Device and method of perfusion for cryopreservation of biological tissues

Abstract

The present invention relates to the technical field of biological specimen preservation, in particular to an automatic perfusion device and perfusion method for cryopreservation of biological tissues. The device comprises a liquid supply module, a perfusion module and a control module, the liquid supply module being connected to the perfusion module by means of a liquid pipeline, and the control module controlling the liquid supply proportion and the liquid supply rate of the liquid supply module. The operation is simple, and after a perfusion or resuscitation operation on any biological specimen is completed, an operator only needs to replace a specimen carrier plate or an external carrying container with a new one, and connects the new specimen carrier plate or the new external carrying container, so as to start a new round of operations. In addition, during the perfusion or resuscitation operation, a data processing module achieves automation control of a temperature source and a peristaltic pump on the basis of a temperature-time working curve, a perfusate flow velocity-time working curve and a perfusate component-time working curve, thus maintaining the continuity and consistency of perfusion and resuscitation operations on biological specimens, and eliminating the effect of human factors on operation results.

Description

A biological tissue cryopreservation perfusion device and perfusion method Technical Field

[0001] The present invention relates to the technical field of biological sample preservation, in particular to an automatic perfusion device and perfusion method for low-temperature preservation of biological tissues. Background Art

[0002] Vitrification, initially successfully used to preserve mouse embryos, involves transforming a substance into a glassy, ​​amorphous form. The molecular motion energy of vitrified products is extremely low, with only smaller units, such as polymer side groups and side chains, able to move. The molecular chains and segments are essentially frozen, exhibiting mechanical properties similar to those of glass. This state, known as the glassy state, is a transition between liquid and solid. During this process, no ice crystals are formed, the molecules do not rearrange, and no violent motion occurs. The physical properties of the glassy state make vitrification a method that can mitigate damage from freezing cells and tissues and enable long-term preservation. Achieving vitrification requires a sufficiently rapid cooling rate. When the temperature drops, the potential barrier becomes sufficiently large, and the viscosity rises to 1014 Pas. At this point, the molecular conformation is frozen, and diffusion-controlled exchanges and chemical reactions with the environment are kinetically hindered. Since most protein degradation pathways are due to molecular mobility, vitrified systems possess excellent structural and chemical stability.

[0003] There are still great difficulties in the vitrification preservation of biological tissues, mainly because the structure of tissues and organs is complex, the connections between cells are diverse, different structures have different requirements for cooling rates, and the penetration of protective agents is also difficult.

[0004] Under low-temperature conditions, the metabolic capacity of living cells decreases significantly, allowing cells and tissues to be preserved for long periods of time. However, direct freezing without protection is lethal to most organisms because the formation of intracellular and extracellular ice crystals leads to changes in the cellular environment and mechanical damage to the cells. Cell damage under rapid cooling rates is attributed to the formation of intracellular ice crystals, while cell damage under slow cooling is caused by the mechanical interaction between intracellular and extracellular ice crystals. The addition of cryoprotectants (CPAs) during the freezing process regulates water transport and inhibits the growth rate of ice crystals, thereby effectively reducing the risk of cell damage and osmotic stress during freezing, improving cryopreservation results, and enabling the long-term preservation of cells, tissues, and organs.

[0005] Cryoprotectants CPAs are essential in the freezing process. CPAs can prevent freezing damage in many ways, such as reducing the solution effect. CPAs are divided into: permeable and non-permeable CPAs. The working principle of permeable CPAs is that the protective agent can penetrate into the interior of the cell, balance the osmotic pressure inside and outside the cell, avoid excessive shrinkage of the cell, and reduce the formation of ice crystals in the cell. Dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, etc. are all commonly used permeable cryoprotectants. Non-permeable CPAs cannot pass through the cell membrane, but can dehydrate the cell by changing the osmotic pressure and reduce the formation of ice crystals in the cell. Non-permeable CPAs include two groups of substances: oligosaccharides (sucrose and trehalose are the most commonly used) and high molecular weight compounds (albumin, polyvinyl pyrrolidone, etc.).

[0006] During the vitrification process, the interaction between the protective agent and the frozen tissue cells determines the formation of ice crystals in the cells. Therefore, the perfusion method of the freezing fluid in the tissue is also an important factor affecting vitrification.

[0007] Currently, during vitrification, the perfusion of tissues or cells with cryoprotectant reagents, as well as the removal of protective agents during tissue or cell freezing and thawing, are all performed manually by operators. One of the drawbacks of manual operation is that the operator's workload must be considered. The concentration range of cryoprotectant solutions is limited, that is, during tissue perfusion, it is impossible to set too many cryoprotectant concentrations. This results in discontinuous contact between the cells of the frozen tissue and the cryoprotectant of different concentrations, which can easily lead to excessive cell shrinkage. Secondly, the time interval between two different concentrations of cryoprotectant solution can also lead to improper cell shrinkage. How to effectively improve the interaction between cryoprotectant and tissue cells, enhance the biological activity of biological tissues during cryopreservation and after thawing, and improve the perfusion method of tissue cells is desirable.

[0008] Summary of the Invention

[0009] The present invention provides a device and method for low-temperature preservation perfusion of biological tissue.

[0010] Firstly,

[0011] The present invention provides an automated biological tissue cryopreservation perfusion device, which includes a liquid supply module, a perfusion module and a control module. The liquid supply module is connected to the perfusion module via a liquid pipeline, and the control module controls the liquid supply ratio and liquid supply rate of the liquid supply module and the temperature regulation in the perfusion module.

[0012] Liquid supply module:

[0013] The liquid supply module includes a reagent container and a peristaltic pump disposed at an opening of the reagent container and connected to the reagent container via a pipeline. The reagent container contains a liquid, which includes a biological tissue preservation solution and a biological tissue cryoprotectant solution. The biological tissue preservation solution can be tissue fluid in the in vivo environment of the biological tissue, or a liquid similar to the tissue fluid in the in vivo environment, to maintain the structural integrity and functional activity of the biological tissue.

[0014] The peristaltic pump is used to transport the liquid in the reagent container into the pipeline, and then mix the liquid through the pipeline and transport it into the perfusion module.

[0015] The number of the peristaltic pumps matches the number of the reagent containers, and the number of the reagent containers is determined according to the type of liquid supply. When the liquid supply types are divided into tissue fluid and freezing fluid, there are preferably two reagent containers, one of which contains tissue fluid and the other contains cryoprotective fluid; when the liquid supply types are classified according to the components of tissue fluid and cryoprotective fluid, the number of containers set is consistent with the number of components.

[0016] After the peristaltic pump extracts the corresponding reagents from the liquid supply container, the reagents are merged into the mixing tube, mixed in the mixing tube, and the evenly mixed liquid is transported to the perfusion module via the pipeline.

[0017] Furthermore, a mixing assembly may be provided downstream of the mixing tube to further mix the mixed liquid.

[0018] In a specific embodiment of the present invention, a pipeline branch is provided downstream of the mixing tube as a detection pipeline, and the detection pipeline is connected to an analysis device for analyzing whether the concentration and ratio of each component in the mixed solution meet the requirements of the control scheme.

[0019] The perfusion module includes a temperature control plate and a flow channel plate that is pressed together with the temperature control plate to form a sealed cavity. A sample loading plate is disposed within the sealed cavity. The sample loading plate is provided with a plurality of sample loading spaces for the biological sample, with adjacent sample loading spaces connected by flow channels. After the mixed solution enters the sample loading spaces, it permeates into the biological sample, displacing the tissue fluid and / or protective fluid in the biological sample according to a concentration gradient. The displaced fluid then exits the perfusion module through the flow channels and is recovered.

[0020] Furthermore, the sample loading plate and the flow channel plate in the perfusion module can be prepared into different structures according to different biological samples.

[0021] In one embodiment of the present invention, the biological sample is a tissue slice, and the sample carrier is a geometrically shaped flat plate, preferably provided with a space for placing the slice. In another embodiment of the present invention, the biological sample is a tissue block, organoid, or organ, and the sample carrier is a carrying container, wherein a liquid flow channel is provided in the carrying container for the entry or exit of tissue fluid or protective fluid. In yet another embodiment of the present invention, the biological sample is a cell-like or virus-like tissue, and the sample carrier is a test tube-like container, which is also provided with a liquid flow channel for the entry or exit of tissue fluid or protective fluid.

[0022] Furthermore, the biological sample of the present invention is selected from cells, viruses, organoids, tissues, eggs, sperm, embryos, and various physiological and pathological organ slices, such as heart, liver, lung, ovary, kidney, cartilage, blood vessels, valves, heart, liver, lung, ovary, kidney or cornea.

[0023] Furthermore, the sample carrier of the present invention can also be installed in the perfusion module in a pull-out manner;

[0024] Furthermore, the sample loading container of the present invention can also be connected to the perfusion module in an external manner;

[0025] Control Module:

[0026] The control module includes a temperature control component, a liquid supply control component and a display component.

[0027] The temperature control assembly includes a cold source bonding surface and a refrigeration component that are bonded to the bottom of the temperature control plate.

[0028] The cold source contact surface contacts an external cold source and exchanges heat with the temperature control plate, thereby controlling the temperature of the perfusion module. Preferably, the temperature control component further includes a temperature sensor for detecting the temperature.

[0029] The refrigeration component includes a refrigeration platform, which includes a plurality of support platforms connected to a cold source. The support platforms are in heat exchange with the cold source. The tops of the support platforms are coplanar to form a refrigeration plane. The surface of the refrigeration plane that contacts the temperature control plate is the cold source contact surface. The perfusion module is placed on the refrigeration plane and is in contact with each support platform to ensure the heat exchange efficiency between the perfusion module and the refrigeration module.

[0030] The liquid supply control component controls the liquid supply rate and liquid supply composition by controlling the working state of the peristaltic pump, that is, the peristaltic rate of each peristaltic pump.

[0031] Different biological samples have different perfusion fluid flow rates, temperatures, and fluid supply components. According to each biological sample, the working curves of the perfusion fluid flow rate and time, and the working curves of the perfusion fluid supply component and time can be drawn. By programming the working curves of different biological samples into corresponding programs, the automatic perfusion of the perfusion fluid can be achieved.

[0032] Furthermore, the liquid supply control assembly further includes a flow rate detector disposed downstream of the mixing tube for monitoring the liquid supply flow rate, and a tissue fluid concentration detector / cryoprotective fluid concentration detector for monitoring the mixed concentration;

[0033] A display component includes an integrated display screen with data processing function, which controls the temperature of the temperature control component and provides real-time feedback of the temperature detected by the temperature sensor. The liquid supply control component provides periodic data for the peristaltic pump control process, so that the peristaltic pump runs automatically during the perfusion process, and feeds back the data detected by the flow rate detector and the tissue fluid concentration detector / cryoprotective fluid concentration detector to the display screen, so that relevant operators can monitor and adjust the flow rate of the liquid supply and the liquid concentration ratio at relevant time nodes; the display component, liquid supply control component and temperature control component are connected to an external power supply.

[0034] Furthermore, the display component is based on a variety of biological samples, and the display component is pre-loaded with corresponding control schemes, the control schemes including working curves and temperature selection schemes for different biological samples, and further, the working curves include working curves of perfusion fluid flow rate and time, and working curves of perfusion fluid supply composition and time.

[0035] The control scheme controls the real-time working status of the refrigeration module and the liquid supply module. In one embodiment of the present invention, the display screen further includes a function selection key for biological samples, and different biological samples correspond to different control schemes.

[0036] Second aspect

[0037] The present invention provides an automatic perfusion method for cryopreservation of biological tissues, the method comprising the following steps:

[0038] S01 selects a corresponding sample loading plate according to the perfused biological sample and places the biological sample in the sample loading plate.

[0039] S02: selecting the biological sample in the display component so that the biological sample corresponds to a corresponding perfusion control scheme, wherein the perfusion control scheme is formed by loading a working curve and a type of reagent solution adapted to the corresponding biological sample into a data processing system;

[0040] Based on the perfusion control scheme, the data processing system controls the operating states of the temperature source and the peristaltic pump, adjusts the output power of the temperature source, and keeps the real-time temperature of the sample carrier consistent with the temperature-time working curve; and adjusts the peristaltic rate of each peristaltic pump to adjust the feed rate of each reagent, so that the feed rate is consistent with the corresponding perfusate working curve.

[0041] S03. Under the control of the perfusion scheme, the peristaltic pump sucks out the liquid in the corresponding reagent container and merges it into the mixing tube. After the liquid in the mixing tube is evenly mixed, it enters the perfusion module through the flow channel, flows and diffuses in the perfusion module to the carrying space or liquid channel of the sample plate, and infiltrates the biological sample.

[0042] Furthermore, a mixing assembly for further mixing the liquid is provided downstream of the mixing tube so that the liquid is fully mixed.

[0043] S04, the excess mixed liquid in the sample loading space or the liquid channel or the liquid seeping from the biological sample flows out through the flow channel outlet and is recovered.

[0044] S05. After the perfusion protocol is completed, the biological sample is removed individually or the sample carrier is removed, placed in a cooling device for cooling, and then stored in liquid nitrogen or a low-temperature storage device.

[0045] Furthermore, the temperature-time working curve is a curve formed by sequentially connecting the temperature values ​​or temperature ranges required for the biological sample at each stage with the time spent in each stage of perfusion and / or infiltration and recovery of the biological sample as the axis.

[0046] When the temperature-time working curve is loaded into the data processing module, the data processing module controls the output power of the temperature control module at each stage based on the temperature values ​​of the temperature-time working curve, so that the temperature of the biological sample meets the settings of the temperature-time working curve. Furthermore, by controlling the operating time of the temperature control module at each stage, the temperature of the space containing the biological sample is controlled so that the temperature of the biological sample meets the settings of the temperature-time working curve.

[0047] Preferably, temperature control is achieved through multi-core semiconductor refrigeration, which realizes cooling by the Peltier effect caused by the passage of current through semiconductor materials, and the temperature-time working curve is achieved through a PID algorithm.

[0048] The perfusate flow rate versus time working curve and the perfusate composition versus time working curve are curves formed by sequentially connecting the feed amount and feed speed of the corresponding reagent solution fed by the peristaltic pump at each stage with the time experienced in each stage of perfusion and / or infiltration and resuscitation of the biological sample as the axis; or the perfusate flow rate versus time working curve and the perfusate composition versus time working curve of the reagent solution are adjusted according to sensor feedback information.

[0049] Depending on the type of biological sample, the type of reagent solution, and the requirements of the perfusion and / or infiltration and resuscitation operations, each reagent solution has its own perfusate flow rate versus time working curve and perfusate composition versus time working curve. Correspondingly, the peristaltic pump that feeds the reagent solution also has its own perfusate flow rate versus time working curve and perfusate composition versus time working curve.

[0050] Preferably, when the perfusate flow rate versus time working curve and the perfusate composition versus time working curve of each peristaltic pump are loaded into the data processing module respectively, the feed amount and feed speed of the corresponding reagent solution at each stage are directly input into the data processing module. The data processing module achieves precise control of the feed amount of the reagent solution by controlling the operating frequency or operating speed of the peristaltic pump based on the feed amount and feed speed of the perfusate flow rate versus time working curve and the perfusate composition versus time working curve, so that each reagent solution meets the settings of the perfusate flow rate versus time working curve and the perfusate composition versus time working curve.

[0051] Preferably, the feed amount of the perfusate flow rate and time working curve is converted into the operating frequency value or the operating speed value of the peristaltic pump. The data processing module adjusts the feed amount of each peristaltic pump by reading the operating frequency value or the operating speed value of the peristaltic pump, thereby achieving precise control of the feed amount of the reagent solution, so that each reagent solution meets the settings of the perfusate flow rate and time working curve and the perfusate composition and time working curve.

[0052] Preferably, at each stage of the perfusion and / or infiltration and resuscitation process of the biological sample, the temperature-time working curve or the perfusion fluid flow rate and time working curve and the perfusion fluid composition and time working curve are divided into curves for each time interval based on the time interval corresponding to each stage. The curves for each time interval are set using the same function so that the working curve is a smooth continuous curve or are set using different functions to meet the requirements of the temperature or temperature change of each biological sample at each stage of perfusion and / or infiltration and resuscitation, and the feed amount and feed speed of each reagent solution at each stage of perfusion and / or infiltration and resuscitation.

[0053] Preferably, the data processing module receives a signal indicating the real-time temperature of the sample loading module, and determines whether the real-time temperature of the sample loading module is consistent with the temperature value corresponding to the temperature-time working curve at the current time point. If it is, the data processing module maintains the output power of the temperature control module according to the temperature-time working curve; if it is not, the data processing module adjusts the output power of the refrigeration module of the temperature control module to compensate for the temperature offset of the sample loading module, so that the real-time temperature of the sample loading module reaches the temperature value corresponding to the temperature-time working curve at the current time point; then, the temperature control module is restored to the output power corresponding to the temperature-time working curve.

[0054] Preferably, the delivery parameters are adjusted according to the difference adjusted by the data processing module so that the feeding amount and feeding speed conform to the settings of the perfusate flow rate and time working curve and the perfusate composition and time working curve.

[0055] By using this biological sample perfusion device, operators can perform large-scale rapid perfusion or resuscitation operations on the same biological sample. After completing the perfusion or resuscitation operation for any biological sample, the operator only needs to replace the new sample carrier or a new external carrier container, connect the sample carrier or external carrier container, and then start a new round of operations. At the same time, during the perfusion or resuscitation operation, the data processing module automatically controls the temperature source and peristaltic pump based on the temperature-time working curve, the perfusion fluid flow rate and time working curve, and the perfusion fluid composition and time working curve. This ensures that the perfusion and resuscitation operations of each biological sample are continuous and consistent, eliminating the influence of human factors on the operation results.

[0056] The biological sample perfusion method of the present invention is suitable for low-temperature vitrification and freezing of large quantities of biological samples. The specifications of the sample carrier or the external carrying container are designed based on the specifications of the low-temperature vitrification and freezing container, so that the sample carrier and the external carrying container are not only carriers for loading biological sample slices during the perfusion operation, but also carriers for loading biological sample slices during the vitrification and freezing operation, and carriers for loading biological sample slices during the biological sample recovery operation. The sample carrier and the external carrying container serve as carriers for carrying biological samples. After the perfusion is completed, they can be directly extracted into a low-temperature refrigeration unit and low-temperature vitrification and freezing can be carried out in the low-temperature refrigeration unit cooled by liquid nitrogen. During the low-temperature vitrification and freezing of large quantities of biological samples, the operator only needs to repeatedly replace the sample carrier or the external carrying container to complete the batch operation, thereby realizing the modular operation of the biological sample perfusion and simplifying the complexity of the batch operation to the greatest extent.

[0057] Compared with the prior art, the automatic perfusion device for cryopreservation of biological tissues provided by the present invention has the following technical advantages:

[0058] 1. The biological sample perfusion device of the present invention has a wide range of applications. It can be used with different types of biological samples and tissues. When operating on different types of biological samples, only the corresponding sample carrier and flow channel plate need to be replaced, without having to replace the entire device. This makes it suitable for large-scale operation of biological samples and tissues.

[0059] 2. The biological sample perfusion device of the present invention continuously distributes cryoprotectant or tissue fluid to the biological sample through the perfusion unit module, thereby avoiding the adverse cell shrinkage caused by discontinuity.

[0060] 3. The biological sample perfusion device of the present invention realizes automated control of biological sample, tissue perfusion, infiltration, and resuscitation operations through a data processing module, eliminating the need for manual configuration of cryoprotectant or tissue fluid of different concentrations, eliminating operational errors caused by human factors, and improving the stability of biological sample, tissue perfusion, infiltration, and resuscitation operations.

[0061] 4. The biological sample perfusion device of the present invention monitors and adjusts the working status of the liquid supply module in real time. The cryoprotectant solution or tissue fluid is more accurate than manual mixing, and a seamless transition from low concentration to high concentration or high concentration to low concentration can be achieved. The replacement of water inside and outside the cells is more thorough and safe, and the formation of ice crystals during the cooling process is prevented to the greatest extent possible, thereby preserving the activity of the sample.

[0062] 5. The biological sample perfusion device of the present invention monitors and adjusts the working charge of the refrigeration unit in real time, thereby achieving precise temperature control throughout the entire perfusion infiltration process of the biological sample and maximally preserving the activity of the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG1 is a schematic diagram of the overall structure of an automatic perfusion device for cryopreservation of biological tissues;

[0064] FIG2 is a schematic structural diagram of a semiconductor refrigeration module in an automatic perfusion device for cryopreservation of biological tissues;

[0065] FIG3 is a schematic structural diagram of a perfusion unit module of an automatic perfusion device for cryopreservation of biological tissues;

[0066] FIG4 is a schematic structural diagram of the flow path surface of an automatic perfusion device for cryopreservation of biological tissues;

[0067] FIG5 is a schematic diagram of the internal cross-sectional structure of a flow channel plate in a biological tissue cryopreservation and perfusion device;

[0068] FIG6 is a schematic structural diagram of the sealing surface of the automatic perfusion device for cryopreservation of biological tissues;

[0069] FIG7 is a schematic structural diagram of a sample carrier in an automatic perfusion device for cryopreservation of biological tissues;

[0070] FIG8 is a schematic structural diagram of a temperature control panel in an automatic perfusion device for cryopreservation of biological tissues;

[0071] FIG9 is a schematic structural diagram of a mixing assembly of an automatic perfusion device for cryopreservation of biological tissues;

[0072] FIG10 is a schematic structural diagram of an automated perfusion method for cryopreservation of biological tissues;

[0073] FIG11 is a schematic flow chart of a method for automatic perfusion of biological tissue cryopreservation;

[0074] The accompanying drawings are marked as follows: 1-housing; 2-semiconductor refrigeration module; 3-peristaltic pump; 4-reagent container; 5-mixing tube; 6-support platform; 7-refrigeration plane; 8-perfusion unit module; 9-temperature control plate; 10-sample loading plate; 11-flow channel plate; 12-sealing plate; 13-cold source fitting surface; 14-sample loading surface; 15-primary positioning edge; 16-positioning space; 17-secondary positioning edge; 18-secondary positioning space; 19-drain port; 20-recovery port; 21-sample loading space; 22-infiltration port; 23-flow channel surface; 24-sealing surface; 25-flow channel ;26-positioning hole;27-sealing groove;28-through hole;29-gasket;30-channel outlet;31-first perfusion channel;32-second perfusion channel;33-third perfusion channel;34-liquid inlet;35-first perfusion channel;36-second perfusion channel;37-liquid inlet channel;38-first channel;39-second channel;40-third channel;41-mixing assembly;42-mixing piece;43-mixing sheet. DETAILED DESCRIPTION

[0075] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0076] Example 1: An automatic perfusion device for cryopreservation of biological tissue

[0077] As shown in Figure 1-9

[0078] An automatic perfusion device for cryopreservation of biological tissues comprises a device body, which includes a housing 1, within which a temperature control assembly 2 and a liquid supply module are housed. In this embodiment, the liquid supply module comprises two peristaltic pumps 3, serving as liquid supply pumps, and two reagent containers 4. The liquid inlet of each peristaltic pump 3 is independently connected to a corresponding reagent container 4, and the liquid outlets of each peristaltic pump 3 are interconnected via a three-way pipe. At the intersection of the liquid outlets, downstream of a mixing tube 5, the three-way pipe is connected to a detection pipe, which is then connected to an analysis device.

[0079] The temperature control component 2 is provided with a refrigeration platform of open design, which serves as a docking space for the cold source. The refrigeration platform is composed of three support platforms 6 connected to the cold source. The support platform 6 is made of metal material and can maintain a high heat exchange efficiency with the cold source. The top of the support platform 6 is coplanarly formed with a refrigeration plane 7, and the perfusion module 8 separated from the device body is placed on the refrigeration plane 7 and connected to each support platform 6 in a surface contact manner. The outer shell is also provided with a flip cover that can be turned over and closed, and the perfusion unit module 8 can be covered inside to achieve the purpose of protection. According to different cooling methods, the temperature control component 2 can also be replaced with a water tank, using a temperature control method of a combination of an ice water bath and a heating rod, and realizing temperature control of the refrigeration module through the working state of an external compressor. At this time, the inside of the water tank is the docking space for the perfusion module to be placed.

[0080] The perfusion unit module 8 includes a temperature control plate 9, a sample loading plate 10, a flow channel plate 11 and a sealing plate 12, wherein:

[0081] The temperature control plate 9 is made of a metal plate structure, and is provided with a cold source fitting surface 13 and a sample carrying surface 14 arranged opposite to each other on the temperature control plate 9. The cold source fitting surface 13 is a continuous plane, and the sample carrying surface 14 is provided with a primary positioning edge 15 protruding from the sample carrying surface 14. The primary positioning edge 15 is arranged around the sample carrying surface 14 to form a positioning space 16; the primary positioning edge 15 is also provided with a secondary positioning edge 17 protruding from the primary positioning edge 15. The secondary positioning edge 17 is provided on the primary positioning edge 15. The positioning edge 17 is arranged around the primary positioning edge 15 to form a secondary positioning space 18; the sample carrying surface 14 of the temperature control plate 9 is provided with a drain port 19 connected to the outside, and an inclined surface inclined with respect to the sample carrying surface 14 is provided in the drain port 19 to facilitate the flow of fluid out of the drain port 19; the housing 1 is also provided with a recovery port 20, which is located below the drain port 19 and is connected to the drain port 19; a pipe is provided at the bottom of the recovery port 20 to connect to a recovery container;

[0082] The sample loading plate 10 is a plate-like structure made of metal material and is installed in the positioning space 16, with the top of the sample loading plate 10 flush with the top of the primary positioning edge 15. The bottom of the sample loading plate 10 is a continuous plane and is in contact with the sample loading surface 14, and the sample loading surface 14 achieves heat exchange through surface contact. The sample loading plate 10 is provided with four sample loading spaces 21 for holding biological samples. The four sample loading spaces 21 are arranged in sequence. The first three sample loading spaces 21 are provided with multiple infiltration ports 22 connected to adjacent sample loading spaces 21, and the last sample loading space 21 is provided with multiple infiltration ports 22 connected to the drainage port 19.

[0083] The flow channel plate 11 is a plate-like structure installed in the secondary positioning space 18. The flow channel plate 11 is provided with a flow channel surface 23 and a sealing surface 24 arranged opposite each other. The flow channel 25 is provided on the flow channel surface 23. The top opening of the flow channel 25 is connected to the outside. The flow channel surface 23 is also provided with a plurality of positioning holes 26, which are arranged around the flow channel 25. The sealing surface 24 is a continuous plane and is provided with a sealing groove 27 for filling a sealing strip. When the sealing surface 24 is pressed against the sample loading plate 10, the sealing groove 27 surrounds the outside of the sample loading space 21.

[0084] The sealing plate 12 has a plate-like structure and is provided with a through hole 28 corresponding to the position of the positioning hole 26. The operator can fix the sealing plate 12 on the flow channel surface 23 with screws to cover the flow channel 25 on the flow channel plate 11 to prevent the fluid from overflowing during the perfusion operation; a gasket 29 is provided between the sealing plate 12 and the flow channel plate 11 to ensure the sealing of the sealing plate 12 to the flow channel 25.

[0085] The flow plate 11 is pressed onto the sample loading plate 10, and the sealing strip is attached to the sample loading plate 10, so that each sample loading space 21 forms a sealed space. The flow plate 11 is provided with eight flow openings 30, which are evenly arranged and connected to the first sample loading space 21. The flow plate 11 is provided with flow channels 25 connected to each flow opening 30. Specifically, the flow channels 25 include a first perfusion channel 31, a second perfusion channel 32, and a third perfusion channel 33, wherein:

[0086] A liquid inlet 34 is provided in the middle of the first perfusion channel 31 , and channel outlets are provided at both ends of the first perfusion channel 31 , which are connected to the middle of the second perfusion channel 32 to form a first perfusion channel 35 ;

[0087] Both ends of the second perfusion channel 32 are provided with channel outlets, which are connected to the middle of the third perfusion channel 33 to form a second perfusion channel 36;

[0088] Both ends of the third perfusion channel 33 are provided with channel outlets, which are connected to the flow channel opening 30. The third perfusion channels 33 are not connected to each other. The inner diameters of the first perfusion channel 31, the second perfusion channel 32, and the third perfusion channel 33 decrease in sequence.

[0089] In this embodiment, a liquid inlet channel 37 is provided in the flow channel plate 11, and the liquid inlet channel 37 includes a first channel 38, a second channel 39 and a third channel 40 that are interconnected, and the inner diameters of the first channel 38, the third channel 40 and the second channel 39 decrease in sequence. The second channel 39 is arranged perpendicular to the flow channel surface 23 and is connected to the third channel 40. The first channel 38 is connected to the liquid inlet 34, and a mixing assembly 41 is inserted into the first channel 38. The mixing assembly 41 includes two mixing pieces 42 arranged side by side, and each mixing piece 42 includes a plurality of mixing sheets 43 with a plate-like structure; adjacent mixing sheets 43 in the same mixing piece 42 are connected end to end, and the corresponding mixing sheets 43 in the two mixing pieces 42 are cross-connected.

[0090] In this embodiment, a data processing module is provided in the housing 1, and a liquid supply control function module, a temperature control function module, a parameter adjustment function module, and an operation control function module are provided in the data processing module.

[0091] In this embodiment, a control module is provided in the housing 1, and the control module includes:

[0092] A temperature sensor for detecting the temperature of the temperature control component 2;

[0093] Taking the biological sample perfusion operation as an example, the working process of the biological sample perfusion device of this embodiment is as follows:

[0094] First, according to the type and quantity of biological samples to be processed, the control scheme preset in the parameter adjustment function module is read, and based on the type and quantity of reagent solutions shown in the control scheme, appropriate reagent solutions are selected and added to the reagent containers 4 respectively.

[0095] A sample carrier plate 10 loaded with a biological sample to be processed is placed on the sample carrier surface 14. The flow plate 11 is pressed against the sample carrier plate 10 to form the perfusion unit module 8. The perfusion unit module 8 is then placed entirely on the cooling platform of the semiconductor cooling module 2, with the cold source contact surface 13 in surface contact with the cooling plane 7. A clamping device is provided on the housing 1 to press the flow plate 11 against the sample carrier plate 10, ensuring a tight seal between the two.

[0096] The parameter function module is called up to obtain the perfusion control plan corresponding to the biological sample, including the reagent solution ratio, the operating curves of each peristaltic pump 3, and the temperature-time operating curve of the semiconductor cooling module 2. The reagent solutions required for this perfusion infiltration operation are placed in the corresponding solution holders. According to the perfusion control plan, each reagent solution is connected to the inlet of the corresponding peristaltic pump 3, and the three-way pipe is connected to the liquid inlet channel 37 of the flow channel plate 11.

[0097] Upon activation of the operation display assembly, the operation control module reads the control scheme selected by the parameter adjustment module and, based on this scheme, outputs control signals to the liquid inlet control module and the temperature control module. Based on these control signals, the liquid inlet control module controls the feed frequency and feed time of each peristaltic pump 3, directing the reagent solution into the mixing junction, i.e., the mixing tube, for initial mixing. During this process, the temperature control module monitors the real-time temperature of the perfusion unit module 8, ensuring that it matches the current time-temperature value on the temperature-time working curve.

[0098] During the cooling process of the sample carrier 10, the temperature probe arranged on the temperature control board 9 monitors the real-time temperature of the temperature control board 9 and transmits the temperature signal to the data processing module. The operation control function module selects the output power of the semiconductor refrigeration module 2 based on the temperature value at the current time of the temperature-time working curve and the actual temperature of the sample carrier 10 at the current time, and sends this output power to the temperature control function module. The temperature control function module controls the operation of the semiconductor refrigeration module 2, compensates for the temperature offset of the sample carrier 10, and quickly makes the actual temperature of the sample carrier 10 reach the temperature value at the current time of the temperature-time working curve. According to the changes in the temperature-time working curve, the data processing module continuously changes the control output according to the stage of the biological sample, and dynamically adjusts the working state of the semiconductor refrigeration module 2 to make the temperature of the biological sample conform to the setting of the temperature-time working curve.

[0099] Subsequently, the operation control module dynamically adjusts the set value of the output power of the semiconductor refrigeration module 2 based on the temperature change rate of the temperature-time working curve within the time period T, and sends this set value to the temperature control module in real time. The temperature control module then controls the operation of the semiconductor refrigeration module 2, causing the actual temperature of the sample carrier 10 to drop to the set temperature within the set time period T in accordance with the set change of the temperature-time working curve. The operation control module then sends a signal to the temperature control module based on the temperature-time working curve to maintain the output power of the semiconductor refrigeration module 2 and the temperature of the sample carrier 10 at the set temperature.

[0100] After the semiconductor refrigeration module 2 has been running for a period of time and the sample carrier 10 has dropped to a suitable temperature, the operation control function module sends a control signal to the liquid inlet control function module according to the working curve of each peristaltic pump 3.

[0101] The liquid inlet control module controls the feed rate, feed composition, and feed time of each peristaltic pump 3 based on the control signal. It starts the peristaltic pump at the starting time set by the working curve. Subsequently, based on the feed amount, feed composition, and single feed capacity of the peristaltic pump 3 set at the current time of the working curve, it sends a pulse signal to control the operating frequency of the peristaltic pump 3, so that the peristaltic pump 3 feed working curve conforms to the feed amount set at the current time. The reagent solution enters the delivery pipeline from the outlet of the peristaltic pump 3, undergoes preliminary mixing at the mixing intersection, namely the mixing tube, and enters the first channel 38 from the liquid inlet 34. After being fully mixed in the first channel 38 by the mixing component 41, it enters the third channel 40 and the second channel 39 in sequence, and slowly flows into the flow channel 25 of the flow channel plate 11. According to the changes in each working curve, the data processing module continuously changes the control output according to the stage of the biological sample, dynamically adjusting the working state of each peristaltic pump 3, so that the feed parameters of each reagent solution conform to the setting of the working curve.

[0102] After the cryoprotectant solution, resulting from the mixing of the reagent solutions, enters the flow channel 25, it is sequentially split through the first perfusion channel 31, the second perfusion channel 32, and the third perfusion channel 33, with equal amounts flowing into the flow channel opening 30 and into the sample loading plate 10. The sample loading plate 10 may be provided with a heat exchange space upstream of the sample loading space, connected to the sample loading space 21. After entering the sample loading plate 10, the cryoprotectant solution first flows through the heat exchange space, exchanging heat with the sample loading plate 10. After reaching the isothermal state of the sample loading plate 10, it then flows into the first sample loading space 21 of the sample loading plate 10, simultaneously perfusing the biological samples within that same sample loading space 21. The multi-stage splitting of the first perfusion channel 31, the second perfusion channel 32, and the third perfusion channel 33 ensures that the cryoprotectant solution is evenly distributed into the sample loading space 21, thereby simultaneously perfusing the biological samples within that space. Through the infiltration port 22 of each sample loading space 21, the cryoprotectant solution uniformly immerses the biological samples within each sample loading space 21. Subsequently, the replaced cell tissue fluid and the remaining cryoprotectant fluid flow out from the infiltration port 22 of the last sample loading space 21 to the drain port 19 and are discharged to the recovery port 20 , and then flow back into the recovery container through the recovery port 20 .

[0103] After completing the perfusion infiltration operation for the first batch of biological samples, the operator removes the sample carrier 10 and directly stores it in an ultra-low temperature storage container. Subsequently, the operator places a new sample carrier 10 loaded with the same biological sample into the temperature control plate 9 and repeats the perfusion infiltration operation to complete the perfusion infiltration operation for a large number of biological samples of the same type.

[0104] Furthermore, when processing very small biological tissues, the device can also incorporate a carrier container connected to the perfusion unit module, allowing the biological tissue to be loaded through a carrier channel within the carrier container. When the carrier container is connected to the sample loading module, the carrier channel communicates with the sample loading space 21. In this case, in addition to the loading function of the sample loading space 21, the sample loading space 21 now serves to divert and cool the mixed solution.

[0105] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A device for automatic perfusion during cryopreservation of biological tissues, comprising a fluid supply module, a perfusion module and a control module, wherein the fluid supply module is connected to the perfusion module via a fluid pipeline, and the control module regulates the ratio of the fluid supply and the rate of fluid supply to the fluid supply module; the perfusion module includes a temperature control plate, a plate for placing samples, a flow channel plate and a sealing plate; a drain channel is formed on the sample placement surface of the temperature control plate and communicates with the external environment of the perfusion module; inside the drain channel there is an inclined surface located at an angle to the surface for placing samples to facilitate the outflow of fluid; a recirculation channel is formed on the body of the device, located below the drain channel and communicating with it;at the bottom of the recirculation channel there is a pipeline connected to the recirculation tank.

2. A device for the automatic perfusion of biological tissue during cryopreservation according to paragraph 1, characterized in that the fluid supply module includes reagent containers and peristaltic pumps located at the openings of said reagent containers and connected to them by means of tubes; the number of peristaltic pumps corresponds to the number of reagent containers, which is determined depending on the variety of supplied liquids.

3. A device for automatic perfusion during cryopreservation of biological tissues according to claim 1, characterized in that a peristaltic pump pumps liquid from the corresponding containers with reagents and directs it into a mixing tube; after thorough mixing in the mixing tube, the liquid enters through a flow channel into the perfusion module, where it is distributed and diffuses into the supporting space or channels of the sample platform, ensuring saturation of the biological sample; a mixing unit is installed downstream of the mixing tube.

4. A device for the automatic perfusion of biological tissue during cryopreservation according to claim 1, characterized in that: the perfusion module includes a temperature control board and a plate with flow channels pressed against said temperature control board to form a sealed cavity. The plate for placing samples is located inside said sealed cavity and has several sample holding spaces intended for placing biological samples; adjacent sample holding spaces are interconnected by flow channels.

5. A device for automatic perfusion of biological tissue during cryopreservation according to paragraph 4, characterized in that the plate for placing samples and the plate with a flow channel inside the perfusion module can have a different design depending on the different biological samples.

6. A device for automatic perfusion of biological tissue during cryopreservation according to paragraph 5, characterized in that the biological sample contains cells, viruses, organelles, tissues, oocytes, spermatozoa, embryos and various physiological or pathological areas of organs, such as the heart, liver, lungs, ovaries, kidneys, cartilage, blood vessels, valves, heart, liver, lungs, ovaries, kidneys or cornea.

7. A method for automatic perfusion of biological tissue during cryopreservation for use with a device for automatic perfusion of biological tissue during cryopreservation according to any of paragraphs 1-6, characterized in that the method includes the following steps: S01. According to the biological sample to be perfused, select an appropriate sample plate and place the biological sample in the plate; S02. In the display module, select the perfusion control scheme appropriate for the biological sample. The specified perfusion control scheme is generated by loading an operating curve adapted to the corresponding biological sample and reagent solution types into the data processing system. Based on the perfusion control circuit, the data processing system regulates the working mode of the heat source and the peristaltic pump, adjusting the output power of the heat source to maintain consistency between the real-time temperature in the sample loading module and the temperature-time curve; adjusts the feed rate of the peristaltic pump to maintain consistency between the feed rate and the corresponding working time curve of the perfusion fluid flow rate; modulates the aspiration behavior of the peristaltic pump in each reagent reservoir to ensure that the aspirated solution matches the corresponding working time curve of the perfusion fluid composition; S03. Under the control of the perfusion program, the peristaltic pump draws fluid from the respective reagent containers and delivers it to the mixing tube; after thorough mixing in the mixing tube, the fluid enters through the flow channel into the perfusion module, where it is distributed and diffused into the carrier space or channels of the sample holder, ensuring saturation of the biological sample; S04. Excess mixture in the sample space or carrier channels, as well as liquid released from the biological sample, are drained through drainage holes connected to the outside of the perfusion module and collected in a receiving container; S05. Upon completion of the perfusion circuit, the biological specimen is removed separately or together with the specimen plate, placed in a cooling device to reduce the temperature, and then transferred for storage in liquid nitrogen or a cryogenic container.

8. A method for automatic perfusion of biological tissue at low temperatures according to claim 7, characterized in that the feed rate obtained from the perfusion fluid flow rate versus time curve is converted into an operating frequency value or operating speed value of a peristaltic pump, wherein the data processing module regulates the feed volume of each peristaltic pump by reading said operating frequency value or operating speed value of said peristaltic pump, thereby ensuring precise control of the feed volume of said reagent solutions to ensure that each reagent solution corresponds to the perfusion fluid flow rate versus time curve.

9. A method for automatic perfusion of biological tissue during cryopreservation according to paragraph8, characterized in that the data processing module receives signals indicating the real-time temperature of the sample holding plate and determines whether the indicated real-time temperature corresponds to the temperature value indicated by the temperature-time curve at the current time; if the determination is positive, the data processing module maintains the output power of the temperature control module in accordance with the indicated temperature-time curve; if the determination is negative, the data processing module adjusts the output power of the cooling module in the temperature control module to compensate for the deviation in the temperature of the sample holding plate, thereby achieving a real-time temperature of the sample holding plate corresponding to the temperature value at the current time on the temperature-time curve; subsequently, the temperature control module is restored to the output power corresponding to the temperature-time curve.

10. A method for automatic perfusion of biological tissue during cryopreservation according to claim 8, characterized in that the biological sample is a biological sample subject to resuscitation, removed from cryogenic storage.