Deep hypothermia storage method for retaining bone tissue activity

Abstract

The invention belongs to the technical field of bone tissue low-temperature storage, and particularly relates to a deep low-temperature storage method for retaining bone tissue activity, which comprises the following steps: A1, acquiring a bone tissue sample, transporting the bone tissue sample to a treatment center in a cold chain manner, pre-treating the bone tissue, cleaning periosteum, putting the bone tissue sample into negative-pressure ultrasonic purified water for cleaning, inactivating with alcohol, flushing with sterile PBS (Phosphate Buffer Solution), and cleaning the bone tissue; a2, preparing a cryoprotectant, immersing the bone tissue into the cryoprotectant, and maintaining the temperature through an ice-water bath; a3, after the cryoprotectant enters the bone tissue, the bone tissue is placed in pre-freezing equipment, and the bone tissue is pre-frozen; a4, the pre-frozen bone tissue is taken out and then put into a liquid nitrogen storage container, and low-temperature storage of the bone tissue is started; through the treatment of the series of methods, the formation of ice crystals can be reduced, and the icing of water in cells and tissues is promoted to become more uniform and smaller, so that the damage to bone tissue cells is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of bone tissue cryogenic storage, and in particular is a deep cryogenic storage method for retaining bone tissue activity. Background Art

[0002] The method of cryogenic storage of active bone tissue is a complex biomedical technology that is mainly used to preserve bone tissue to maintain its biological activity and structural integrity. This process involves freezing the bone tissue, thereby effectively slowing down the metabolic processes of cells and tissues.

[0003] In the medical field, the preservation of bone tissue is of vital importance for bone transplantation surgery, tissue engineering research and the treatment of certain diseases. At present, the commonly used methods for bone tissue preservation are refrigeration and freeze-drying. Although the refrigeration method is relatively simple to operate, the preservation time is short and can generally only be maintained for a few days to a few weeks. In this process, the activity of bone tissue will gradually decrease. Although the freeze-drying method can prolong the preservation time, it may damage the cell structure of bone tissue during the drying process, resulting in a significant decrease in the activity of bone tissue.

[0004] Therefore, developing a deep-cryogenic storage method that can effectively preserve the activity of bone tissue has become an urgent need in the current medical field. This method must not only be able to preserve bone tissue for a long time at extremely low temperatures, but also ensure that the cellular structure and biological activity of bone tissue are restored to the greatest extent during the recovery process, providing a high-quality bone tissue source for bone transplantation surgery and tissue engineering research.

[0005] When storing bone tissue on the market, in order to increase the freezing rate of bone tissue, the obtained bone tissue is directly immersed in liquid nitrogen. Although the effect of quick freezing is achieved, the freezing rate is too fast, resulting in the formation of larger ice crystals and damage to the bones.

[0006] To this end, the present invention provides a deep-cryogenic storage method for retaining the activity of bone tissue. Summary of the invention

[0007] In order to make up for the deficiencies of the prior art, at least one technical problem raised in the background technology is solved.

[0008] The technical solution adopted by the present invention to solve the technical problem is: a deep low temperature storage method for retaining bone tissue activity according to the present invention comprises the following steps: A1. Obtain bone tissue samples, place the bone tissue in a sterile balanced salt solution, and rinse with sterile PBS to clean the bone tissue; A2. Prepare cryoprotectant, then immerse the bone tissue in the cryoprotectant and maintain the temperature in an ice water bath; A3. After the cryoprotectant enters the bone tissue, the bone tissue is placed in a pre-freezing device to pre-freeze the bone tissue; A4. Take out the pre-frozen bone tissue and place it in a liquid nitrogen storage container to start low-temperature storage of the bone tissue.

[0009] Preferably, in step A4, the detailed steps of placing the bone tissue into the liquid nitrogen storage container are: B1. Place the bone tissue into the accommodating net bag, assemble the accommodating net bag with the closing plate, and then assemble the closing plate with the accommodating shell; B2. Connect the external pipe to the external liquid nitrogen storage chamber, introduce the liquid nitrogen into the inner container, and discharge the air in the inner container through the control valve; B3. When the liquid nitrogen level in the container reaches a desired height, the container is controlled to expand inward until the liquid nitrogen submerges the bone tissue; B4. When removing bone tissue, first open the control valve and then extract the liquid nitrogen in the container through the external pipe, and finally separate the sealing plate and the container net bag.

[0010] Preferably, the liquid nitrogen storage container in step A4 comprises an outer accommodating portion, an inner accommodating portion and a feeding mechanism; The accommodating part includes an accommodating shell, a closing plate and an accommodating net bag, wherein the closing plate is connected to the accommodating shell through internal and external threads, and the accommodating net bag is used to accommodate bone tissue and is detachably connected to the bottom surface of the closing plate; The inner container comprises a heat preservation tube, a sealing tube and a storage liner, wherein the heat preservation tube is arranged inside the storage shell, the sealing tube is arranged inside the heat preservation tube, and the storage liner is arranged inside the sealing tube; A hollow chamber is reserved between the accommodating liner and the closing tube, and the accommodating liner is made of elastic material; The feeding mechanism includes a control valve, an external pipe and a nitrogen inlet pipe. The control valve is arranged on the upper end surface of the closing plate, and one end of the control valve passes through the closing plate through a guide tube. The external pipe is fixedly installed on the outer wall of the containing shell. One end of the nitrogen inlet pipe passes through the bottom of the closing cylinder and extends to the interior of the containing liner. The other end of the nitrogen inlet pipe is connected to the external pipe.

[0011] Preferably, a bearing ring is fixedly mounted on the inner wall of the heat-insulating cylinder, and the sealing cylinder and the accommodating liner are both fixedly mounted on the bottom of the bearing ring; A hard support ring is fixedly installed on the outer wall of one side of the accommodating liner close to the sealing cylinder; An adapter box is arranged between the accommodating shell and the insulation cylinder, an elastic bin is fixedly installed inside the adapter box, a connecting pipe is fixedly installed on the outer wall of the elastic bin, an electromagnetic valve is arranged in the connecting pipe, and one end of the connecting pipe away from the elastic bin extends to between the closing cylinder and the accommodating liner.

[0012] Preferably, a temperature control chamber is provided in the transfer box, and the nitrogen inlet pipe runs through the temperature control chamber; A control cylinder is fixedly installed on one side of the temperature control chamber, a control plug is elastically installed in the inner cavity of the control cylinder, and the inner cavity of the control cylinder is connected with the temperature control chamber; An extrusion rod for extruding the elastic bin is fixedly mounted on one end of the control plug.

[0013] Preferably, a sealing cover is fixedly mounted on the upper end surface of the carrying ring, and a mounting frame is fixedly mounted on the upper end surface of the carrying ring, a control shaft is rotatably mounted on the side wall of the mounting frame, and a transmission cylinder is arranged on the outer wall of the control shaft; An adding block is arranged at the bottom of the bearing ring, and the upper end surface of the adding block is connected to the radial outer wall of the transmission cylinder through a rope.

[0014] Preferably, a mounting arm is fixedly mounted on the outer wall of the mounting frame, a control ratchet is fixedly mounted on the axial end of the control shaft, a transmission plate is rotatably mounted on the outer wall of the mounting arm, an adjustment rod for engaging with the control ratchet is elastically mounted on the outer wall of the transmission plate, and a control ratchet rod engaging with the control ratchet is elastically mounted on the bottom of the mounting arm.

[0015] Preferably, a support arm is fixedly mounted on the outer wall of the mounting arm, an adjustment block is slidably mounted on the outer wall of the support arm, and one end of the adjustment block is connected to the outer wall of the support arm through an elastic member; The outer wall of the transmission plate is provided with a waist-shaped groove, and the adjusting block is slidably matched with the waist-shaped groove through a pin shaft.

[0016] Preferably, a transmission block is elastically mounted on the outer wall of the control shaft, and a plurality of transmission blocks are provided, and the plurality of transmission blocks are evenly arranged in a ring shape along the axis of the control shaft; The plurality of transmission blocks have a tendency to move towards the axis of the control shaft in real time.

[0017] Preferably, a magnetic generator is fixedly mounted on the outer wall of the heat preservation cylinder; The adjusting block is an iron block, and the sliding direction of the adjusting block is controlled by the difference between the magnetic force of the magnetic generator on the adjusting block and the elastic force of the elastic member on the adjusting block; The transmission block is an iron block. The magnetic force generated by the magnetic generator attracts the transmission block to press against the inner wall of the transmission cylinder.

[0018] The beneficial effects of the present invention are as follows: 1. The present invention pre-freezes the bone tissue in an environment between -20°C and -80°C before immersing the bone tissue in liquid nitrogen for storage, thereby reducing the formation of ice crystals and promoting the freezing of water in cells and tissues to become more uniform and smaller, thereby reducing damage to bone tissue cells, reducing the formation of ice crystals and promoting the freezing of water in cells and tissues to become more uniform and smaller, thereby reducing damage to bone tissue cells, thereby effectively improving the quality of bone tissue storage; 2. The present invention provides a containing net bag. After the liquid nitrogen is injected into the containing liner, the liquid nitrogen is in full contact with the pre-frozen bone tissue. Compared with the prior art in which the liquid nitrogen and the bone tissue are isolated during the storage period, the freezing rate can be increased, thereby preserving the activity of the bone tissue and improving the freezing efficiency. At the same time, an elastic bin is provided to control the expansion of the containing liner. After the liquid nitrogen is injected into the containing liner, a heat-insulating gas is injected into the hollow chamber to control the expansion of the containing liner inward, thereby controlling the liquid nitrogen level to rise until the liquid nitrogen submerges the bone tissue. Compared with the prior art in which the liquid nitrogen is directly injected to the liquid level to submerge the bone tissue, the use of liquid nitrogen can be effectively reduced, thereby reducing the use cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below in conjunction with the accompanying drawings.

[0020] Figure 1 is a flow chart of the deep cryogenic storage method of the present invention; Figure 2 is a flow chart of placing bone tissue into a liquid nitrogen storage container in the present invention; Figure 3 It is a schematic diagram of the overall structure of the present invention; Figure 4 It is a schematic diagram of the installation of the net bag in the present invention; Figure 5 It is a schematic diagram of the installation of the heat preservation cylinder in the present invention; Figure 6 It is a schematic diagram of the installation of the sealing cylinder in the present invention; Figure 7 It is a schematic diagram of the structure of the liner in the present invention; Figure 8 It is a structural schematic diagram of the transmission cylinder in the present invention; Fig. 9 It is a structural schematic diagram of the transmission block in the present invention; Fig.10 It is a schematic diagram of the installation of the adjustment block in the present invention; Fig.11 is a cross-sectional view of the transfer box of the present invention; Fig.12 It is a cross-sectional view of the elastic bin in the present invention.

[0021] In the figure: 1. accommodating shell; 2. closing plate; 3. control valve; 4. accommodating net bag; 5. closing cover; 6. insulation cylinder; 7. magnetic generator; 8. external pipe; 9. closing cylinder; 10. adapter box; 11. mounting frame; 12. nitrogen inlet pipe; 13. transmission cylinder; 14. adding block; 15. bearing ring; 16. accommodating liner; 17. hard support ring; 18. control ratchet; 19. mounting arm; 20. support arm; 21. control shaft; 22. adjusting rod; 23. transmission plate; 24. adjusting block; 25. control ratchet rod; 26. connecting pipe; 27. temperature control chamber; 28. extrusion rod; 29. ​​elastic bin; 30. control plug; 31. control cylinder; 32. transmission block. DETAILED DESCRIPTION

[0022] 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.

[0023] like Figure 1 As shown, a deep cryogenic storage method for retaining bone tissue activity according to the present invention comprises the following steps: A1. Obtain bone tissue samples, place the bone tissue in a sterile balanced salt solution, and rinse with sterile PBS to clean the bone tissue; A2, preparing a cryoprotectant, then immersing the bone tissue in the cryoprotectant, and maintaining the temperature by an ice water bath. In this embodiment, the cryoprotectant is selected from glycerol and dimethyl sulfoxide; A3. After the cryoprotectant enters the bone tissue, the bone tissue is placed in a pre-freezing device to pre-freeze the bone tissue; Among them, the pre-freezing temperature is between -20℃ and -80℃. Through pre-freezing, the formation of ice crystals is reduced and the freezing of water in cells and tissues becomes more uniform and smaller, thereby reducing damage to bone tissue cells.

[0024] A4. Take out the pre-frozen bone tissue and place it in a liquid nitrogen storage container to start low-temperature storage of the bone tissue.

[0025] In addition, it reduces the formation of ice crystals and causes the water in cells and tissues to freeze more evenly and in smaller sizes, thereby reducing damage to bone tissue cells.

[0026] like Figure 2 As shown, in step A4, the detailed steps of placing the bone tissue into the liquid nitrogen storage container are: B1. Place the bone tissue into the accommodating net bag 4, assemble the accommodating net bag 4 with the closing plate 2, and then assemble the closing plate 2 with the accommodating shell 1; B2. Connect the external pipe 8 to the external liquid nitrogen storage chamber, introduce the liquid nitrogen into the interior of the accommodating liner 16, and at this time, discharge the air in the accommodating liner 16 through the control valve 3; B3, when the liquid nitrogen level in the accommodating liner 16 reaches a desired height, the accommodating liner 16 is controlled to expand inwards until the liquid nitrogen submerges the bone tissue; B4. When removing the bone tissue, first open the control valve 3 and then extract the liquid nitrogen in the accommodating liner 16 through the external pipe 8, and finally separate the sealing plate 2 and the accommodating net bag 4.

[0027] like Figure 3-12 As shown, the liquid nitrogen storage container of step A4 includes an outer containing part, an inner containing part and a feeding mechanism, the containing part includes a containing shell 1, a closing plate 2 and a containing net bag 4, the closing plate 2 is connected to the containing shell 1 through internal and external threads, and a sealing layer is provided at the contact position between the closing plate 2 and the containing shell 1. Since the storage temperature is relatively low, the material of the sealing layer is selected to be vinyl methyl silicone rubber.

[0028] The accommodating net bag 4 is used to accommodate bone tissue and is detachably connected to the bottom surface of the closing plate 2. The accommodating net bag 4 is connected to the bottom surface of the closing plate 2 through threads. When in use, the bone tissue is first placed in the accommodating net bag 4, and then the accommodating net bag 4 is inserted into the accommodating shell 1, and is connected to the accommodating shell 1 through the closing plate 2 to close the upper end opening of the accommodating shell 1.

[0029] The inner storage part includes a heat preservation tube 6, a sealing tube 9 and a storage liner 16. The heat preservation tube 6 is arranged inside the storage shell 1, and the heat preservation tube 6 is a double-layer vacuum heat preservation tube.

[0030] The sealing tube 9 is arranged inside the heat preservation tube 6, and the accommodating liner 16 is arranged inside the sealing tube 9, wherein the accommodating liner 16 is used to store liquid nitrogen after the accommodating net bag 4 is inserted. Since the liquid nitrogen needs to contact with the accommodating liner 16, the accommodating liner 16 is made of vinyl methyl silicone rubber.

[0031] After the liquid nitrogen is injected into the container 16, the liquid nitrogen is in full contact with the pre-frozen bone tissue. Compared with the portable bone tissue low-temperature storage and transportation device in patent CN118556676B, the freezing rate can be increased by isolating the liquid nitrogen from the bone tissue during the storage period, thereby preserving the activity of the bone tissue and improving the freezing efficiency.

[0032] A hollow chamber is reserved between the containing liner 16 and the sealing tube 9, and the containing liner 16 is made of elastic material. After liquid nitrogen is injected into the containing liner 16, a heat-insulating gas (carbon dioxide is selected) is injected into the hollow chamber to control the containing liner 16 to expand inward, thereby controlling the liquid nitrogen level to rise until the liquid nitrogen submerges the bone tissue. Compared with the prior art in which liquid nitrogen is directly injected to the liquid surface to submerge the bone tissue, the use of liquid nitrogen can be effectively reduced, thereby reducing the use cost.

[0033] In this embodiment, in order to facilitate the detection of the liquid nitrogen level, a page detector (optionally a sonar page detector or an endoscopic detector) is arranged in the accommodating shell 1.

[0034] The feeding mechanism includes a control valve 3, an external pipe 8 and a nitrogen inlet pipe 12. The control valve 3 is arranged on the upper end surface of the closing plate 2, and one end thereof passes through the closing plate 2 through a conduit. An air pressure detector is arranged outside the control valve 3 for real-time detection of the air pressure inside the accommodating liner 16. At the same time, the control valve 3 is used to control the air intake and exhaust inside the accommodating liner 16.

[0035] The external pipe 8 is fixedly mounted on the outer wall of the housing 1, one end of the nitrogen inlet pipe 12 passes through the bottom of the sealing cylinder 9 and extends to the interior of the accommodating liner 16, and the other end of the nitrogen inlet pipe 12 is connected to the external pipe 8, and the external pipe 8 is used to connect to an external liquid nitrogen storage chamber, and after the connection, liquid nitrogen is injected into the accommodating liner 16.

[0036] When bone tissue needs to be removed, the control valve 3 is first opened, and then the liquid nitrogen in the container 16 is discharged through the external pipe 8. After the liquid nitrogen is discharged, the temperature in the container 16 slowly rises. At this time, the container net bag 4 is taken out to prevent the staff from accidentally touching the liquid nitrogen and causing frostbite, thereby improving the safety of use.

[0037] As a preferred embodiment of the present invention, a carrying ring 15 is fixedly installed on the inner wall of the insulation cylinder 6, and the closing cylinder 9 and the accommodating inner liner 16 are fixedly installed on the bottom of the carrying ring 15. The carrying ring 15 provides an installation position for the closing cylinder 9 and the accommodating inner liner 16, and the hollow chamber between the closing cylinder 9 and the accommodating inner liner 16 is closed by the carrying ring 15.

[0038] A hard support ring 17 is fixedly mounted on the outer wall of one side of the accommodating liner 16 close to the sealing tube 9. By providing the hard support ring 17, the accommodating liner 16 can be kept as uniformly deformed as possible during the expansion and contraction of the accommodating liner 16.

[0039] An adapter box 10 is arranged between the accommodating shell 1 and the heat-insulating cylinder 6 . An elastic bin 29 is fixedly installed inside the adapter box 10 . The outer portion of the elastic bin 29 is made of rubber material.

[0040] A connecting pipe 26 is fixedly mounted on the outer wall of the elastic chamber 29 , and a solenoid valve is arranged in the connecting pipe 26 , and the conduction and closing of the inner cavity of the connecting pipe 26 are controlled by the solenoid valve.

[0041] One end of the connecting tube 26 away from the elastic chamber 29 extends to between the closed tube 9 and the accommodating inner liner 16, wherein the elastic chamber 29 stores heat-insulating gas. When it is necessary to control the expansion of the accommodating inner liner 16, the elastic chamber 29 is squeezed, and then the solenoid valve is controlled to be turned on. At this time, the heat-insulating gas enters the hollow chamber between the closed tube 9 and the accommodating inner liner 16 through the connecting tube 26, thereby controlling the expansion of the accommodating inner liner 16.

[0042] When it is necessary to control the contraction of the accommodating liner 16, the squeezing of the elastic chamber 29 is stopped, and the heat-insulating gas is sucked into the inner cavity of the elastic chamber 29 for the next use.

[0043] A temperature control chamber 27 is provided in the adapter box 10 , and the nitrogen inlet pipe 12 passes through the temperature control chamber 27 . The outer wall of the adapter box 10 is made of a heat-insulating material to reduce the loss of cold energy.

[0044] A control cylinder 31 is fixedly installed on one side of the temperature control chamber 27, and a control plug 30 is elastically installed in the inner cavity of the control cylinder 31, and the inner cavity of the control cylinder 31 is connected with the temperature control chamber 27. When liquid nitrogen enters the nitrogen inlet pipe 12, the external temperature of the nitrogen inlet pipe 12 decreases, resulting in a decrease in the internal temperature and pressure of the temperature control chamber 27. At this time, the control plug 30 slides toward one side of the temperature control chamber 27.

[0045] An extrusion rod 28 for extruding the elastic bin 29 is fixedly installed at one end of the control plug 30, wherein the outer wall of the extrusion rod 28 fits with the outer wall of the elastic bin 29. When the control plug 30 slides toward the temperature control chamber 27, the extrusion rod 28 slides synchronously, thereby squeezing the elastic bin 29, so that the heat-insulating gas tends to enter the hollow chamber between the closing tube 9 and the accommodating inner liner 16.

[0046] After the liquid nitrogen is located inside the nitrogen inlet pipe 12, the outer wall absorbs heat, and the pressure in the temperature control chamber 27 is controlled to decrease, thereby serving as a power source to control the expansion of the inner liner 16, thereby improving the utilization efficiency of the cooling capacity.

[0047] As a preferred embodiment of the present invention, a sealing cover 5 is fixedly mounted on the upper end surface of the carrying ring 15, and a mounting frame 11 is fixedly mounted on the upper end surface of the carrying ring 15, and the upper end surface of the carrying ring 15 is sealed by the sealing cover 5 to form a sealed chamber.

[0048] A control shaft 21 is rotatably mounted on the side wall of the mounting frame 11, and a transmission cylinder 13 is disposed on the outer wall of the control shaft 21, wherein a gap is reserved between the control shaft 21 and the transmission cylinder 13, and a bearing is disposed on the outer wall of the control shaft 21, and is connected to the transmission cylinder 13 via the bearing.

[0049] An adding block 14 is provided at the bottom of the carrying ring 15, and the upper end surface of the adding block 14 is connected to the radial outer wall of the transmission cylinder 13 through a rope. After the heat-insulating gas inside the elastic bin 29 is completely injected into the hollow chamber between the closing cylinder 9 and the accommodating liner 16, if it is necessary to continue to increase the liquid nitrogen level and reduce the friction between the control shaft 21 and the transmission cylinder 13, the gravity of the adding block 14 is used to control the adding block 14 to enter the liquid nitrogen, and then continue to control the liquid nitrogen level to rise until the liquid nitrogen submerges the bone tissue.

[0050] When the adding block 14 needs to be recovered, the friction between the control shaft 21 and the transmission cylinder 13 is increased. At this time, the control shaft 21 is rotated to drive the transmission cylinder 13 to rotate, thereby winding the rope to realize the pulling of the adding block 14.

[0051] A mounting arm 19 is fixedly mounted on the outer wall of the mounting frame 11 , and a control ratchet 18 is fixedly mounted on the axial end of the control shaft 21 . Rotating the control ratchet 18 drives the control shaft 21 to rotate synchronously.

[0052] A transmission plate 23 is rotatably mounted on the outer wall of the mounting arm 19 , and an adjustment rod 22 for engaging with the control ratchet 18 is elastically mounted on the outer wall of the transmission plate 23 , wherein the adjustment rod 22 is connected to the transmission plate 23 via a torsion spring.

[0053] A control ratchet rod 25 engaged with the control ratchet 18 is elastically installed at the bottom of the mounting arm 19. The control ratchet rod 25 is connected to the mounting arm 19 through a torsion spring. The elastic force of the torsion spring drives the control ratchet rod 25 to have a tendency to engage with the control ratchet 18 in real time, thereby allowing the control ratchet 18 to rotate in one direction.

[0054] By reciprocating the transmission plate 23, the adjusting rod 22 is driven to slide back and forth along the outer wall of the control ratchet 18. After the adjusting rod 22 is engaged with the control ratchet 18, the control ratchet 18 is pushed to rotate. Then, after the friction between the control shaft 21 and the transmission cylinder 13 is increased, the transmission cylinder 13 is driven to rotate by the rotation of the control shaft 21, thereby realizing the lifting of the adding block 14.

[0055] A support arm 20 is fixedly mounted on the outer wall of the mounting arm 19 , and an adjustment block 24 is slidably mounted on the outer wall of the support arm 20 , and a sliding track of the adjustment block 24 is defined by the support arm 20 .

[0056] One end of the adjustment block 24 is connected to the outer wall of the support arm 20 through an elastic member. After the adjustment block 24 is slid, the elastic force of the elastic member controls the adjustment block 24 to reset.

[0057] The outer wall of the transmission plate 23 is provided with a waist-shaped groove, and the adjustment block 24 is slidably matched with the waist-shaped groove through a pin shaft. By reciprocatingly sliding the adjustment block 24, the transmission plate 23 is driven to reciprocate and deflect, thereby driving the control ratchet 18 to rotate.

[0058] As a preferred embodiment of the present invention, a transmission block 32 is elastically mounted on the outer wall of the control shaft 21. A plurality of transmission blocks 32 are provided, and a plurality of transmission blocks 32 are evenly arranged in a ring shape along the axis of the control shaft 21. A spring is provided on the outer wall of the transmission block 32, and the other end of the spring is connected to the outer wall of the control shaft 21.

[0059] The plurality of transmission blocks 32 have a tendency to move toward the axis of the control shaft 21 in real time. After the transmission blocks 32 are separated from the inner wall of the transmission cylinder 13 , the transmission cylinder 13 can rotate freely to facilitate the release and insertion of the adding blocks 14 .

[0060] When the adding block 14 needs to be lifted, the transmission block 32 is slid until the transmission block 32 presses against the inner wall of the transmission cylinder 13. At this time, the control shaft 21 is rotated to drive the transmission cylinder 13 to rotate, and the adding block 14 is pulled up by the winding rope.

[0061] A magnetic generator 7 is fixedly mounted on the outer wall of the heat preservation tube 6. The magnetic generator 7 is a common electromagnet. The magnetic field strength of the magnetic generator 7 is controlled by controlling the current.

[0062] The adjusting block 24 is an iron block, and the sliding direction of the adjusting block 24 is controlled by the difference between the magnetic force of the magnetic generator 7 on the adjusting block 24 and the elastic force of the elastic member on the adjusting block 24 .

[0063] By adjusting the magnetic field strength of the magnetic generator 7, the adjustment block 24 is controlled to slide back and forth in coordination with the elastic force of the elastic member, and the transmission plate 23 is deflected back and forth.

[0064] The transmission block 32 is an iron block. The magnetic force generated by the magnetic generator 7 attracts the transmission block 32 to press against the inner wall of the transmission cylinder 13. After the magnetic generator 7 is energized, a magnetic force is generated, which in turn attracts the transmission block 32, so that the transmission block 32 presses against the inner wall of the transmission cylinder 13, so that the control shaft 21 drives the transmission cylinder 13 to rotate.

[0065] When the adding block 14 needs to be put into the liquid nitrogen, the magnetic generator 7 is powered off, at which time the transmission block 32 loses its magnetic attraction and moves away from the transmission cylinder 13, and the adding block 14 slides down due to gravity.

[0066] 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.

[0067] 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.

[0068] 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 deep cryogenic storage method for preserving bone tissue activity, characterized in that: The following steps are involved: A1. Obtain bone tissue samples, place the bone tissue in a sterile balanced salt solution, and rinse with sterile PBS to clean the bone tissue; A2. Prepare cryoprotectant, immerse the bone tissue in the cryoprotectant, and maintain the temperature in an ice water bath; A3. After the cryoprotectant enters the bone tissue, the bone tissue is placed in a pre-freezing device to pre-freeze the bone tissue; A4. Take out the pre-frozen bone tissue and place it in a liquid nitrogen storage container to start low-temperature storage of the bone tissue.

2. A method for deep cryopreservation of bone tissue for preserving its activity according to claim 1, characterized in that: In step A4, detailed steps for placing bone tissue into a liquid nitrogen storage container: B1. Place the bone tissue into the accommodating net bag (4), assemble the accommodating net bag (4) with the closing plate (2), and then assemble the closing plate (2) with the accommodating shell (1); B2, connecting the external pipe (8) to the external liquid nitrogen storage chamber, introducing the liquid nitrogen into the interior of the accommodating inner container (16), and exhausting the air in the accommodating inner container (16) through the control valve (3); B3, when the liquid nitrogen level in the accommodating liner (16) reaches a desired height, controlling the accommodating liner (16) to expand inwards until the liquid nitrogen submerges the bone tissue; B4. When removing bone tissue, first open the control valve (3), then draw out the liquid nitrogen in the accommodating liner (16) through the external pipe (8), and finally separate the sealing plate (2) and the accommodating net bag (4).

3. A method for deep cryopreservation of bone tissue for preserving its activity according to claim 2, characterized in that: The liquid nitrogen storage container in step A4 includes an outer accommodating portion, an inner accommodating portion and a feeding mechanism; The accommodating portion comprises an accommodating shell (1), a closing plate (2) and an accommodating net bag (4); the closing plate (2) is connected to the accommodating shell (1) by means of internal and external threads; the accommodating net bag (4) is used to accommodate bone tissue and is detachably connected to the bottom surface of the closing plate (2); The inner storage portion comprises a heat preservation tube (6), a sealing tube (9) and a storage liner (16); the heat preservation tube (6) is arranged inside the storage shell (1); the sealing tube (9) is arranged inside the heat preservation tube (6); and the storage liner (16) is arranged inside the sealing tube (9); A hollow chamber is reserved between the accommodating inner container (16) and the sealing cylinder (9), and the accommodating inner container (16) is made of elastic material; The feeding mechanism comprises a control valve (3), an external pipe (8) and a nitrogen inlet pipe (12); the control valve (3) is arranged on the upper end surface of the closing plate (2), and one end thereof passes through the closing plate (2) through a guide tube; the external pipe (8) is fixedly mounted on the outer wall of the containing shell (1); one end of the nitrogen inlet pipe (12) passes through the bottom of the closing cylinder (9) and extends to the interior of the containing liner (16); the other end of the nitrogen inlet pipe (12) is connected to the external pipe (8).

4. A method for deep cryopreservation of bone tissue for preserving its activity according to claim 3, characterized in that: A bearing ring (15) is fixedly mounted on the inner wall of the heat-insulating cylinder (6), and the sealing cylinder (9) and the accommodating liner (16) are both fixedly mounted on the bottom of the bearing ring (15); A hard support ring (17) is fixedly mounted on the outer wall of the accommodating inner container (16) on one side close to the sealing cylinder (9); An adapter box (10) is provided between the housing shell (1) and the heat-insulating cylinder (6); an elastic chamber (29) is fixedly installed inside the adapter box (10); a connecting pipe (26) is fixedly installed on the outer wall of the elastic chamber (29); a solenoid valve is provided inside the connecting pipe (26); and one end of the connecting pipe (26) away from the elastic chamber (29) extends to between the sealing cylinder (9) and the housing liner (16).

5. A method for deep cryopreservation of bone tissue for preserving its activity according to claim 4, characterized in that: A temperature control chamber (27) is provided in the adapter box (10), and the nitrogen inlet pipe (12) passes through the temperature control chamber (27); A control cylinder (31) is fixedly mounted on one side of the temperature control chamber (27); a control plug (30) is elastically mounted in the inner cavity of the control cylinder (31); and the inner cavity of the control cylinder (31) is in communication with the temperature control chamber (27); A squeezing rod (28) for squeezing the elastic bin (29) is fixedly mounted on one end of the control plug (30).

6. A method for deep cryopreservation of bone tissue for preserving its activity according to claim 5, characterized in that: A sealing cover (5) is fixedly mounted on the upper end surface of the bearing ring (15), and a mounting frame (11) is fixedly mounted on the upper end surface of the bearing ring (15); a control shaft (21) is rotatably mounted on the side wall of the mounting frame (11), and a transmission cylinder (13) is provided on the outer wall of the control shaft (21); An adding block (14) is provided at the bottom of the bearing ring (15), and an upper end surface of the adding block (14) is connected to the radial outer wall of the transmission cylinder (13) via a rope.

7. A method for deep cryopreservation of bone tissue for retaining activity according to claim 6, characterized in that: A mounting arm (19) is fixedly mounted on the outer wall of the mounting frame (11); a control ratchet (18) is fixedly mounted on the axial end of the control shaft (21); a transmission plate (23) is rotatably mounted on the outer wall of the mounting arm (19); an adjustment rod (22) for engaging with the control ratchet (18) is elastically mounted on the outer wall of the transmission plate (23); and a control ratchet rod (25) for engaging with the control ratchet (18) is elastically mounted on the bottom of the mounting arm (19).

8. A method for deep cryopreservation of bone tissue for retaining activity according to claim 7, characterized in that: A support arm (20) is fixedly mounted on the outer wall of the mounting arm (19), an adjustment block (24) is slidably mounted on the outer wall of the support arm (20), and one end of the adjustment block (24) is connected to the outer wall of the support arm (20) via an elastic member; The outer wall of the transmission plate (23) is provided with a waist-shaped groove, and the adjustment block (24) is slidably matched with the waist-shaped groove via a pin shaft.

9. A method for deep cryopreservation of bone tissue for preserving its activity according to claim 8, characterized in that: A transmission block (32) is elastically mounted on the outer wall of the control shaft (21), a plurality of the transmission blocks (32) are provided, and a plurality of the transmission blocks (32) are evenly arranged in a ring shape along the axis of the control shaft (21); The plurality of transmission blocks (32) have a tendency to move toward the axis of the control shaft (21).

10. A deep cryopreservation method for preserving bone tissue activity according to claim 9, characterized in that: A magnetic generator (7) is fixedly mounted on the outer wall of the heat-insulating cylinder (6); The adjustment block (24) is an iron block, and the sliding direction of the adjustment block (24) is controlled by the difference between the magnetic force of the magnetic generator (7) on the adjustment block (24) and the elastic force of the elastic member on the adjustment block (24); The transmission block (32) is an iron block, and the magnetic force generated by the magnetic generator (7) attracts the transmission block (32) to press against the inner wall of the transmission cylinder (13).

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