Automatic freezing device, automatic freezing method, and freezing container
Patent Information
- Application Number
- JP2022013066
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2022-01-31
- Publication Date
- 2024-12-19
AI Technical Summary
Existing cryopreservation methods risk erroneously recovering frozen objects and leaving residual liquid, placing a heavy burden on workers due to the need for precise manual handling.
An automatic freezing apparatus and method that includes a container with a bottom opening smaller than the object, using an operating unit to inject and drain liquids, and a refrigerant reservoir, reducing manual handling and ensuring complete liquid removal.
The apparatus and method prevent erroneous recovery of frozen objects and residual liquids, reducing worker burden by automating the cryopreservation process.
Smart Images
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Abstract
Description
[Technical Field]
[0001] The present invention relates to an automatic freezing treatment device, an automatic freezing treatment method, and a freezing container. [Background technology]
[0002] Techniques for cryopreserving cells (subjects to be frozen), such as fertilized embryos and eggs, have been developed as a useful technique in infertility treatments and other treatments. In cell cryopreservation, the target cells, such as embryos and eggs, are placed in a freezing container and subjected to a pre-freezing process. The cells placed in the freezing container are then rapidly frozen using liquid nitrogen. In the pre-freezing process, the cells are immersed in an equilibration solution (ES) and a vitrification solution (VS). This replaces the cellular fluid inside the cells and vitrifies them.
[0003] When pre-freezing is performed manually, the embryologist uses a glass Pasteurizer connected to a mouthpiece by a tube, and traps the target at the tip by aspirating and discharging with their mouth, then transports it to an equilibration solution, transports it to a vitrification solution, and washes it. Regarding pre-freezing, for example, Patent Document 1 (U.S. Patent No. 9,826,733) discloses a technique for injecting an equilibration solution into a cavity where an embryo has been placed, recovering the equilibration solution, injecting a vitrification solution, and recovering the vitrification solution. [Prior art documents] [Patent documents]
[0004] [Patent Document 1] U.S. Patent No. 9,826,733 Summary of the Invention [Problem to be solved by the invention]
[0005] In the technology described in Patent Document 1, there is a risk that the frozen object (such as an embryo) may also be recovered when recovering the equilibration solution or vitrification solution. On the other hand, if an operator such as an embryologist is afraid of recovering the frozen object and does not sufficiently recover the liquid that should be recovered during the recovery process, there is a concern that the liquid that should be recovered may remain in the frozen object as a foreign body. This requires the operator to work carefully, which places a heavy burden on the operator.
[0006] The present invention was devised in consideration of the above-mentioned circumstances, and its purpose is to provide a technology for reducing the burden on workers in pre-processing frozen objects during cryopreservation, while avoiding situations in which frozen objects are mistakenly recovered and where liquid that should be recovered remains in the frozen objects. [Means for solving the problem]
[0007] An automatic freezing treatment device according to one aspect of the present disclosure comprises a container for containing an object to be frozen, an operating unit that operates to inject and discharge liquid into the container and move the container, and a refrigerant storage unit that stores a refrigerant for freezing the object to be frozen, wherein the container has an opening at the bottom that is smaller than the size of the object to be frozen, and the operating unit injects liquid into the container, then discharges the liquid from the container through the opening and moves the container to the refrigerant storage unit.
[0008] An automatic freezing method according to one aspect of the present disclosure includes the steps of injecting a liquid into a container having an opening at the bottom that is smaller than the size of the object to be frozen, discharging the liquid from the container through the opening after the liquid has been injected, and moving the container to a refrigerant storage section after the liquid has been discharged.
[0009] A freezing container according to one aspect of the present disclosure is a freezing container for pre-treating an object to be frozen by immersing it in a liquid, and includes a placement portion that allows the liquid to pass through and on which the object to be frozen can be placed. [Effects of the Invention]
[0010] According to certain aspects of the present disclosure, during cryopreservation, situations in which the frozen object is erroneously retrieved and situations in which liquid that should be retrieved remains at the frozen object are avoided, while the burden on the worker in pre-processing the frozen object is reduced. [Brief explanation of the drawings]
[0011] [Figure 1] 1 is a diagram showing a schematic diagram of the overall configuration of a freezing device 100. FIG. [Figure 2] 2 is a diagram showing the configuration of a tube 200. FIG. [Figure 3] 1A and 1B are diagrams showing the structure of a cap 300. FIG. [Figure 4] FIG. 1 is a diagram showing an example of a block diagram of a freezing device 100. [Figure 5] 1 is a flowchart of a process performed in a freezing device 100. [Figure 6] 6 is a flowchart of a subroutine of the processing shown in FIG. 5. [Figure 7] 6 is a flowchart of a subroutine of the processing shown in FIG. 5. [Figure 8] 1 is a diagram showing a state of a freezing device 100. FIG. [Figure 9] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 10] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 11] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 12] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 13] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 14] 1 is a diagram showing a state of a freezing device 100. FIG. [Figure 15] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 16] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 17] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 18]1 is a diagram showing the state of a freezing device 100. FIG. [Figure 19] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 20] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 21] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 22] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 23] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 24] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 25] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 26] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 27] 1 is a diagram showing the state of a freezing device 100. FIG. [Figure 28] FIG. 1 is a diagram schematically illustrating the overall configuration of a freezing device 100A. [Figure 29] 10 is a flowchart of a process performed in a freezing device 100A. [Figure 30] 30 is a flowchart of a subroutine of the process of FIG. 29. [Figure 31] 30 is a flowchart of a subroutine of the process of FIG. 29. [Figure 32] FIG. 10 is a diagram for explaining the state of the freezing device 100A. [Figure 33] FIG. 10 is a diagram for explaining the state of the freezing device 100A. [Figure 34] FIG. 10 is a diagram for explaining the state of the freezing device 100A. [Figure 35] 10A and 10B are diagrams showing the configuration of a modified example of the tube 200. DETAILED DESCRIPTION OF THE INVENTION
[0012] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. In the drawings, the same or corresponding parts are designated by the same reference numerals, and description thereof will not be repeated.
[0013] [Embodiment 1] <Automatic freezing treatment device> 1 is a diagram showing a schematic diagram of the overall configuration of a freezing device 100. The freezing device 100 is an example of an automatic freezing treatment device. The freezing device 100 freezes the object to be frozen, such as a fertilized embryo or an ovum, after pre-treatment.
[0014] 1, the freezing apparatus 100 includes a cap holder 10, a tube holder 20, an ES reservoir 30, a VS reservoir 40, a liquid nitrogen tank 50, an electric pipetter 70, and a case 101. The case 101 covers the outer shell of the freezing apparatus 100.
[0015] One or more caps 300 are placed in the cap holder 10. One or more tubes 200 are placed in the tube holder 20. The tubes 200 are an example of a freezing container.
[0016] The ES reservoir 30 contains an ES. The VS reservoir 40 contains a VS. Each of the ES and VS is an example of a liquid used in pre-processing an object to be frozen. The liquid nitrogen tank 50 contains liquid nitrogen. Liquid nitrogen is an example of a refrigerant for freezing. In the liquid nitrogen tank 50, one or more freezing canes 60 are immersed in the refrigerant. Each freezing cane 60 contains one tube 200.
[0017] The electric pipettor 70 includes one or more nozzles 71 and sends air to one or more tubes 200 via each of the one or more nozzles 71. The air is sent using, for example, a pump 151, which will be described later with reference to FIG. 4. FIG. 1 shows an X-axis and a Y-axis. The X-axis represents the direction in which the cap holder 10, the tube holder 20, the ES reservoir 30, the VS reservoir 40, and the liquid nitrogen tank 50 are arranged. The Y-axis represents the vertical direction. The position of the electric pipettor 70 can be controlled so that the position of the one or more nozzles 71 moves in the X-axis and Y-axis directions, as will be described later with reference to FIG. 4 and the like.
[0018] <Tube> FIG. 2 is a diagram showing the configuration of the tube 200. The tube 200 includes a base 201 and a bottom 202. The base 201 has a columnar shape. More specifically, the base 201 is tapered so that its diameter decreases from the top end 203 to the bottom 202. This improves mold releasability when the tube 200 is manufactured by injection molding and also improves the degree of adhesion between the cap 300 or the tips 401, 402 and the tube 200. The bottom 202 has a mesh structure. The base 201 and the bottom 202 are made of a synthetic resin such as polypropylene. For convenience, FIG. 1 illustrates the tube 200 as if the base 201 were not tapered. The base 201 of the tube 200 may be tapered as shown in FIG. 2 or not tapered as shown in FIG. 1.
[0019] The size of the gaps in the stitches of the bottom 202 is smaller than the size of the object to be frozen. In one implementation example, the object to be frozen is a fertilized egg, the size of the fertilized egg is about 140 μm, and each of the vertical and horizontal lengths of the gaps is smaller than 140 μm (for example, about several tens of μm). Each of the vertical and horizontal lengths of the gaps may be half or less the size of the fertilized egg (for example, about 50 to 70 μm).
[0020] The gaps in the weave of the bottom 202 are an example of openings in the tube 200. As long as openings are formed in the bottom 202 of the tube 200, it is not necessary for the bottom 202 to have a weave structure.
[0021] The bottom 202 of the tube 200 allows liquids such as ES and VS to pass through while retaining the target 900 to be frozen. This eliminates the need for an operator to manually remove the liquid from the tube 200 when the target 900 to be frozen is immersed in the liquid in the tube 200. This can prevent an operator from removing enough liquid from the tube 200 due to fear of removing the target 900 to be frozen.
[0022] <Cap> FIG. 3 is a diagram showing the structure of cap 300. It includes base 301 and bottom 302. Base 301 is tapered so that its diameter decreases from top end 303 to bottom 302. This improves mold releasability when cap 300 is manufactured by injection molding and also improves the degree of adhesion between tube 200 and cap 300. Base 301 and bottom 302 are made of a synthetic resin such as polypropylene. A recess may be formed on the outer side of base 301, and a protrusion may be formed on the inner side of tube 200 near top end 203. This reliably prevents cap 300, which is fixed to tube 200, from separating from tube 200.
[0023] The bottom 302 has a mesh structure. The mesh of the bottom 302 may have a structure similar to that of the mesh of the bottom 202 of the tube 200. The gaps between the mesh of the bottom 302 are an example of openings in the cap 300. The cap 300 does not need to have a mesh structure in the bottom 302 as long as it has openings.
[0024] The outer diameter of the base 301 near the bottom 302 is approximately equal to the inner diameter of the base 201 of the tube 200 near the top end 203. That is, the cap 300 covers the tube 200 by fitting the area near the bottom 302 onto the top end 203 of the tube 200.
[0025] A thread may be formed on the outer surface of the base 301 near the bottom 302 and on the inner surface of the tube 200 near the top end 203 of the base 201. The cap 300 may be fixed to the tube 200 by being screwed onto the tube 200 according to the thread structure.
[0026] <control block> 4 is a diagram showing an example of a block diagram of freezing device 100. Freezing device 100 includes a control device 110. Control device 110 includes a control unit 120, an input unit 131, and a display unit 132. Control unit 120 includes a CPU (Central Processing Unit) 111, a ROM (Read Only Memory) 112, and a RAM (Random Access Memory) 113.
[0027] The CPU 111 performs overall control of the freezing device 100. The CPU 111 loads a program stored in the ROM 112 into the RAM 113 and executes it. The ROM 112 stores a program describing the processing procedures of the freezing device 100. The RAM 113 serves as a working area when the CPU 111 executes a program, and temporarily stores the program, data used when the program is executed, and the like.
[0028] Input unit 131 accepts inputs from a user, including instructions for freezing device 100. Input unit 131 is, for example, a keyboard, a mouse, or a touch panel. Display unit 132 is a display device that displays various screens, and displays the processing status of freezing device 100. Display unit 132 includes, for example, a display such as a liquid crystal display or an organic EL (Electro Luminescence) display.
[0029] The freezing device 100 further includes a pump 151 , an X-axis direction motor 161 , a Y-axis direction motor 162 , and an eject motor 171 .
[0030] The pump 151 is driven to discharge and suck air through the nozzle 71. The X-axis direction motor 161 is driven to move the electric pipettor 70 in the X-axis direction. The Y-axis direction motor 162 is driven to move the nozzle 71 of the electric pipettor 70 in the Y-axis direction. The freezing apparatus 100 is equipped with members that constitute a mechanism for connecting the electric pipettor 70 to the X-axis direction motor 161 and the Y-axis direction motor 162.
[0031] The eject motor 171 drives the caps 300 attached to the tips of one or more nozzles 71 to detach them from the nozzles 71. The electric pipetter 70 is equipped with members that constitute a mechanism for detaching the caps 300 from the nozzles 71.
[0032] In the freezing device 100, the control device 110, the pump 151, the X-axis direction motor 161, the Y-axis direction motor 162, and the eject motor 171 are housed in a case 101, for example.
[0033] <Processing flow> FIG. 5 is a flowchart of a process performed in freezing device 100. In one implementation example, freezing device 100 performs the process of FIG. 5 by having CPU 111 execute a given program. Each of FIGS. 6 and 7 is a flowchart of a subroutine of the process shown in FIG. 5. Each of FIGS. 8 to 28 is a diagram showing the state of freezing device 100. The process of FIG. 5 starts in a state in which a target 900 to be frozen is contained in tube 200, as shown in FIG. 8. The target 900 to be frozen is placed in tube 200 manually or by freezing device 100.
[0034] Referring to FIG. 5, in step SA10, the freezing apparatus 100 attaches the caps 300 to the nozzles 71. In one implementation example, the freezing apparatus 100 drives the Y-axis motor 162 to change the position of the nozzles 71 from the position shown in FIG. 8 to the position shown in FIG. 9. As a result, each of the one or more nozzles 71 is inserted into each of the one or more caps 300 placed on the cap holder 10. Thereafter, the freezing apparatus 100 drives the Y-axis motor 162 to change the position of the nozzles 71 from the position shown in FIG. 9 to the position shown in FIG. 10. As a result, each of the one or more nozzles 71 with the caps 300 attached is pulled up.
[0035] In step SA20, freezing apparatus 100 performs equilibration of freezing target 900. Figure 6 shows the subroutine of step SA20.
[0036] 6, in step SA210, the freezing apparatus 100 moves the tube 200 to the ES reservoir 30. An example of the movement of the tube 200 to the ES reservoir 30 will be described with reference to FIGS.
[0037] The freezing device 100 moves the electric pipetter 70, with the cap 300 attached to the nozzle 71 as shown in FIG. 10, to above the tube holder 20 (FIG. 11).
[0038] Thereafter, the freezing device 100 moves the nozzle 71 downward (FIG. 12). As a result, the cap 300 is fixed to the tube 200 in a state where the cap 300 is inserted into the upper end 203 of the tube 200. In other words, the cap 300 and the tube 200 are attached to the nozzle 71.
[0039] Thereafter, the freezing apparatus 100 moves the nozzle 71 upward (FIG. 13), and moves the electric pipetter 70 to above the ES reservoir 30 (FIG. 14).Then, the freezing apparatus 100 moves the nozzle 71 downward to a position where the bottom 202 of the tube 200 faces the ES in the ES reservoir 30 (FIG. 15).
[0040] 6, in step SA220, the freezing apparatus 100 injects the ES into the tube 200. The injection of the ES into the tube 200 will be described with reference to FIGS.
[0041] When the bottom 202 of the tube 200 is positioned facing the ES as shown in Fig. 15, the freezing apparatus 100 causes the nozzle 71 to suck air. This causes the ES 31 to be injected into the tube 200 through the opening in the bottom 202 as shown in Fig. 16. The ES 31 injected into the tube 200 remains within the tube 200 due to surface tension.
[0042] Returning to Fig. 6, in step SA230, freezing apparatus 100 discharges ES from tube 200. Thereafter, freezing apparatus 100 returns control to Fig. 5. Discharge of ES will be described with reference to Figs. 16 and 17.
[0043] As shown in Fig. 16, the freezing device 100 discharges air from the nozzle 71 into the tube 200 when the ES 31 has been poured into the tube 200. This causes the ES to be discharged from the gaps in the mesh of the bottom 202 of the tube 200, as shown in Fig. 17. In this sense, the bottom 202 is an example of a placement portion that allows liquid to pass through and on which the object to be frozen can be placed. The freezing device 100 may further include a container for storing the ES discharged from the tube 200.
[0044] Returning to Fig. 5, in step SA30, freezing apparatus 100 vitrifies freezing target 900. Fig. 7 shows a subroutine of step SA30.
[0045] 7, in step SA310, freezing apparatus 100 moves tube 200 to VS reservoir 40. An example of the movement of tube 200 to VS reservoir 40 will be described with reference to FIGS.
[0046] After discharging the ES from the tube 200 (FIG. 17), the freezing apparatus 100 moves the electric pipettor 70 upward (FIG. 18) and then moves it to above the VS reservoir 40 (FIG. 19).The freezing apparatus 100 then moves the nozzle 71 downward to a position where the bottom 202 of the tube 200 faces the VS in the VS reservoir 40 (FIG. 20).
[0047] 7, in step SA320, the freezing apparatus 100 injects VS into the tube 200. The injection of VS into the tube 200 will be described with reference to FIGS.
[0048] When the bottom 202 of the tube 200 is positioned facing the VS as shown in Fig. 20, the freezing apparatus 100 causes the nozzle 71 to suck air. This causes the VS41 to be injected into the tube 200 through the opening in the bottom 202 as shown in Fig. 21. The VS41 injected into the tube 200 remains within the tube 200 due to surface tension.
[0049] Returning to Figure 7, in step SA330, freezing apparatus 100 discharges VS from tube 200. Freezing apparatus 100 then returns control to Figure 5. Discharge of VS will be described with reference to Figures 21 and 22.
[0050] 21, the freezing apparatus 100 discharges air from the nozzle 71 into the tube 200 while the VS 41 is being poured into the tube 200. This causes the VS to be discharged from the tube 200 as shown in FIG. 22. The freezing apparatus 100 may further include a container for containing the discharged VS.
[0051] 5, in step SA40, freezing apparatus 100 freezes object to be frozen 900 in tube 200. Thereafter, freezing apparatus 100 ends the processing in FIG.
[0052] The freezing of the freezing target 900 in step SA40 will be described with reference to FIGS.
[0053] After the VS is discharged from the tube 200 (FIG. 22), the freezing apparatus 100 raises the nozzle 71, to which the tube 200 and cap 300 are attached (FIG. 23), and moves the electric pipetter 70 so that the tube 200 is positioned above the freezing cane 60 in the liquid nitrogen tank 50 (FIG. 24). The freezing apparatus 100 then lowers the nozzle 71 (FIG. 25). This causes the tube 200 to be housed in the freezing cane 60.
[0054] Thereafter, the freezing device 100 drives the eject motor 171 while pulling the nozzle 71 upward, thereby detaching the cap 300 from the nozzle 71 (FIG. 26).
[0055] A lid 61 is then attached to the freezing cane 60 (FIG. 27). Attachment of the lid 61 may be done manually or by the freezing device 100.
[0056] In this manner, the freezing device 100 freezes the object to be frozen. In the processes described with reference to FIGS. 6 to 27, the liquid (ES or VS) was injected into the tube 200 by suction using the nozzle 71. However, the method of injecting the liquid is not limited to this. For example, the liquid may be injected into the tube 200 by immersing the tube 200 in a tank containing the liquid. In this case, the size of the openings (gaps between the meshes) in the bottom 202 may be larger than the size at which the liquid remains in the tube 200 due to surface tension. Also, in the processes described with reference to FIGS. 6 to 27, the size of the openings may be larger than the size at which the liquid remains in the tube 200 due to surface tension.
[0057] In the processes described with reference to FIGS. 6 to 27, the liquid (ES or VS) was discharged from the tube 200 by ejecting air using the nozzle 71. However, the method of discharge is not limited to this. For example, the opening of the bottom 202 may be adjusted so that the liquid is discharged from the tube 200 by pulling the tube 200, which is immersed in the tank, out of the tank. The liquid may be discharged from the tube 200 after a given time has elapsed since the liquid was poured into the tube 200.
[0058] [Embodiment 2] <Automatic freezing treatment device> 28 is a diagram showing a schematic diagram of the overall configuration of freezing device 100A. Freezing device 100A is an example of an automatic freezing processing device. The following describes the freezing device 100A, mainly focusing on changes made to freezing device 100.
[0059] Compared to the freezing apparatus 100 shown in Fig. 1, the freezing apparatus 100A further includes a tip holder 80 and a container 90. The tip holder 80 includes an ES tip holder 81 and a VS tip holder 82. The ES tip holder 81 houses an ES tip 401. The VS tip holder 82 houses a VS tip 402.
[0060] <Processing flow> Fig. 29 is a flowchart of the processing carried out in freezing apparatus 100A. Fig. 30 and Fig. 31 are flowcharts of subroutines of the processing of Fig. 29. Fig. 32 to Fig. 34 are diagrams for explaining the state of freezing apparatus 100A. In one implementation example, freezing apparatus 100A carries out the processing of Fig. 29 by CPU 111 executing a given program. The processing of Fig. 29 starts in a state where the object to be frozen 900 is contained in tube 200, as shown in Fig. 28.
[0061] 29, in step SB10, freezing apparatus 100A performs equilibration of freezing target 900. FIG. 30 shows the subroutine of step SB10.
[0062] 30, in step SB110, freezing apparatus 100A attaches tip 401 to nozzle 71. More specifically, freezing apparatus 100A moves electric pipetter 70 above ES tip holder 81, and then lowers nozzle 71 to a position where nozzle 71 can be fitted into tip 401. As a result, tip 401 is attached to nozzle 71.
[0063] In step SB120, the freezing apparatus 100A injects the ES into the tube 200. More specifically, the freezing apparatus 100A moves the nozzle 71 equipped with the tip 401 upward, moves the electric pipetter 70 to the ES reservoir 30, lowers the nozzle 71, and then drives the pump 151. This causes the tip 401 to aspirate the ES. The freezing apparatus 100A then moves the nozzle 71 upward, moves the electric pipetter 70 to the tube holder 20, and lowers the nozzle 71.
[0064] Then, the freezing apparatus 100 drives the pump 151. This causes the tip 401 to discharge the ES into the tube 200, and the ES is injected into the tube 200. In FIG. 32, the ES injected into the tube 200 is shown as "ES31." In FIG. 32, the arrow A1 indicates the direction in which the ES is discharged.
[0065] Returning to FIG. 30 , in step SB130, freezing apparatus 100A agitates the liquid inside tube 200. In one implementation example, freezing apparatus 100A drives pump 151 to cause tip 401 to discharge air into tube 200. The discharged air agitates the liquid inside tube 200. Freezing apparatus 100A may agitate the liquid inside tube 200 by causing tip 401 to repeatedly discharge and suck air. Freezing apparatus 100A may agitate the liquid inside tube 200 by rocking tube holder 20.
[0066] In step SB140, the freezing apparatus 100A discharges the ES from the tube 200. In one implementation example, the freezing apparatus 100 lowers the nozzle 71 to insert the tip 401 into the top of the tube 200, as shown in FIG. 33. This seals the tube 200 with the tip 401 and the electric pipettor 70. In this state, the freezing apparatus 100 sends air into the tube 200 via the tip 401. This causes the ES in the tube 200 to be discharged through the opening in the bottom 202. In FIG. 33, arrow A2 indicates the direction in which the air is sent. Arrow A3 indicates the direction in which the ES is discharged.
[0067] Returning to Figure 30, in step SB150, freezing apparatus 100A detaches the tip 401 from the nozzle 71. More specifically, freezing apparatus 100A moves the nozzle 71 upward, moves the electric pipetter 70 to above the container 90, and then drives the eject motor 171 to detach the tip 401 from the nozzle 71. The detached tip 401 is stored in the container 90. After step SB150, freezing apparatus 100A returns control to Figure 29.
[0068] Returning to Fig. 29, in step SB20, freezing apparatus 100 vitrifies freezing target 900. Fig. 31 shows the subroutine of step SB20.
[0069] 31 , in step SB210, freezing apparatus 100A attaches tip 402 to nozzle 71. More specifically, freezing apparatus 100A moves electric pipetter 70 above VS tip holder 82, and then moves nozzle 71 to a position where tip 402 can be fitted in. As a result, tip 402 is attached to nozzle 71.
[0070] In step SB220, the freezing apparatus 100A injects VS into the tube 200. More specifically, the freezing apparatus 100A moves the nozzle 71, to which the tip 402 is attached, upward, moves the electric pipetter 70 to the VS reservoir 40, lowers the nozzle 71, and then drives the pump 151. This causes the tip 402 to aspirate the VS. The freezing apparatus 100A then moves the nozzle 71 upward, moves the electric pipetter 70 to the tube holder 20, and lowers the nozzle 71. The freezing apparatus 100A then drives the pump 151. This causes the tip 402 to dispense VS into the tube 200.
[0071] In step SB230, freezing apparatus 100A agitates the liquid inside tube 200, similar to step SB130.
[0072] In step SB240, freezing apparatus 100A discharges VS from tube 200, similar to step SB140.
[0073] In step SB250, similar to step SB150, freezing apparatus 100A detaches tip 402 from nozzle 71. Thereafter, freezing apparatus 100A returns the control to FIG.
[0074] 29, in step SB30, the freezing apparatus 100A attaches the cap 300 to the tube 200. In one implementation example, the freezing apparatus 100A attaches the cap 300, which is attached to the nozzle 71, to the tube 200 as described with reference to FIGS.
[0075] In step SB40, freezing apparatus 100A freezes freezing target 900 in tube 200. Thereafter, freezing apparatus 100A ends the processing of FIG.
[0076] In one implementation example, in step SB40, freezing apparatus 100A accommodates tube 200 and cap 300 attached to nozzle 71 in freezing cane 60, as described with reference to FIGS. 22 to 25. This state is also shown in FIG. 34. Then, freezing apparatus 100A drives eject motor 171 to detach cap 300 from nozzle 71. Then, freezing apparatus 100A returns nozzle 71 to the position shown in FIG. 28. A lid 61 is attached to freezing cane 60.
[0077] 28 to 34, a liquid (ES or VS) is injected into the tube 200. The injected liquid does not leak from the opening in the bottom 202 of the tube 200 due to surface tension. Thereafter, air is discharged into the tube 200, thereby discharging the liquid from the tube 200 through the opening in the bottom 202. The liquid may be discharged from the tube 200 after a given time has elapsed since the liquid was injected into the tube 200.
[0078] [Variations] Fig. 35 is a diagram showing a modified example of the tube 200. As shown in Fig. 35, supports 291, 292 may be attached to the bottom 202 of the tube 200. The supports 291, 292 are made of, for example, the same material as the base 201 of the tube 200. This reliably prevents the mesh structure (or porous structure) of the bottom 202 from being damaged by volume changes caused by temperature changes such as freezing. Supports similar to the supports 291, 292 may also be provided on the bottom 302 of the cap 300.
[0079] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.
[0080] (Item 1) An automatic freezing treatment device according to one embodiment comprises a container for containing an object to be frozen, an operating unit that operates to inject and discharge liquid into the container and to move the container, and a refrigerant storage unit that stores a refrigerant for freezing the object to be frozen, wherein the container has an opening at the bottom that is smaller than the size of the object to be frozen, and the operating unit may inject liquid into the container, then discharge the liquid from the container through the opening and move the container to the refrigerant storage unit.
[0081] According to the automatic freezing treatment device described in paragraph 1, during cryopreservation, situations in which the object to be frozen is erroneously recovered and situations in which liquid that should be recovered remains in the object to be frozen are avoided, while the burden on the operator in pre-processing the object to be frozen is reduced.
[0082] (Item 2) In the automatic freezing treatment device described in item 1, injecting liquid into the container may include injecting the liquid into the container from above the container, and discharging the liquid from the container may include sending air into the container from above.
[0083] According to the automatic freezing treatment device described in item 2, the liquid moves into the container by its own weight, thereby ensuring that the liquid is poured into the container.
[0084] (Item 3) In the automatic freezing treatment device described in item 2, injecting the liquid into the container from above the container may include injecting the liquid using a tip.
[0085] According to the automatic freezing treatment device described in the third aspect, the liquid is more reliably poured into the container. (Item 4) In the automatic freezing treatment device described in item 1, pouring liquid into the container may include sucking the liquid into the container from a tank that contains the liquid.
[0086] According to the automatic freezing treatment device described in item 4, the liquid is reliably poured into the container by suction force.
[0087] (Item 5) In the automatic freezing treatment device described in any one of Items 1 to 4, the operating unit may perform an operation to stir the liquid in the container during the period from when the liquid is poured into the container until when the liquid is discharged from the container.
[0088] According to the automatic freezing treatment device described in item 5, the liquid acts more reliably on the object to be frozen in the container.
[0089] (Item 6) The automatic freezing treatment device according to item 5 may further include a pump that discharges air into the container for the operation.
[0090] According to the automatic freezing treatment device described in item 6, agitation of the liquid can be easily achieved. (7th Item) In the automatic freezing treatment device according to any one of the first to sixth items, the operating section may attach a cap to the container.
[0091] According to the automatic freezing treatment device described in item 7, the object to be frozen in the container can be more reliably prevented from leaking out of the container.
[0092] (Item 8) In the automatic freezing treatment device described in item 7, the cap may have an opening that is smaller in size than the object to be frozen.
[0093] According to the automatic freezing treatment device described in item 8, liquid can be poured into the container with the cap attached, while preventing the object to be frozen from leaking out of the container.
[0094] (Item 9) In the automatic freezing treatment device described in items 1 to 8, injecting a liquid into the container may include injecting an equilibration liquid into the container, and after the equilibration liquid is discharged from the container, injecting a vitrification liquid into the container.
[0095] According to the automatic freezing treatment device described in item 9, the burden on the operator is reduced in both equilibration and vitrification of the frozen object.
[0096] (Item 10) An automatic freezing treatment method according to one embodiment may include the steps of injecting a liquid into a container having an opening at the bottom that is smaller than the size of the object to be frozen, discharging the liquid from the container through the opening after the liquid has been injected, and moving the container to a refrigerant storage section after the liquid has been discharged.
[0097] According to the automatic freezing processing method described in paragraph 10, during cryopreservation, situations in which the frozen object is erroneously recovered and situations in which liquid that should be recovered remains at the frozen object are avoided, while the burden on the operator in pre-processing the frozen object is reduced.
[0098] (Item 11) A freezing container according to one embodiment is a freezing container for immersing an object to be frozen in a liquid to pre-treat it for freezing, and may include a placement portion that allows the liquid to pass through and on which the object to be frozen can be placed.
[0099] The freezing container described in paragraph 11 reduces the burden on workers in pre-processing the frozen object while preventing the frozen object from being erroneously retrieved and the liquid that should be retrieved from the frozen object from remaining in the frozen object during cryopreservation.
[0100] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present disclosure is defined by the claims, not by the description of the above embodiments, and is intended to include all modifications within the meaning and scope of the claims. Furthermore, it is intended that each technique in the embodiments can be implemented alone or, if necessary, in combination with other techniques in the embodiments to the extent possible. [Explanation of symbols]
[0101] 10 cap holder, 20 tube holder, 30 ES reservoir, 40 VS reservoir, 50 liquid nitrogen tank, 60 freezing cane, 61 lid, 70 electronic pipettor, 71 nozzle, 80 tip holder, 81 ES tip holder, 82 VS tip holder, 90 container, 100, 100A freezing device, 101 case, 200 tube, 201, 301 base, 202, 302 bottom, 203, 303 top, 300 cap, 401, 402 tip, 900 freezing target.
Claims
1. a container for containing an object to be frozen; an actuation unit that operates to inject and discharge liquid into and from the container and to move the container; a refrigerant containing section that contains a refrigerant for freezing an object to be frozen, The container has an opening at the bottom that is smaller than the size of the object to be frozen, The actuation unit is After injecting a liquid into the container, the liquid is discharged from the container through the opening; An automatic freezing treatment device that moves the container to the refrigerant containing section.
2. Injecting the liquid into the container includes injecting the liquid into the container from above the container; The automatic freezing treatment device of claim 1 , wherein discharging the liquid from the container includes blowing air into the container from above.
3. The automatic freezing treatment device according to claim 2 , wherein injecting the liquid into the container from above the container includes injecting the liquid using a tip.
4. The automatic freezing treatment device of claim 1 , wherein injecting the liquid into the container comprises aspirating the liquid into the container from a tank containing the liquid.
5. The automatic freezing treatment device according to any one of claims 1 to 4, wherein the operating unit performs an operation to stir the liquid in the container between the time when the liquid is poured into the container and the time when the liquid is discharged from the container.
6. The automatic freeze treatment device of claim 5 further comprising a pump that expels air into the container for said operation.
7. The automatic freezing treatment device according to any one of claims 1 to 6, wherein the operating unit attaches a cap to the container.
8. The automatic freezing treatment device according to claim 7 , wherein the cap has an opening smaller in size than the size of the object to be frozen.
9. Injecting a liquid into the container comprises: injecting an equilibration fluid into the container; The automatic freezing treatment device according to any one of claims 1 to 8, further comprising: injecting a vitrification liquid into the container after the equilibration liquid has been discharged from the container.
10. Pouring liquid into a container having an opening at the bottom that is smaller than the size of the object to be frozen; After the liquid is poured, the liquid is discharged from the container through the opening; and after the liquid is discharged, moving the container to a refrigerant container.
11. A freezing container for pre-treating a frozen object by immersing it in a liquid, A freezing container comprising a placement portion through which the liquid can pass and on which the object to be frozen can be placed.