CN109221081B - Multi-tissue vitrification in-situ storage device - Google Patents
Multi-tissue vitrification in-situ storage device Download PDFInfo
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- CN109221081B CN109221081B CN201811041589.4A CN201811041589A CN109221081B CN 109221081 B CN109221081 B CN 109221081B CN 201811041589 A CN201811041589 A CN 201811041589A CN 109221081 B CN109221081 B CN 109221081B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
- A01N1/0268—Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen
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Abstract
The invention discloses a multi-tissue vitrification in-situ preservation device which comprises a preservation box, wherein a cover plate is arranged above the preservation box, and a water bath is arranged below the preservation box; the storage box in be equipped with fixed knot structure, storage box top input structure and output structure, the input structure for first injection syringe and second injection syringe, first injection syringe and second injection syringe and storage box between be equipped with the hybrid chamber, fixed knot structure be the carrier support, carrier support top is equipped with the tissue carrier. The carrier supports are arranged to be matched, and the distance between the carrier supports and the tissue can be finely adjusted by rotating the carrier to change the tension of the tissue and ensure that the concentration of the solution around the tissue is uniform and consistent in the perfusion process. Utilize this application to carry out the research of different concentration protective agent to the effect influence is preserved to tissue vitrification, its advantage is in can preserving multiunit sample simultaneously, and can fully guarantee the uniformity of all the other experimental conditions for the experimental result is more accurate reliable.
Description
Technical Field
The invention relates to the field of biological tissue preservation, in particular to a multi-tissue vitrification in-situ preservation device.
Background
The human body needs various tissues in the body to work normally and coordinately when maintaining the health state, and once a certain tissue is diseased, the normal life of people is influenced and even the life is threatened. The blood vessel is used as a pipeline for human blood transportation, and once damaged, the blood vessel seriously endangers the life safety of a patient. The transplantation of blood vessels is an effective means for repairing severely damaged blood vessels, but the source of blood vessels is limited and the development of artificial blood vessels is not mature, so the clinical application of the blood vessel transplantation operation is greatly limited by the supply and demand. The trachea is used as a pipeline connecting the larynx and the bronchus, is not only a passage of air, but also has the effects of defending, removing foreign matters and the like. In patients with severe tracheal disease, a tracheotomy is required, and if the length of the resection exceeds 6cm, a tracheal transplantation is required, wherein preservation of the transplanted trachea is a major problem. The movement of human skeleton depends on the traction of tendon, the injury of tendon will seriously affect the normal movement of limb, the allogeneic tendon transplantation is a method for repairing tendon injury, especially the defect of multiple tendons is ideal, and a key problem to be considered in allogeneic transplantation is how to effectively preserve the transplanted tendon. In storing the above-mentioned tissues, in-situ storage of the tissues is realized in consideration of mechanical properties such as viscoelasticity.
Slow freezing is currently the most common method of biological tissue preservation, enabling the tissue to remain viable after long-term storage. However, this preservation method has many short plates, and slow freezing causes ice crystal formation and growth in solution, which causes cell damage and leads to tissue death by inactivation.
When the vitrification method is used for freezing and preserving living tissues such as blood vessels, large ice crystals which damage organelles are not formed by liquid inside and outside cells, and the cell structure is not damaged, so the vitrification method is mostly adopted for preserving the biological tissues in tissue engineering. However, the vitrifaction preservation requires the addition of a high-concentration protective agent, which is likely to damage tissues by a solution due to the excessive concentration of the surrounding protective agent, so that the protective agent needs to be added step by step, but the step-by-step addition process is complex and tedious, and the removal process of the protective agent also causes inconvenience to experimenters.
In addition, the existing way for preserving large-size tissues such as blood vessels is to put the treated tissues into a low-temperature cryopreservation tube or a cryopreservation bag, so that the tissues cannot maintain pretightening force, and the tissues are curled and bent after being frozen, thereby causing the problems of the damage of tissue structure, mechanics and biological characteristics and the like. In addition, the existing preservation technology adopts a method of gradually adding a protective agent, the temperature reduction process is program temperature reduction, the duration is long, the operation is complicated, and the preservation effect of the tissue is seriously influenced.
Disclosure of Invention
The invention aims to solve the technical problem that the existing mode for preserving large-size tissues such as blood vessels is to treat the tissues and put the tissues into a low-temperature cryopreservation tube or a cryopreservation bag, the tissues cannot maintain pretightening force, and the tissues are curled and bent after being frozen, so that the problems of damage to tissue structures, mechanics and biological characteristics and the like are caused. In addition, the existing preservation technology adopts a method of gradually adding a protective agent, the temperature reduction process is program temperature reduction, the duration is long, the operation is complicated, and the preservation effect of the tissue is seriously influenced.
In order to solve the technical problems, the invention adopts the following technical means:
a multi-tissue vitrification in-situ preservation device comprises a preservation box, wherein a cover plate is arranged above the preservation box, and a water bath is arranged below the preservation box; the storage box in be equipped with fixed knot structure, storage box top input structure and output structure, the input structure for first injection syringe and second injection syringe, first injection syringe and second injection syringe and storage box between be equipped with the hybrid chamber, fixed knot structure be the carrier support, carrier support top is equipped with the tissue carrier. Through setting up the carrier support, make it cooperate to the distance between the two is finely tuned to the rotatory carrier of accessible, with the tension that changes the tissue, through setting up the pre-mixing effect to solution through the hybrid chamber, make the solution concentration that gets into in the storage box even unanimous, thereby guarantee that the perfusion in-process organizes the even unanimity of solution concentration all around.
Preferably, the further technical scheme of the invention is as follows:
one side of the tissue carrier is provided with a rotary screw thread, and the rotary screw thread is provided with a rotary screw cap matched with the rotary screw thread in size. The complete sealing of the device can be achieved by rotating the thread which cooperates with a corresponding nut.
The tissue carrier is a through carrier, the through carrier is provided with a through outer ring and a through inner ring, and a through cavity is arranged in a circular ring of the through inner ring. The hollow tissue carrier is used for bearing blood vessels and air pipes, and protective agents can be filled into the blood vessels and the air pipes.
The tissue carrier is a clamp type carrier, and the clamp type carrier is provided with an inlay. The clamp type tissue carrier is used for clamping tendon.
The cover plate and the preservation box are provided with a sealing structure, the sealing structure is a groove arranged on the cover plate and on the periphery of the preservation box, the groove is matched with the groove on the cover plate.
The input structure for set up the protective agent entry above the apron, the protective agent entry passes through hose connection mixing chamber one end, the other end of mixing chamber passes through hose connection first injection syringe and second injection syringe, first injection syringe and second injection syringe are equipped with the syringe pump respectively. The first injection syringe and the second injection syringe are respectively added with a protective agent and a culture medium, and injection speeds are respectively set, so that a solution with a certain concentration can be obtained, and solutions with different concentrations can be obtained by changing the injection speeds.
The output structure is a protective agent outlet above the cover plate, and the protective agent outlet is connected with an extraction injector through a hose.
The bottom of the water bath is distributed with a fret-shaped water bath pipeline, one end of the fret-shaped water bath pipeline is provided with a water bath pipeline inlet, and the other end of the fret-shaped water bath pipeline is provided with a water bath pipeline outlet. Realize the circulation flow of the liquid in the whole water bath pipeline so as to pre-cool the preservation box and maintain the optimal temperature in the adding process of the protective agent
The materials of the preservation box, the water bath and the cover plate are silicon glass or polydimethylsiloxane or polyimide or polymethyl methacrylate or parylene or polytetrafluoroethylene, and the material of the tissue carrier is polytetrafluoroethylene which has good biocompatibility.
The use method of the multi-tissue vitrification in-situ preservation device comprises a tissue preservation method and a tissue taking-out method, wherein the tissue preservation method comprises the steps of selecting a tissue carrier, cooling a preservation box, adjusting the distance between the carriers, injecting and extracting a protective agent and a culture medium, and sealing the preservation box, and the tissue taking-out method comprises the steps of unfreezing the preservation box, extracting the protective agent and taking out the preserved tissue, and is characterized by comprising the following specific steps:
step 1: selecting a tissue carrier, and selecting a corresponding through carrier or a clamp carrier to be arranged on a carrier bracket in the storage box according to whether the stored tissue is a blood vessel or a trachea or a tendon;
step 2: cooling the preservation box, introducing liquid with a certain temperature into a water bath pipeline inlet of the fret-shaped water bath pipeline, and extracting at the same speed at a water bath pipeline outlet of the fret-shaped water bath pipeline to pre-cool the preservation box;
and step 3: adjusting the distance between the carriers, rotating the tissue carriers to adjust the distance between the carriers, wherein the distance is equal to the length of the tissue to be preserved before being cut off, so that the tissue maintains pretightening force during vitrification preservation, and the in-situ preservation of the tissue is realized;
and 4, step 4: injecting and extracting a protective agent and a culture medium, wherein a first injection injector, a second injection injector and an extraction injector are respectively controlled by different injection pumps to control the injection speed and the extraction speed, the protective agent and the culture medium are respectively added into the first injection injector and the second injection injector, the injection pumps are started to continuously inject the protective agent, the first injection injector and the second injection injector are firstly injected into a mixing cavity, and the mixing cavity performs pre-mixing action on the solution, so that the concentration of the solution entering a storage box is uniform and consistent, and the concentration of the solution around tissues in the perfusion process is ensured to be uniform and consistent;
and 5: sealing the preservation box, sealing the protective agent inlet and outlet on the cover plate by using a screw cap after the protective agent is added, and putting the preservation box into liquid nitrogen for freezing preservation;
step 6: the preservation box is unfrozen, the preservation box is taken out of liquid nitrogen and placed into the water bath, a water bath pipeline inlet is formed in one end of the fret-shaped water bath pipeline, liquid with a certain temperature is introduced into the water bath pipeline inlet, a water bath pipeline outlet is formed in the other end of the fret-shaped water bath pipeline, and the water bath pipeline outlet is used for extracting the liquid at the same speed;
and 7: extracting the protective agent, taking down screw caps on an inlet and an outlet of the protective agent, respectively connecting a first injection syringe, a second injection syringe and an extraction syringe with an inlet and an outlet of the protective agent, and setting parameters of an injection pump so that the injection speed and the extraction speed are the same;
and 8: the preserved tissue is removed, the cover plate is removed, and the preserved tissue is removed from the tissue carrier for use.
In the tissue vitrification in-situ preservation process, the tissue carrier can keep the original geometric shape and certain tension of the tissue during vitrification, and the tissue is prevented from being damaged in mechanical property and function; according to the embodiment of the application, the protective agent is added and removed by adopting a continuous perfusion method, and the concentration of the protective agent is uniformly increased and reduced, so that the solution damage of tissues caused by the fact that the concentration of the surrounding solution changes too fast is avoided; the water bath pipeline designed in the embodiment of the application is distributed in the water bath in a meandering shape, and can fully ensure the temperature constancy in the adding process of the protective agent and the temperature uniformity of the preservation box.
Furthermore, the tissue carrier in the embodiment of the present application is divided into two types, namely, a through type and a clamp type, which can be respectively used for bearing the blood vessel, the trachea and the tendon, and the through type carrier can perfuse a protective agent into the blood vessel or the trachea; the other end of the two carriers is provided with a thread, and the distance between the carriers can be adjusted according to the requirement to change the tension of the tissue.
Drawings
FIG. 1 is a schematic diagram of an overall system architecture provided by an embodiment of the present invention;
FIG. 2 is a schematic structural view of an integrated multi-tissue vitrification in-situ preservation device provided in an embodiment of the present invention;
FIG. 3 is a schematic diagram of a cover plate according to an embodiment of the present invention;
FIG. 4 is a schematic structural diagram of a storage case according to an embodiment of the present invention;
FIG. 5 is a schematic view of a through-type tissue carrier according to an embodiment of the present invention;
FIG. 6 is a schematic view of a clamp-type tissue carrier according to an embodiment of the present invention;
FIG. 7 is a schematic diagram of a water bath configuration provided by an embodiment of the present invention;
FIG. 8 is a block diagram of a method of use of the present invention.
Description of reference numerals: 1-cover plate; 2-preservation box; 3-water bath; 4-a through-type carrier; 5-clamp type carrier; 6-first injection syringe; 7-a second injection syringe; 8-extracting the syringe; 9-a mixing chamber; 10-protectant inlet; 11-a protectant outlet; 12-a syringe pump; 13-a tongue; 14-a groove; 15-a water bath pipeline in a shape of a Chinese character hui; 16-water bath pipeline inlet; 17-outlet of water bath pipeline; 18-a carrier support; 19-a hollow cavity; 20-rotating the thread; 21-a central through outer ring; 22-inlay.
Detailed Description
The present invention will be further described with reference to the following examples.
Specific example 1:
referring to fig. 1, 3 and 7, the multi-tissue vitrification in-situ preservation device comprises a preservation box 2, wherein a cover plate 1 is arranged above the preservation box 2, and a water bath 3 is arranged below the preservation box 2; the storage box 2 is internally provided with a fixed structure, an input structure and an output structure are arranged above the storage box 2, the input structure is a first injection injector 6 and a second injection injector 7, a mixing chamber 9 is arranged between the first injection injector 6 and the second injection injector 7 and the storage box 2, the fixed structure is a carrier support 18, a tissue carrier is arranged above the carrier support 18, a sealing structure is arranged between the cover plate 1 and the storage box 2, the sealing structure is that a convex groove 13 is arranged on the cover plate 1, a groove 14 which is matched with the convex groove 13 and is arranged on the cover plate 1 and is arranged around the storage box 2 is arranged on the cover plate 1, the input structure is a protective agent inlet 10 arranged above the cover plate 1, the protective agent inlet 10 is connected with one end of the mixing chamber 9 through a hose, the other end of the mixing chamber 9 is connected with the first injection injector 6 and the second injection injector 7 through hoses, the first injection injector 6 and the second injector 7 are, the output structure is a protective agent outlet 11 above the cover plate 1, the protective agent outlet 11 is connected with an extraction injector 8 through a hose, the bottom of the water bath 3 is distributed with a fret-shaped water bath pipeline 15, one end of the fret-shaped water bath pipeline 15 is provided with a water bath pipeline inlet 16, the other end of the fret-shaped water bath pipeline 15 is provided with a water bath pipeline outlet 17, the storage box 2, the water bath 3 and the cover plate 1 are made of silicon glass, polydimethylsiloxane, polyimide, polymethyl methacrylate, parylene C or polytetrafluoroethylene, and the tissue carrier is made of polytetrafluoroethylene and has good biocompatibility.
Referring to fig. 2, 4 and 5, the multi-tissue vitrification in-situ preservation device of the present invention is shown, wherein a rotary screw 20 is provided on one side of the tissue carrier, a rotary nut with a size matching the rotary screw 20 is provided on the rotary screw, the tissue carrier is a through carrier 4, the through carrier is provided with a through outer ring 21 and a through inner ring, and a through cavity 19 is provided in the circular ring of the through inner ring.
Referring to fig. 2, 4 and 6, the multi-tissue vitrification in-situ preservation device of the present invention is shown, wherein a rotary screw 20 is disposed on one side of a tissue carrier, a rotary nut with a size matching the rotary screw 20 is disposed on the rotary screw 20, the tissue carrier is a clamp-type carrier 5, and the clamp-type carrier 5 is disposed with an inlay 22.
Specific example 2:
referring to fig. 8, the application method of the multi-tissue vitrification in-situ preservation device of the invention comprises a tissue preservation method and a tissue taking-out method, wherein the tissue preservation method comprises the steps of selecting a tissue carrier, cooling the preservation box 2, adjusting the distance between the carriers, injecting and extracting a protective agent and a culture medium, and sealing the preservation box 2, and the tissue taking-out method comprises the steps of unfreezing the preservation box 2, extracting the protective agent and taking out the preserved tissue, and is characterized by comprising the following specific steps:
step 1: selecting a tissue carrier, and selecting a corresponding through carrier 4 or clamp carrier 5 to be arranged on a carrier bracket 18 in the storage box 2 according to whether the stored tissue is a blood vessel or a trachea or a tendon;
step 2: cooling the preservation box 2, introducing liquid with a certain temperature into a water bath pipeline inlet 16 of the meander-shaped water bath pipeline 15, and extracting at the same speed at a water bath pipeline outlet 17 of the meander-shaped water bath pipeline 15 to pre-cool the preservation box 2;
and step 3: adjusting the distance between the carriers, rotating the tissue carriers to adjust the distance between the carriers, wherein the distance is equal to the length of the tissue to be preserved before being cut off, so that the tissue maintains pretightening force during vitrification preservation, and the in-situ preservation of the tissue is realized;
and 4, step 4: injecting and extracting a protective agent and a culture medium, wherein the injection speed and the extraction speed of the first injection injector 6, the second injection injector 7 and the extraction injector 8 are respectively controlled by different injection pumps 12, the protective agent and the culture medium are respectively added into the first injection injector 6 and the second injection injector 7, the injection pumps 12 are started to continuously inject the protective agent, the first injection injector 6 and the second injection injector 7 are firstly injected into the mixing cavity 9, and the mixing cavity 9 performs pre-mixing action on the solution, so that the concentration of the solution entering the preservation box 2 is uniform and consistent, and the concentration of the solution around the tissue is ensured to be uniform and consistent in the perfusion process;
and 5: sealing the preservation box 2, sealing the protective agent inlet and outlet on the cover plate 1 by using a screw cap after the protective agent is added, and putting the preservation box 2 into liquid nitrogen for freezing preservation;
step 6: the preservation box 2 is unfrozen, the preservation box 2 is taken out of liquid nitrogen and is placed in the water bath 3, a water bath pipeline inlet 16 is arranged at one end of the fret-shaped water bath pipeline 15, liquid with a certain temperature is introduced into the water bath pipeline inlet 16, a water bath pipeline outlet 17 is arranged at the other end of the fret-shaped water bath pipeline 15, and the water bath pipeline outlet 17 performs extraction at the same speed;
and 7: extracting the protective agent, taking down screw caps on an inlet and an outlet of the protective agent, respectively connecting a first injection syringe 6, a second injection syringe 7 and an extraction syringe 8 with an inlet 10 and an outlet of the protective agent, and setting parameters of an injection pump 12 to ensure that the injection speed and the extraction speed are the same;
and 8: the preserved tissue is removed, the cover plate 1 is removed, and the preserved tissue is removed from the tissue carrier for use.
Specific example 3:
FIG. 1 is a schematic structural diagram of a multi-tissue vitrification in-situ preservation system provided by an embodiment of the present invention. As shown in fig. 1, the embodiment of the invention discloses a multi-tissue vitrification in-situ storage device, which comprises a cover plate 1, a storage box 2, a water bath 3, a tissue carrier, two injection syringes with respective controlled speeds, an extraction syringe 8 and a mixing chamber 9; after the injection injectors 6 and 7 are connected in parallel, the injection injectors are connected with a protective agent inlet 10; the extraction syringe 8 is connected with the protective agent outlet 11; a mixing chamber 9 is connected between the injection syringe and the protective agent inlet 10.
The tissue to be preserved is a tendon.
The present examples first prepare the medium and protective agent solution for the vitrification preservation of tendon according to the conventional experimental method existing in the prior art.
Because the permeability of the cell membrane of the cell to the protective agent is different at different temperatures, a certain temperature needs to be maintained in the protective agent adding process in order to ensure the optimal effect of the protective agent. And placing the prepared culture medium and the protective agent solution in a refrigerator at 4 ℃ for pre-cooling, introducing water at 4 ℃ from an inlet of the water bath pipeline, and extracting at the same speed from an outlet of the water bath pipeline.
In vitro tendon will shrink and curl, and the tendon will be damaged in histology, biomechanics and the like after being frozen and stored in this state. And the tendon is a ribbon structure. Therefore, in this embodiment, we select the clamp-type tissue carrier to carry the tendon, so as to maintain the pre-tightening force of the tendon and achieve the in-situ preservation of the tendon.
In the addition process of the protective agent, the concentration of the protective agent needs to be gradually increased in order to prevent the cells from being damaged by the solution due to the too fast change of the concentration of the solution. The programmable injection pump 12 is used for respectively controlling the injection speed of the protective agent and the injection speed of the culture medium, so that the concentration of the protective agent can be accurately controlled. In addition, the culture medium and the protective agent are injected into the mixing cavity 9, so that the solution can be mixed in advance, and the uniform concentration of the solution around the tendon in the perfusion process is ensured. Specifically, in this embodiment, the cover plate 1 is covered, two injection syringes with respective speeds controlled by the injection pump 12 are connected in parallel and then connected to the mixing chamber 9, the two injection syringes are pre-filled with the prepared culture medium and protective agent, the other end of the mixing chamber 9 is connected to the protective agent inlet 10 of the cover plate 1, and the extraction syringe 8 is connected to the protective agent outlet 11. The speed control of the injector by the injection pump 12 can be realized by software programming, so that the respective injection speeds of the two injection injectors can be accurately controlled, different ratios of solution concentrations can be realized, and the total injection speed and the extraction speed can be accurately controlled to be always consistent, thereby ensuring the balance of the solution pressure in the preservation box 2.
In order to prevent the tissue from being infected due to the contact with the external environment and ensure that the protective agent cannot leak in the preservation process, after the protective agent is added, the protective agent inlet and outlet are sealed by a screw cap, and then the preservation box 2 is placed in liquid nitrogen for preservation.
After being stored for 24 hours or even longer, the embodiment of the application carries out the rewarming and protective agent removing processes:
successful vitrification cryopreservation should avoid devitrification during the rewarming process in addition to avoiding solidification during the cooling down process, so that devitrification is avoided during the heating process at a sufficiently fast heating rate. In this application, the water bath pipeline is the fret shape, can guarantee 2 thermally equivalent of save box, the quick rise of temperature. Specifically, the preservation box 2 is taken out of liquid nitrogen and placed in a water bath 3, and water with the temperature of 37 ℃ is introduced from an inlet of a water bath pipeline to rewarm the device.
The concentration of the protectant surrounding the tissue is gradually increased as the protectant is added to prevent damage to the tissue from the surrounding solution due to too rapid a change in concentration of the surrounding solution, and likewise, the protectant is gradually decreased as the protectant is removed. Therefore, after the rewarming is finished, the screw cap on the protective agent inlet and outlet is unscrewed, and the two ends of the protective agent inlet and outlet are respectively connected with the injection syringe and the extraction syringe 8, wherein the injection syringe is controlled by the injection pump 12, the culture medium is filled in the injection syringe, and the parameters of the injection pump 12 are set so that the injection speed and the extraction speed are equal.
After the protective agent is removed, the cover plate 1 is opened, and the tendons which are subjected to vitrification preservation are taken down from the tissue carrier for use.
Specific example 4:
the structure of the multiple-tissue vitrification in-situ preservation device is shown in figures 1-7. This embodiment is similar to embodiment 1, except that the tissue stored in this embodiment is a blood vessel.
The embodiment of the application firstly carries out the freezing process:
the culture medium and the protective agent solution for vitrification preservation of blood vessels are prepared according to conventional experimental methods known in the art.
And placing the prepared culture medium and the protective agent into a refrigerator at the temperature of-4 ℃ for pre-cooling. Ethanol at-4 ℃ is introduced from the inlet of the water bath pipeline, and the ethanol is extracted from the outlet at the same speed.
Because the blood vessel in the isolated state can shrink and curl, the mechanical properties of the blood vessel such as viscoelasticity and the like are damaged after the blood vessel is frozen and stored in the isolated state. In order to prevent the blood vessel from shrinking and adhering, a protective agent needs to be infused into the blood vessel to maintain a certain positive pressure in the blood vessel. Therefore, in the embodiment, the through-type tissue carrier is selected to bear the blood vessel, so that the protective agent can be filled into the blood vessel, the pretightening force of the blood vessel can be maintained, and the in-situ preservation of the blood vessel is realized.
Covering the cover plate 1, connecting two injection syringes with the speeds respectively controlled by the injection pump 12 in parallel and then connecting the two injection syringes with the mixing chamber 9, wherein the prepared culture medium and the protective agent are respectively pre-filled in the two injection syringes, the other end of the mixing chamber 9 is connected with a protective agent inlet 10 on the cover plate 1, and the extraction syringe 8 is connected with a protective agent outlet 11. The parameters of the injection pump 12 are set to realize different ratios of solution concentration, and the total injection speed and the extraction speed are controlled to be always kept consistent, so that the balance of the solution pressure in the preservation box 2 is ensured.
After the protective agent is added, the protective agent inlet and outlet are sealed by nuts, and then the preservation box 2 is placed in liquid nitrogen for preservation.
After being stored for 24 hours or even longer, the embodiment of the application carries out the rewarming and protective agent removing processes:
and taking the preservation box 2 out of the liquid nitrogen and putting the preservation box into a water bath 3, and introducing water with the temperature of 37 ℃ from an inlet of the water bath pipeline to rewarm the device.
Screwing off the screw cap on the protective agent inlet and outlet, and connecting the injection syringe and the extraction syringe 8 with the speed controlled by the injection pump 12 at two ends respectively, wherein the injection syringe is filled with the culture medium, and the parameters of the injection pump 12 are set so that the injection speed and the extraction speed are equal.
After the protective agent is removed, the cover plate 1 is opened, and the blood vessel which is subjected to vitrification preservation is taken down from the tissue carrier for use.
Specific example 5:
the embodiment of the application is used for researching the influence of different protective agent concentrations on the preservation effect.
Specifically, three storage cases 2 are placed side by side in a water bath 3 as shown in fig. 1 and 2, with a blood vessel as a subject of study.
And (3) introducing ethanol with the temperature of-4 ℃ from a water bath pipeline inlet 16 to pre-cool the storage boxes 2, installing the three groups of hollow tissue carriers in the three storage boxes 2, and fixing the three groups of blood vessels.
Covering the cover plate 1, wherein protective agent inlets 10 on the three cover plates 1 are respectively connected with three groups of injection syringes, correspondingly, three protective agent outlets 11 are respectively connected with three extraction syringes 8, the injection and extraction speeds of the injection syringes and the extraction syringes 8 are controlled by an injection pump 12, and the injection speed and the extraction speed of each group are kept consistent; the concentration of the protective agent in each group of injection syringes was different to study the effect of different concentrations of protective agent on preservation.
After the protective agent is added, the protective agent inlet and outlet are sealed by nuts, and the three groups of storage boxes 2 are simultaneously placed in liquid nitrogen for storage.
After 24 hours of preservation, the three groups of preservation boxes 2 are taken out and put into a water bath 3 for rewarming.
After the temperature recovery is finished, the screw caps on the protective agent inlet and outlet are unscrewed, the protective agent inlet and outlet on each group of cover plates 1 are respectively connected with an injection injector and an extraction injector 8, the speed of the injection injector is controlled by an injection pump 12, a culture medium is filled in the injection injector, and the parameters of the injection pump 12 are set so that the injection speed and the extraction speed are equal.
After the protective agent is removed, the cover plate 1 is opened, and the blood vessel which is subjected to vitrification preservation is taken down from the tissue carrier. And respectively detecting the performances of cell activity, viscoelasticity and the like of each group of blood vessels so as to obtain the optimal protective agent concentration for the vitrification preservation of the blood vessels.
Utilize this application to carry out the research of different concentration protective agent to the effect influence is preserved to tissue vitrification, its advantage is in can preserving multiunit sample simultaneously, and can fully guarantee the uniformity of all the other experimental conditions for the experimental result is more accurate reliable.
Since the above description is only a specific embodiment of the present invention, but the protection of the present invention is not limited thereto, any equivalent changes or substitutions of the technical features of the present invention which can be conceived by those skilled in the art are included in the protection scope of the present invention.
Claims (8)
1. A multi-tissue vitrification in-situ preservation device comprises a preservation box, wherein a cover plate is arranged above the preservation box, and a water bath is arranged below the preservation box; the method is characterized in that: preserve the box in be equipped with fixed knot structure, preserve box top input structure and output structure, the input structure for first injection syringe and second injection syringe, first injection syringe and second injection syringe and preserve and be equipped with the hybrid chamber between the box, fixed knot structure be the carrier support, carrier support top is equipped with the tissue carrier, the tissue carrier be well logical type carrier, well logical carrier is equipped with well logical outer ring and well logical inner ring, is equipped with well logical cavity in the ring of well logical inner ring, the tissue carrier be clamp type carrier, clamp type carrier is equipped with the inlay.
2. The multi-tissue vitrification in-situ preservation device according to claim 1, wherein: one side of the tissue carrier is provided with a rotary screw thread, and the rotary screw thread is provided with a rotary screw cap matched with the rotary screw thread in size.
3. The multi-tissue vitrification in-situ preservation device according to claim 1, wherein: the cover plate and the preservation box are provided with a sealing structure, the sealing structure is a groove arranged on the cover plate and on the periphery of the preservation box, the groove is matched with the groove on the cover plate.
4. The multi-tissue vitrification in-situ preservation device according to claim 1, wherein: the input structure for set up the protective agent entry above the apron, the protective agent entry passes through hose connection mixing chamber one end, the other end of mixing chamber passes through hose connection first injection syringe and second injection syringe, first injection syringe and second injection syringe are equipped with the syringe pump respectively.
5. The multi-tissue vitrification in-situ preservation device according to claim 1, wherein: the output structure is a protective agent outlet above the cover plate, and the protective agent outlet is connected with an extraction injector through a hose.
6. The multi-tissue vitrification in-situ preservation device according to claim 1, wherein: the bottom of the water bath is distributed with a fret-shaped water bath pipeline, one end of the fret-shaped water bath pipeline is provided with a water bath pipeline inlet, and the other end of the fret-shaped water bath pipeline is provided with a water bath pipeline outlet.
7. The multi-tissue vitrification in-situ preservation device according to claim 1, wherein: the materials of the preservation box, the water bath and the cover plate are silicon glass or polydimethylsiloxane or polyimide or polymethyl methacrylate or parylene or polytetrafluoroethylene, and the material of the tissue carrier is polytetrafluoroethylene which has good biocompatibility.
8. The use method of the multi-tissue vitrification in-situ preservation device according to claim 1, which consists of a tissue preservation method and a tissue taking method, wherein the tissue preservation method comprises the steps of selecting a tissue carrier, cooling a preservation box, adjusting the distance between the carriers, injecting and extracting a protective agent and a culture medium, sealing the preservation box, and the tissue taking method comprises the steps of unfreezing the preservation box, extracting the protective agent and taking out preserved tissues, and is characterized in that the method comprises the following specific steps:
step 1: selecting a tissue carrier, and selecting a corresponding through carrier or a clamp carrier to be arranged on a carrier bracket in the storage box according to whether the stored tissue is a blood vessel or a trachea or a tendon;
step 2: cooling the preservation box, introducing liquid with a certain temperature into a water bath pipeline inlet of the fret-shaped water bath pipeline, and extracting at the same speed at a water bath pipeline outlet of the fret-shaped water bath pipeline to pre-cool the preservation box;
and step 3: adjusting the distance between the carriers, rotating the tissue carriers to adjust the distance between the carriers, wherein the distance is equal to the length of the tissue to be preserved before being cut off, so that the tissue maintains pretightening force during vitrification preservation, and the in-situ preservation of the tissue is realized;
and 4, step 4: injecting and extracting a protective agent and a culture medium, wherein a first injection injector, a second injection injector and an extraction injector are respectively controlled by different injection pumps to control the injection speed and the extraction speed, the protective agent and the culture medium are respectively added into the first injection injector and the second injection injector, the injection pumps are started to continuously inject the protective agent, the first injection injector and the second injection injector are firstly injected into a mixing cavity, and the mixing cavity performs pre-mixing action on the solution, so that the concentration of the solution entering a storage box is uniform and consistent, and the concentration of the solution around tissues in the perfusion process is ensured to be uniform and consistent;
and 5: sealing the preservation box, sealing the protective agent inlet and outlet on the cover plate by using a screw cap after the protective agent is added, and putting the preservation box into liquid nitrogen for freezing preservation;
step 6: the preservation box is unfrozen, the preservation box is taken out of liquid nitrogen and placed into the water bath, a water bath pipeline inlet is formed in one end of the fret-shaped water bath pipeline, liquid with a certain temperature is introduced into the water bath pipeline inlet, a water bath pipeline outlet is formed in the other end of the fret-shaped water bath pipeline, and the water bath pipeline outlet is used for extracting the liquid at the same speed;
and 7: extracting the protective agent, taking down screw caps on an inlet and an outlet of the protective agent, respectively connecting a first injection syringe, a second injection syringe and an extraction syringe with an inlet and an outlet of the protective agent, and setting parameters of an injection pump so that the injection speed and the extraction speed are the same;
and 8: the preserved tissue is removed, the cover plate is removed, and the preserved tissue is removed from the tissue carrier for use.
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| US8961904B2 (en) | 2013-07-16 | 2015-02-24 | Premium Genetics (Uk) Ltd. | Microfluidic chip |
| US11796449B2 (en) | 2013-10-30 | 2023-10-24 | Abs Global, Inc. | Microfluidic system and method with focused energy apparatus |
| US10180388B2 (en) | 2015-02-19 | 2019-01-15 | 1087 Systems, Inc. | Scanning infrared measurement system |
| CN109892321A (en) * | 2019-04-17 | 2019-06-18 | 中国科学技术大学 | A kind of method of large volume biological sample cryo-conservation |
| CN117413819A (en) * | 2019-04-18 | 2024-01-19 | 艾步思国际有限责任公司 | System and process for continuous addition of cryoprotectant |
| US11628439B2 (en) | 2020-01-13 | 2023-04-18 | Abs Global, Inc. | Single-sheath microfluidic chip |
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