top end of the rotating shaft, and a gear 2 is fixed on the bottom end of the rotating shaft, and the gear 1 and gear 2 are meshed with each other.

[0016] Preferably, an elastic telescopic sleeve is sealingly fixed to the lower end surface of the sealing cover, a spring 1 is fixed inside the elastic telescopic sleeve, and the vertical rod sealingly slides through the center hole of the elastic telescopic sleeve.

[0017] Preferably, a cavity is provided in the middle of the vertical rod, the bottom of the cavity is connected with the lifting ring, and a movable rod is slidably installed inside the cavity, a spring 2 is fixed on the top of the movable rod, a vertical plate is fixed on the bottom end of the movable rod, a magnetic block 1 is fixed on the side of the vertical plate, a movable plate is provided on the side of the lifting ring, a knocking block is fixed on the bottom side wall of the movable plate, connecting plates are fixed on both sides of the movable plate, a guide rod is fixed on the connecting plate, the guide rod is slidably set in a sliding hole opened on the lifting ring, a spring 3 is installed on the guide rod to reset it, a plurality of magnetic blocks 2 are fixed at equal intervals on the upper side wall of the connecting plate, the magnetic blocks 1 and 2 repel each other, an L-shaped support rod is fixed on the side of the movable rod, a through groove is opened on the upper side wall of the vertical rod, and the support rod is slidably set in the through groove.

[0018] Preferably, a fixed block is fixed on the top side wall of the inner barrel, a limiting guide rail is vertically fixed on the inner wall of the outer barrel, the fixed block is slidably arranged in the middle of the limiting guide rail, a bolt is threadedly connected to the top of the outer barrel, and the bottom end of the bolt is threadedly connected to a threaded hole arranged in the middle of the fixed block.

[0019] Preferably, an electromagnetic block, a control device and a battery are installed in the middle of the base, and the electromagnetic block can attract the inner barrel after being energized.

[0020] Preferably, a top of the cavity is provided with an injection port for adding liquid nitrogen.

[0021] Preferably, a mounting hole is provided at the bottom of the hanging ring, and a limiting sleeve is fixed below the mounting hole.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1. The present invention discloses a method for long-term low-temperature preservation of autologous bone tissue. When the bone tissue needs to be taken out, the motor drives the sealing ring 1 to move upward, and at the same time drives the outer barrel to move upward. The inner barrel is retained at the bottom of the second cavity under the influence of its own gravity and the freezing medium. The outer barrel drives the bone tissue to move above the liquid surface of the freezing medium through the sealing cover and the net bag, thereby separating the bone tissue and the freezing medium. After standing for a period of time, the freezing medium attached to the net bag and the bone tissue falls freely, so that a large amount of freezing medium attached to the preservation container can be removed, thereby reducing the workload of subsequent cleaning of the freezing medium on the surface of the bone tissue and reducing the work intensity of medical staff; in addition, the exposure time of the bone tissue to the air can be shortened, so as to protect the biological function of the bone tissue, reduce damage, and maintain its quality; and the liquid nitrogen in the gap is pressed into the cavity 1 through the through hole through the sealing ring 1, so as to recover the liquid nitrogen and avoid leakage of liquid nitrogen when taking out the preservation container, thereby saving the use cost.

[0024] 2. The present invention discloses a method for long-term low-temperature preservation of autologous bone tissue. When the outer barrel moves upward and the position of the inner barrel remains unchanged, negative pressure is formed between the outer barrel and the inner barrel. At this time, the sealing plug is rotated to open, and the pressure difference between the inside and outside of the preserver causes air to quickly enter the barrel through the air hole after the sealing cover is opened to balance the pressure in the barrel, thereby facilitating the subsequent removal of the sealing cover. In addition, when the air flow passes through the air hole, it drives the impeller to rotate, and then drives the second gear to rotate through the rotating shaft. Then, the second gear drives the first gear to rotate, and then drives the vertical rod, the hanging ring, and the net bag and bone tissue suspended below to rotate. The centrifugal force generated during the rotation further accelerates the removal of the attached cryopreservation medium, thereby improving the efficiency of removing the cryopreservation medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below in conjunction with the accompanying drawings.

[0026] Figure 1 It is a schematic flow chart of the method of the present invention;

[0027] Figure 2 is a stereogram of the present invention;

[0028] Figure 3 is a cross-sectional view of the present invention;

[0029] Figure 4 is another angled cross-sectional view of the present invention;

[0030] Figure 5 yes Figure 4 A schematic diagram of the structure enlargement in the middle;

[0031] Figure 6 It is a partial structural schematic diagram of the present invention;

[0032] Figure 7 yes Figure 6A magnified schematic diagram of the structure at B in the middle;

[0033] Figure 8 yes Figure 6 A magnified schematic diagram of the structure at C in the middle;

[0034] Fig. 9 It is a schematic diagram of the connection position of the bolt and the fixing block of the present invention.

[0035] In the figure: 1. base; 2. tank body; 3. outer barrel; 4. sealing cover; 5. inner barrel; 6. cavity one; 7. cavity two; 8. through hole; 9. screw rod; 10. sealing ring one; 11. electromagnetic block; 12. vertical rod; 13. lifting ring; 14. air hole; 15. impeller; 16. rotating shaft; 17. gear one; 18. elastic telescopic sleeve; 19. spring one; 20. gear two; 21. sealing plug; 22. spring two; 23. movable rod; 24. through groove; 25. support rod; 26. vertical plate; 27. magnetic block one; 28. movable plate; 29. ​​connecting plate; 30. guide rod; 31. spring three; 32. magnetic block two; 33. mounting hole; 34. limiting sleeve; 35. fixing block; 36. bolt; 37. sealing ring two; 38. spring four; 39. knocking block. DETAILED DESCRIPTION

[0036] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0037] like Figure 1 As shown, the method for long-term low-temperature preservation of autologous bone tissue of the present invention comprises the following steps:

[0038] S1. Obtain bone tissue samples and flush the bone tissue with saline or phosphate buffer to remove blood and other residues;

[0039] S2, prepare a freezing medium, pour the freezing medium into the inner barrel 5 of the preservation device, wrap the bone tissue sample with a gauze net bag and hang it under the middle sealing cover 4 of the outer barrel 3, so that the outer barrel 3 and the inner barrel 5 can slide relative to each other, and then the bone tissue sample is sunk into the freezing medium for preservation. The freezing medium uses glycerol or dimethyl sulfoxide. Usually, the freezing medium is used to protect the tissue and prevent the formation of harmful ice crystals during the freezing process, thereby avoiding tissue damage;

[0040] S3, placing the preservation device in a freezing device to pre-freeze the bone tissue;

[0041] S4. After the prefreezing is completed, the preserver is taken out and placed in a liquid nitrogen storage tank to preserve the bone tissue at low temperature. During the prefreezing process, the bone tissue is gradually frozen in a low temperature environment of -80°C, which helps to slow down the temperature change and thus avoid damage to the internal tissue;

[0042] S5, when taking out the bone tissue sample, the bone tissue sample is separated from the freezing medium by lifting the outer barrel 3, so that the freezing medium attached to the surface of the bone tissue sample can drip freely, thereby reducing the amount of freezing medium attached;

[0043] S6. Open the sealing cover 4 on the top of the outer barrel 3 to take out the bone tissue sample.

[0044] Embodiment 1: Figures 2 to 9 As shown, the storage device in step S2 includes an outer barrel 3 and an inner barrel 5; the upper and lower ends of the outer barrel 3 are respectively provided with openings, the top of the inner barrel 5 is open, and the inner barrel 5 is sealingly slidably installed in the lower opening of the outer barrel 3, the sealing cover 4 is threadedly connected to the upper opening of the outer barrel 3, the bottom of the sealing cover 4 is provided with a vertical rod 12, the bottom of the vertical rod 12 is fixed with a hanging ring 13, and the bone tissue sample is suspended below the hanging ring 13 through a net bag;

[0045] During operation, the freezing medium is first placed in the inner barrel 5, and then the bone tissue is placed in the net bag, and the net bag is hung on the ring 13. The bone tissue is wrapped with a gauze net bag to make it easier to operate and remove in the freezing medium. The experimenter can easily extract the entire net bag from the freezing medium to avoid direct contact with the bone tissue and reduce the risk of contamination during operation; the gauze net bag can help the tissue remain suspended and prevent it from contacting the surface of the container, thereby reducing mechanical damage; then the sealing cover 4 is picked up by the handle, the net bag and bone tissue are slowly placed in the freezing medium, and then the sealing cover 4 is tightened, and then the preserver is placed in the freezing equipment to gradually freeze the bone tissue; when the bone tissue needs to be removed, the outer barrel 3 is first lifted, and the outer barrel 3 drives the net bag and bone tissue to move above the liquid surface of the freezing medium, so that the freezing medium falls freely, thereby reducing the amount of attached freezing medium, so as to reduce the difficulty of subsequent cleaning, and then the sealing cover 4 is opened to remove the bone tissue.

[0046] In step S4, the liquid nitrogen storage tank includes a base 1 and a tank body 2, the outer wall of the tank body 2 has an insulation layer, the tank body 2 is fixed to the top of the base 1, a cavity 2 7 for placing a retainer is provided in the middle of the tank body 2, a sealing ring 10 is installed in the cavity 2 7 for sealing and movably, a through hole is provided in the middle of the sealing ring 10 for allowing the inner barrel 5 to pass through, a stepped groove is provided on the upper part of the through hole, the bottom of the outer barrel 3 is clamped in the stepped groove, there is a gap between the outer barrel 3 and the inner wall of the cavity 2 7, a screw rod 9 is rotatably installed in the gap, the sealing ring 10 is threadedly connected to the screw rod 9, a motor for driving the screw rod 9 to rotate is fixed on the top of the tank body 2, a cavity 6 for storing liquid nitrogen is provided in the middle of the side wall of the tank body 2, a through hole 8 is provided in the middle of the cavity 1 6 and the top of the cavity 2 7, a sealing ring 2 37 is installed in the cavity 6 for sealing and sliding, and a spring 4 38 is installed at the bottom of the sealing ring 2 37 for resetting it;

[0047] During operation, initially, the motor drives the screw rod 9 to rotate through the forward transmission, drives the sealing ring 10 to move to the top of the cavity 2 7, and closes the through hole 8 through the sealing ring. Then, a sufficient amount of liquid nitrogen is injected into the cavity 1 6, and the liquid nitrogen pushes the sealing ring 2 37 to move downward, and the spring 4 38 is compressed. Then, the preserving device that has completed the pre-freezing work is put into the top opening of the cavity 2 7, and the bottom of the outer barrel 3 is clamped in the stepped groove. Then, the motor drives the screw rod 9 to flip, drives the sealing ring 10 to move downward to the bottom of the cavity 2 7, and the preserving device is put into the cavity 2 7. After the sealing ring 10 is shifted, it no longer blocks the through hole 8. At this time, the spring 4 38 rebounds and pushes the sealing ring 2 37 upward, and then the liquid nitrogen is pressed into the gap between the outer barrel 3 and the inner cavity 2 through the through hole 8, so that the liquid nitrogen surrounds the outside of the outer barrel 3 to create a continuous low-temperature environment, so as to preserve the bone tissue at a low temperature for a long time.

[0048] When the bone tissue needs to be removed, the motor drives the sealing ring 10 to move upward, and at the same time drives the outer barrel 3 to move upward. The inner barrel 5 is retained at the bottom of the cavity 2 7 under the influence of its own gravity and the freezing medium. The outer barrel 3 drives the bone tissue to move above the liquid surface of the freezing medium through the sealing cover 4 and the net bag, thereby separating the bone tissue and the freezing medium. After standing for a period of time, the freezing medium attached to the net bag and the bone tissue falls freely. In this way, a large amount of freezing medium attached to the preservation container can be removed, thereby reducing the workload of subsequent cleaning of the freezing medium on the surface of the bone tissue and reducing the work intensity of medical staff; in addition, the time that the bone tissue is exposed to the air can be shortened, so as to protect the biological function of the bone tissue, reduce damage, and maintain its quality; and the liquid nitrogen in the gap is pressed into the cavity 1 through the through hole 8 through the sealing ring 10, so as to recover the liquid nitrogen and avoid leakage of liquid nitrogen when taking out the preservation container, thereby saving the use cost.

[0049] The vertical rod 12 is rotatably mounted at the middle position of the lower end surface of the sealing cover 4, a gear 17 is fixed on the vertical rod 12, an air hole 14 is opened on the sealing cover 4, a sealing plug 21 is threadedly connected to the top of the air hole 14, a bracket is fixed below the air hole 14, a rotating shaft 16 is rotatably mounted in the middle of the bracket, the top of the rotating shaft 16 extends to the inside of the air hole 14, an impeller 15 is fixed on the top of the rotating shaft 16, a gear 20 is fixed on the bottom of the rotating shaft 16, and the gear 17 and the gear 20 are meshed with each other; during operation, when the outer barrel 3 moves up and the position of the inner barrel 5 remains unchanged, the outer barrel 3 and the inner barrel 5 are meshed with each other. Negative pressure is formed in the barrel, and the sealing plug 21 is rotated to open at this time. The pressure difference between the inside and outside of the preserver causes the air to quickly enter the barrel through the air hole 14 after the sealing cover 4 is opened, so as to balance the pressure in the barrel, thereby facilitating the subsequent removal of the sealing cover 4; in addition, when the air flow passes through the air hole 14, it drives the impeller 15 to rotate, and then drives the gear 2 20 to rotate through the rotating shaft 16, and then the gear 2 20 drives the gear 1 17 to rotate, and then drives the vertical rod 12, the ring 13, and the net bag and bone tissue suspended below to rotate. The centrifugal force generated during the rotation process further accelerates the removal of the attached cryopreservation medium, thereby improving the efficiency of removing the cryopreservation medium.

[0050] An elastic telescopic sleeve 18 is sealed and fixed on the lower end surface of the sealing cover 4, a spring 19 is fixed inside the elastic telescopic sleeve 18, and the vertical rod 12 seals and slides through the center hole of the elastic telescopic sleeve 18; when working, the elastic telescopic sleeve 18 is covered on the outside of the gear 1 17, the gear 2 20, the impeller 15 and other structures, so that the related structures will not be contaminated by the freezing medium; in addition, the inhaled air is supplemented into the elastic telescopic sleeve 18 to avoid direct contact between air and bone tissue, so as to protect the biological function of the bone tissue and maintain its quality.

[0051] A cavity is provided in the middle of the vertical rod 12, and the bottom of the cavity is connected to the lifting ring 13. A movable rod 23 is slidably installed inside the cavity. A spring 22 is fixed on the top of the movable rod 23, and a vertical plate 26 is fixed on the bottom end of the movable rod 23. A magnetic block 27 is fixed on the side of the vertical plate 26. A movable plate 28 is provided on the side of the lifting ring 13, and a knocking block 39 is fixed on the bottom side wall of the movable plate 28. Connecting plates 29 are fixed on both sides of the movable plate 28, and guide rods 30 are fixed on the connecting plates 29. The guide rods 30 are slidably set in the sliding holes opened on the lifting ring 13, and springs 31 are installed on the guide rods 30 to reset them. A plurality of magnetic blocks 232 are fixed at equal intervals on the upper side wall of the connecting plate 29, and the magnetic blocks 1 27 and 2 32 repel each other. An L-shaped support rod 25 is fixed on the side of the movable rod 23, and a through groove 24 is opened on the upper side wall of the vertical rod 12, and the support rod 25 is slidably set in the through groove 24;

[0052] During operation, when air enters the elastic telescopic sleeve 18, the elastic telescopic sleeve 18 expands downward, the spring 19 is stretched, and the spring 22 pushes the movable rod 23, the vertical plate 26 and the magnetic block 1 27 downward. When the magnetic block 1 27 moves to align with the magnetic block 2 32, the two repel each other, and the movable plate 28 is limited by the guide rod 30, the connecting plate 29 and the sliding hole, thereby pushing the movable rod 23 to move horizontally outward. At this time, the spring 31 is compressed. When the magnetic block 1 27 continues to move and is misaligned with the magnetic block 2 32, the movable plate 28 is driven to reset by the rebound of the spring 31. The movable plate 28 knocks on the upper outer wall of the net bag through the knocking block 39 at the bottom, so that the net bag shakes, thereby assisting in removing the attached cryopreservation medium. When bone tissue needs to be removed , by rotating the sealing cover 4, the sealing cover 4 is removed from the top of the outer barrel 3. During the rotation of the sealing cover 4, the net bag and the bone tissue are further driven to rotate so as to remove the attached cryopreservation medium; when the sealing cover 4 is removed, the negative pressure in the barrel disappears, and under the rebound action of the spring 19, the elastic telescopic sleeve 18 is driven to close and the air inside it is discharged. During the closing process of the elastic telescopic sleeve 18, the support rod 25 is pushed to move, and then the movable rod 23 is driven to move upward through the support rod 25, and then the movable plate 28 and the magnetic block 1 27 are driven to move upward, and the magnetic block 1 27 can be aligned with the magnetic block 2 32 again, so that the net bag can be knocked again, and a plurality of magnetic blocks 2 32 are provided to increase the knocking frequency and further improve the effect of removing the attached cryopreservation medium.

[0053] A fixing block 35 is fixed on the top side wall of the inner barrel 5, and a limiting guide rail is fixed vertically on the inner wall of the outer barrel 3. The fixing block 35 is slidably arranged in the middle of the limiting guide rail. A bolt 36 is threadedly connected to the top of the outer barrel 3, and the bottom end of the bolt 36 is threadedly connected to the threaded hole set in the middle of the fixing block 35. During operation, the limiting guide rail and the fixing block 35 cooperate to limit the position of the inner barrel 5, so that the threaded hole on the fixing block 35 is aligned with the bolt 36. In this way, the bolt 36 is threadedly connected inside the threaded hole to fix the outer barrel 3 and the inner barrel 5 together, which is convenient for placing the bone tissue in the cryopreservation medium for pre-freezing treatment in the early stage, and also convenient for placing the pre-freezing container into the tank body 2. Before the outer barrel 3 is driven upward by the sealing ring, the bolt 36 is rotated to disengage the bottom end of the bolt 36 from the threaded hole, so that the outer barrel 3 and the inner barrel 5 are separated.

[0054] An electromagnetic block 11, a control device and a battery are installed in the middle of the base 1. The electromagnetic block 11 can attract the inner barrel 5 after being energized. When working, the battery provides power to the electromagnetic block 11 and the motor, and the control device controls the electromagnetic block 11 and the motor to work. When the outer barrel 3 needs to be lifted, the electromagnetic block 11 is energized. At this time, the electromagnetic block 11 generates suction to attract the inner barrel 5, so that the inner barrel 5 will not move up synchronously with the outer barrel 3.

[0055] An injection port for adding liquid nitrogen is provided at the top of the cavity 1 6; during operation, liquid nitrogen can be added to the cavity 1 6 through the injection hole. An ultrasonic liquid level meter can also be installed in the base 1 to detect the inside of the device. When the sealing ring 10 is located at the bottom of the cavity 2 7, the space between the outer barrel 3 and the inner wall of the cavity 2 7 is filled with liquid nitrogen. At this time, the ultrasonic liquid level meter is used to detect the remaining amount of liquid nitrogen in the cavity 1 6 to determine whether the remaining amount of liquid nitrogen in the device is sufficient so as to replenish the liquid nitrogen in time.

[0056] Embodiment 2: Figure 8 As shown, compared with Example 1, another implementation of the present invention is: a mounting hole 33 is opened at the bottom of the hanging ring 13, and a limiting sleeve 34 is fixed below the mounting hole 33; when working, the top of the net bag is inserted into the limiting sleeve 34 and the mounting hole 33 from bottom to top, and then the net bag is tied to the hanging ring 13, and the swing amplitude of the net bag is limited by the limiting sleeve 34 to avoid collision between bone tissue and the inner wall of the container during rotation to cause damage.

[0057] Working principle: first put the cryopreservation medium into the outer barrel 3 and the inner barrel 5, then put the bone tissue into the net bag, and hang the net bag on the hanging ring 13, and use the gauze net bag to wrap the bone tissue, so that it is easier to operate and take out in the cryopreservation medium. The experimenter can easily extract the entire net bag from the cryopreservation medium to avoid direct contact with the bone tissue and reduce the risk of contamination during operation; the gauze net bag can help the tissue stay suspended and prevent it from contacting the surface of the container, thereby reducing mechanical damage; then pick up the sealing cover 4 by the handle, slowly put the net bag and bone tissue into the cryopreservation medium, tighten the sealing cover 4, and then put the preserver into the freezing device to gradually freeze the bone tissue;

[0058] The pre-freezing container is placed from the top opening of the cavity 2 7, and the bottom of the outer barrel 3 is clamped in the stepped groove. Then, the motor drives the screw rod 9 to flip, and drives the sealing ring 10 to move down to the bottom of the cavity 2 7, so that the container is put into the cavity 2 7. After the sealing ring 10 is moved, it no longer blocks the through hole 8. At this time, the spring 4 38 rebounds and pushes the sealing ring 2 37 upward, and then the liquid nitrogen is pressed into the gap between the outer barrel 3 and the inner cavity 2 through the through hole 8, so that the liquid nitrogen surrounds the outside of the outer barrel 3 to create a continuous low-temperature environment, so as to preserve the bone tissue at low temperature for a long time.

[0059] When the bone tissue needs to be removed, the motor drives the sealing ring 10 to move upward, and at the same time drives the outer barrel 3 to move upward. The inner barrel 5 is retained at the bottom of the cavity 2 7 under the influence of its own gravity and the freezing medium. The outer barrel 3 drives the bone tissue to move above the freezing medium liquid level through the sealing cover 4 and the net bag, thereby separating the bone tissue and the freezing medium. After standing for a period of time, the freezing medium attached to the net bag and the bone tissue falls freely. In this way, a large amount of freezing medium attached to the preservation device can be removed inside the preservation device, thereby reducing the workload of subsequent cleaning of the freezing medium on the surface of the bone tissue and reducing the work intensity of medical staff; in addition, the time that the bone tissue is exposed to the air can be shortened, so as to protect the biological function of the bone tissue, reduce damage, and maintain its quality;

[0060] When the outer barrel 3 moves upward and the position of the inner barrel 5 remains unchanged, negative pressure is formed between the outer barrel 3 and the inner barrel 5. At this time, the sealing plug 21 is rotated to open, and the pressure difference between the inside and outside of the storage container causes the air to quickly enter the barrel through the air hole 14 after the sealing cover 4 is opened, so as to balance the pressure in the barrel, thereby facilitating the subsequent removal of the sealing cover 4; in addition, when the air flow passes through the air hole 14, it drives the impeller 15 to rotate, and then drives the gear 2 20 to rotate through the rotating shaft 16, and then the gear 2 20 drives the gear 1 17 to rotate, and then drives the vertical rod 12, the ring 13, and the net bag and bone tissue suspended below to rotate, and the centrifugal force generated during the rotation process further accelerates the removal of the attached cryopreservation medium, thereby improving the efficiency of removing the cryopreservation medium;

[0061] When air enters the elastic telescopic sleeve 18, the elastic telescopic sleeve 18 expands downward, the spring 19 is stretched, and the spring 22 pushes the movable rod 23, the vertical plate 26 and the magnetic block 1 27 downward. When the magnetic block 1 27 moves to align with the magnetic block 2 32, the two repel each other, and the movable plate 28 is limited by the guide rod 30, the connecting plate 29 and the sliding hole, thereby pushing the movable rod 23 to move horizontally outward. At this time, the spring 31 is compressed. When the magnetic block 1 27 continues to move and is misaligned with the magnetic block 2 32, the movable plate 28 is driven to reset by the rebound of the spring 31. The movable plate 28 knocks on the upper outer wall of the net bag through the knocking block 39 at the bottom, so that the net bag shakes, thereby assisting in removing the attached cryopreservation medium; when the bone tissue needs to be removed, the By rotating the sealing cover 4, the sealing cover 4 is removed from the top of the outer barrel 3. During the rotation of the sealing cover 4, the net bag and the bone tissue are further driven to rotate so as to remove the attached cryopreservation medium. After the sealing cover 4 is removed, the negative pressure in the barrel disappears. Under the rebound action of the spring 19, the elastic telescopic sleeve 18 is driven to close and the air inside it is discharged. During the closing process of the elastic telescopic sleeve 18, the support rod 25 is pushed to move, and then the movable rod 23 is driven to move upward through the support rod 25, and then the movable plate 28 and the magnetic block 1 27 are driven to move upward. The magnetic block 1 27 can be aligned with the magnetic block 2 32 again, so that the net bag can be knocked again, and a plurality of magnetic blocks 2 32 are provided to increase the knocking frequency and further improve the effect of removing the attached cryopreservation medium.

[0062] The above-mentioned front, back, left, right, top and bottom are all based on the figures in the specification. Figure 1 As a benchmark, according to the person's observation perspective, the side of the device facing the observer is defined as the front, the left side of the observer is defined as the left, and so on.

[0063] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the scope of protection of the present invention.

[0064] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for long-term storage of autologous bone tissue at low temperature, characterized in that: The following steps are involved: S1. Obtain bone tissue samples and flush the bone tissue with saline or phosphate buffer to remove blood and other residues; S2, preparing a cryopreservation medium, pouring the cryopreservation medium into the inner barrel (5) of the preservation device, wrapping the bone tissue sample with a gauze net bag and hanging it under the middle sealing cover (4) of the outer barrel (3), so that the outer barrel (3) and the inner barrel (5) can slide relative to each other, thereby sinking the bone tissue sample into the cryopreservation medium for preservation; S3, placing the preservation device in a freezing device to pre-freeze the bone tissue; S4. After the prefreezing is completed, the preservation device is taken out and placed in a liquid nitrogen storage tank to preserve the bone tissue at low temperature; S5. When taking out the bone tissue sample, the bone tissue sample is separated from the cryopreservation medium by lifting the outer barrel (3), so that the cryopreservation medium attached to the surface of the bone tissue sample can drip freely, thereby reducing the amount of cryopreservation medium attached; S6. Open the top sealing cover (4) of the outer barrel (3) and take out the bone tissue sample; In step S2, the storage container comprises an outer barrel (3) and an inner barrel (5); the outer barrel (3) is provided with openings at both upper and lower ends, the inner barrel (5) is open at the top, and the inner barrel (5) is sealingly slidably mounted in the lower opening of the outer barrel (3); the sealing cover (4) is threadedly connected to the upper opening of the outer barrel (3); a vertical rod (12) is provided at the bottom of the sealing cover (4); a hanging ring (13) is fixed at the bottom of the vertical rod (12), and the bone tissue sample is hung below the hanging ring (13) through a net bag; In step S4, the liquid nitrogen storage tank comprises a base (1) and a tank body (2), wherein the tank body (2) is fixed to the top of the base (1), and a second cavity (7) for placing the storage container is provided in the middle of the tank body (2), and a sealing ring (10) is movably installed in the second cavity (7), and a through hole is provided in the middle of the sealing ring (10) for allowing the inner barrel (5) to pass through, and a stepped groove is provided on the upper part of the through hole, and the bottom of the outer barrel (3) is clamped in the stepped groove, and there is a gap between the outer barrel (3) and the inner wall of the second cavity (7). A screw (9) is rotatably installed in the gap, the sealing ring (10) is threadedly connected to the screw (9), a motor for driving the screw (9) to rotate is fixed on the top of the tank body (2), a cavity (6) for storing liquid nitrogen is opened in the middle of the side wall of the tank body (2), a through hole (8) is provided in the middle of the top of the cavity (6) and the cavity (7), a sealing ring (37) is sealingly and slidably installed inside the cavity (6), and a spring (38) for returning the sealing ring (37) is installed at the bottom of the sealing ring (37).

2. The method for long-term low-temperature storage of autologous bone tissue according to claim 1, characterized in that: The vertical rod (12) is rotatably mounted at the middle position of the lower end surface of the sealing cover (4); a gear 1 (17) is fixed on the vertical rod (12); an air hole (14) is opened on the sealing cover (4); a sealing plug (21) is threadedly connected to the top of the air hole (14); a bracket is fixed below the air hole (14); a rotating shaft (16) is rotatably mounted in the middle of the bracket; the top end of the rotating shaft (16) extends into the interior of the air hole (14); an impeller (15) is fixed on the top end of the rotating shaft (16); a gear 2 (20) is fixed on the bottom end of the rotating shaft (16); the gear 1 (17) and the gear 2 (20) are meshed with each other.

3. The method for long-term low-temperature storage of autologous bone tissue according to claim 2, characterized in that: An elastic telescopic sleeve (18) is sealingly fixed to the lower end surface of the sealing cover (4), a spring 1 (19) is fixed inside the elastic telescopic sleeve (18), and the vertical rod (12) is sealingly slidable and penetrates the center hole of the elastic telescopic sleeve (18).

4. The method for long-term low-temperature storage of autologous bone tissue according to claim 3, characterized in that: A cavity is provided in the middle of the vertical rod (12), the bottom of the cavity is communicated with the hanging ring (13), a movable rod (23) is slidably installed in the cavity, a spring 2 (22) is fixed on the top of the movable rod (23), a vertical plate (26) is fixed on the bottom end of the movable rod (23), a magnetic block 1 (27) is fixed on the side of the vertical plate (26), a movable plate (28) is provided on the side of the hanging ring (13), a knocking block (39) is fixed on the bottom side wall of the movable plate (28), connecting plates (29) are respectively fixed on both sides of the movable plate (28), and the connecting plates A guide rod (30) is fixed on (29), and the guide rod (30) is slidably set in a sliding hole opened on the lifting ring (13). A spring three (31) is installed on the guide rod (30) to reset it. A plurality of magnetic blocks (32) are fixed at equal intervals on the upper side wall of the connecting plate (29), and the magnetic blocks (27) and (32) repel each other. An L-shaped support rod (25) is fixed on the side of the movable rod (23), and a through groove (24) is opened on the upper side wall of the vertical rod (12), and the support rod (25) is slidably set in the through groove (24).

5. The method for long-term low-temperature storage of autologous bone tissue according to claim 4, characterized in that: A fixing block (35) is fixed on the top side wall of the inner barrel (5); a limit guide rail is vertically fixed on the inner wall of the outer barrel (3); the fixing block (35) is slidably arranged in the middle of the limit guide rail; a bolt (36) is threadedly connected to the top of the outer barrel (3); and the bottom end of the bolt (36) is threadedly connected to a threaded hole arranged in the middle of the fixing block (35).

6. The method for long-term low-temperature storage of autologous bone tissue according to claim 5, characterized in that: An electromagnetic block (11), a control device and a storage battery are installed in the middle of the base (1); the electromagnetic block (11) is capable of attracting the inner barrel (5) when energized.

7. The method for long-term low-temperature storage of autologous bone tissue according to claim 6, characterized in that: The top of the cavity 1 (6) is provided with an injection port for adding liquid nitrogen.

8. The method for long-term low-temperature storage of autologous bone tissue according to claim 7, characterized in that: A mounting hole (33) is provided at the bottom of the hanging ring (13), and a limiting sleeve (34) is fixed below the mounting hole (33).

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