Cell freezing pretreatment plate

Abstract

This cell cryopreservation pretreatment operation plate 1 is provided with a tabular operation part 2, a first recessed part 14 for accommodating a treatment solution therein, a second recessed part 15 for accommodating a treatment solution therein, a third recessed part 16 for accommodating a treatment solution therein, and a fourth recessed part 18 for accommodating a waste treatment solution therein. The surface of the tabular operation part 2 has, in order to hold a treatment solution, a first circular compartment 21, a second circular compartment 22, a third circular compartment 23, an expanded first circular compartment 61 enclosing the first circular compartment 21, an expanded second circular compartment 62 enclosing the second circular compartment 22, and an expanded third circular compartment 63 enclosing the third circular compartment 23.

Description

[Technical field]

[0001] The present invention relates to a cell pre-freezing treatment plate used for cryopreserving biological cells such as mammalian ova, eggs such as embryos, sperm, hematopoietic stem cells, pluripotent stem cells, and other stem cells. [Background technology]

[0002] Cryopreservation of mammalian embryos allows the preservation of genetic resources of specific lineages or breeds, is effective in maintaining endangered animal species, and is also useful in the treatment of human infertility.

[0003] As a method for cryopreserving mammalian embryos, JP 2000-189155 A (Patent Document 1) proposes attaching mammalian embryos or eggs to the inner surface of a cryopreservation container such as a sterilized freezing straw, freezing vial, or freezing tube with a minimum amount of vitrification liquid sufficient to encapsulate the embryos or eggs, sealing the cryopreservation container, and contacting the container with liquid nitrogen to rapidly cool it.

[0004] The present inventors have also proposed Patent Document 2 (JP Patent Publication 2002-315573, WO 02-085110 A1). The egg freezing storage device 1 in Patent Document 2 comprises a main body 2 made of a cold-resistant material, an egg attachment / retention strip 3 attached to one end of the main body 2 and made of a flexible, transparent, liquid nitrogen-resistant material, and a cylindrical member 4 made of a cold-resistant material and having one end closed, which is detachably attached to the main body 2 so as to be able to encapsulate the egg attachment / retention strip 3.

[0005] Japanese Patent Application Laid-Open No. 2017-118884 (Patent Document 3) proposes a cell treatment container for use in treating cells with a treatment liquid, such as for treating embryos immediately before transplantation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication 2000-189155 [Patent Document 2] Patent Publication No. 2002-315573 (WO 02-085110 A1) [Patent Document 3] Patent Publication No. 2017-118884 (WO 2016-190322 A1) Summary of the Invention [Problem to be solved by the invention]

[0007] The vitrification method is used for the above-mentioned freezing and preservation of eggs. The vitrification method utilizes the principle that ice crystals are unlikely to form even below freezing due to the freezing point depression of an aqueous solution containing a cryoprotectant such as glycerol or ethylene glycol. When this solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals, and the water becomes a glass-like solid without crystallizing, which is called vitrification. In order to freeze eggs by the vitrification method, pretreatment of the eggs is required. The treatment container in Patent Document 3 is not for pretreatment for vitrification freezing, but is a container for treating embryos immediately before implantation.

[0008] Conventionally, there have been no dedicated containers for the pretreatment of vitrification. Therefore, each clinic currently uses regular petri dishes, multi-well petri dishes, center well dishes, etc., to minimize operational errors and shorten the time required for transplantation.

[0009] An object of the present invention is to provide a cell pre-freezing treatment plate which allows pre-treatment for vitrification to be easily performed with fewer procedural errors. [Means for solving the problem]

[0010] The above objectives are achieved by the following: A cell pre-freezing treatment plate, the plate comprising a work flat section, a first recess for storing a low-concentration treatment liquid containing a low concentration of a cell membrane-permeable cryoprotectant, a second recess for storing a high-concentration treatment liquid containing a high concentration of a cell membrane-permeable cryoprotectant, a third recess for storing a high-concentration treatment liquid containing a high concentration of a cell membrane-permeable cryoprotectant, and a fourth recess for storing a waste liquid, and further comprising a cell membrane-permeable cryoprotectant-free treatment liquid on the surface of the work flat section. The work plate has a first circular partition for placing a low-concentration treatment liquid, a second circular partition for placing the low-concentration treatment liquid, and a third circular partition for placing the low-concentration treatment liquid, the first circular partition, the second circular partition, and the third circular partition being adjacent to each other, and further, a surface of the work plate has an enlarged first circular partition surrounding the first circular partition, an enlarged second circular partition surrounding the second circular partition, and an enlarged third circular partition surrounding the third circular partition. The outer edges of the enlarged first circular partition, the enlarged second circular partition, and the enlarged third circular partition are annular grooves, the outer edges of the first circular partition, the second circular partition, and the enlarged third circular partition are annular grooves, the enlarged first circular partition surrounds the first circular partition in a state of contact with the inner side and outer edge of the enlarged first circular partition, the enlarged second circular partition surrounds the second circular partition in a state of contact with the inner side and outer edge of the enlarged second circular partition, and the enlarged third circular partition surrounds the third circular partition in a state of contact with the inner side and outer edge of the enlarged third circular partition. A plate for pre-freezing cells.

[0011] The above object is also achieved by the following: A cell pre-freezing processing working plate, the plate comprising a working flat portion, a first recess for accommodating a processing liquid, a second recess for accommodating a processing liquid, a third recess for accommodating a processing liquid, and a fourth recess for accommodating a waste processing liquid, the surface of the working flat portion further comprising a first circular partition for placing a processing liquid, a second circular partition for placing a processing liquid, and a third circular partition for placing a processing liquid, the first circular partition, the second circular partition, and the third circular partition being adjacent to each other, the surface of the working flat portion further comprising an enlarged first circular partition surrounding the first circular partition, an enlarged second circular partition surrounding the second circular partition, and an enlarged third circular partition surrounding the third circular partition, The outer edges of the enlarged first circular partition, the enlarged second circular partition, and the enlarged third circular partition are annular grooves, the outer edges of the first circular partition, the second circular partition, and the enlarged third circular partition are annular grooves, the enlarged first circular partition surrounds the first circular partition in a state of contact with the inner side and outer edge of the enlarged first circular partition, the enlarged second circular partition surrounds the second circular partition in a state of contact with the inner side and outer edge of the enlarged second circular partition, and the enlarged third circular partition surrounds the third circular partition in a state of contact with the inner side and outer edge of the enlarged third circular partition. A plate for pre-freezing cells. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a front view of a cell pre-freezing treatment working plate having a basic structure of the cell pre-freezing treatment working plate of the present invention. [Diagram 2] FIG. 2 is a cross-sectional view taken along line AA in FIG. [Diagram 3] FIG. 3 is a cross-sectional view taken along line BB in FIG. [Figure 4] FIG. 4 is a perspective view of the cell pre-freezing treatment plate shown in FIG. [Diagram 5] FIG. 5 is a front view of the cell freezing pretreatment plate shown in FIG. 1 with a lid placed thereon. [Figure 6] FIG. 6 is a perspective view of a lid placed on the cell pre-freezing treatment plate shown in FIG. [Figure 7] FIG. 7 is an enlarged view of the flat portion of the cell pre-freezing treatment plate shown in FIG. [Figure 8] FIG. 8 is an enlarged cross-sectional view taken along line CC in FIG. [Figure 9] FIG. 9 is an enlarged cross-sectional view taken along line DD in FIG. [Figure 10] FIG. 10 is a front view of another example of a cell pre-freezing treatment working plate having the basic structure of the cell pre-freezing treatment working plate of the present invention. [Figure 11] FIG. 11 is a front view of another example of a cell pre-freezing treatment working plate having the basic structure of the cell pre-freezing treatment working plate of the present invention. [Figure 12] FIG. 12 is a front view of a cell pre-freezing treatment plate according to an embodiment of the present invention. [Figure 13] FIG. 13 is a front view of a cell pre-freezing treatment plate according to another embodiment of the present invention. [Figure 14] FIG. 14 is a front view of a cell pre-freezing treatment plate according to another embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The cell pre-freezing treatment plate of the present invention will be described with reference to the embodiment shown in the drawings.

[0014] A cell pre-freezing treatment working plate having the basic configuration of the cell pre-freezing treatment working plate of the present invention comprises a working flat portion 2, a first recess 14 for storing a low-concentration treatment liquid containing a low concentration of a cell membrane-permeable cryoprotectant, a second recess 15 for storing a high-concentration treatment liquid containing a high concentration of a cell membrane-permeable cryoprotectant, a third recess 16 for storing a high-concentration treatment liquid containing a high concentration of a cell membrane-permeable cryoprotectant, and a fourth recess 18 for storing a waste liquid, and further comprises, on the surface of the working flat portion 2, a first circular partition 21 for placing a treatment liquid not containing a cell membrane-permeable cryoprotectant, a second circular partition for placing a low-concentration treatment liquid, and a third circular partition for placing a low-concentration treatment liquid, and the first circular partition 21, the second circular partition 22, and the third circular partition 23 are adjacent to each other.

[0015] Furthermore, a cell freezing pretreatment working plate having the basic structure of the cell freezing pretreatment working plate of the present invention may be as follows. The cell freezing pretreatment working plate of this embodiment comprises a working plate portion 2, a first recess 14 for storing processing liquid, a second recess 15 for storing processing liquid, a third recess 16 for storing processing liquid, and a fourth recess 18 for storing waste processing liquid, and further comprises, on the surface of the working plate portion 2, a first circular partition 21 for placing processing liquid, a second circular partition 22 for placing processing liquid, and a third circular partition 23 for placing processing liquid, and the first circular partition 21, the second circular partition 22 and the third circular partition 23 are located close to each other.

[0016] Furthermore, a cell freezing pretreatment working plate having the basic structure of the cell freezing pretreatment working plate of the present invention may be as follows.

[0017] This cell pre-freezing treatment working plate comprises a working flat plate portion 2, a first recess 14 for storing a treatment liquid, a second recess 15 for storing a treatment liquid, a third recess 16 for storing a treatment liquid, and a fourth recess 18 for storing a waste treatment liquid. Furthermore, the surface of the working flat plate portion 2 is provided with a first circular partition 21 for placing a treatment liquid, a second circular partition 22 for placing a treatment liquid, and a third circular partition 23 for placing a treatment liquid, and the first circular partition 21, the second circular partition 22, and the third circular partition 23 are arranged close to one another. The work plate portion 2 is in contact with the first circular compartment 21, and further comprises a first display portion 26 having an indication of the name and amount of the processing liquid to be placed in the first circular compartment 21, a second display portion 27 having an indication of the name and amount of the processing liquid to be placed in the second circular compartment 22, and a third display portion 28 having an indication of the name and amount of the processing liquid to be placed in the third circular compartment 23, and further the cell freezing pretreatment work plate 1 has a fourth display portion 29 having an indication of the name and amount of the processing liquid stored in the first recess 14.

[0018] 1 to 4 includes a lower annular wall 4, an annular side wall 3 extending upward from the lower annular wall 4, a bottom portion 6 located within the annular side wall 3, and an upper surface portion 12 provided at one end of the lower annular wall 4. In the plate 1 of this embodiment, the lower annular wall 4 is substantially rectangular, and is larger than the annular side wall 3. On the upper surface of the lower annular wall 4 in the portion that protrudes from the annular side wall 3, the above-mentioned horizontally elongated upper surface portion 12 and a narrow U-shaped flat portion 13 that connects both sides and the lower end of the innermost portion of the annular side wall 3 to the upper end of the lower annular wall 4 are provided.

[0019] The lower annular wall 4 is a rectangular annular wall with curved corners. One end of the lower annular wall, specifically the lower end (near side) in FIG. 1, is provided with an upper surface portion 12 that is connected to the upper end of the lower annular wall 4. The upper surface portion 12 is flat, and characters or the like can be written on the surface with a writing implement. In addition, it is preferable that the surface of the upper surface portion 12 is a rough surface, specifically a matte surface. This makes it easier to write with a writing implement or the like, and also makes it easier to see the written characters or the like.

[0020] The cell freezing pretreatment working plate 1 also has an annular side wall 3 extending upward from the lower annular wall 4 except for the upper surface portion 12. A narrow U-shaped flat portion 13 is provided connecting both sides and the lower end of the innermost portion of the annular side wall 3 to the upper end of the lower annular wall 4. Therefore, a step is formed between the lower annular wall 4 and the annular side wall 3 by the upper surface portion 12 and the flat portion 13. The upper surface of this step serves as a mounting portion for the lid 5. As shown in FIG. 3, the annular side wall 3 is formed higher than the upper surface portion 12. In this embodiment, the annular side wall 3 is a rectangular annular wall with curved corners as shown in FIGS. 1 to 4. Inside the annular side wall 3, there are provided a working flat plate portion 2, a first recess 14 for storing processing liquid, a second recess 15 for storing processing liquid, a third recess 16 for storing processing liquid, and a fourth recess 18 for storing waste processing liquid.

[0021] 4, in this cell freezing pretreatment working plate 1, the working plate 2 is provided at a position slightly below the upper end of the annular side wall 3, and is a substantially rectangular plate. In this embodiment, a second plate 17 is provided, and a first recess 14 is formed in the second plate 17. Specifically, the second plate 17 is formed so as to be adjacent to the working plate 2, and the upper surface thereof is at substantially the same height as the upper surface of the working plate 2.

[0022] A first recess 14 is formed in the center of the second flat plate portion 17. The first recess 14 has a flat, small circular bottom surface and tapered side surfaces that decrease in diameter toward the bottom surface. Therefore, the first recess 14 has an inverted truncated cone shape (the cross-sectional area decreases toward the bottom surface). The first recess 14 is a recess for storing a cell treatment solution, and more specifically, a recess for storing a low-concentration treatment solution that contains a low concentration of a cell membrane-permeable cryoprotectant.

[0023] As shown in Figs. 1 to 4, the cell pre-freezing treatment working plate 1 has a first protruding portion 41 protruding upward from the bottom 6 inside the annular side wall 3, and a second recess 15 is formed in the first protruding portion 41. Furthermore, the cell pre-freezing treatment working plate 1 of this embodiment has a second protruding portion 42 protruding upward from the bottom 6 inside the annular side wall 3, and a third recess 16 is formed in the second protruding portion 42. The second recess 15 and the third recess 16 have a flat, small circular bottom surface and a tapered side surface that decreases in diameter toward the bottom surface. Therefore, the second recess 15 and the third recess 16 are inverted truncated cone shape (the cross-sectional area decreases toward the bottom surface). The height of the annular side wall 3, in other words the distance between the upper end of the annular side wall 3 and the upper ends of the working plate portion 2, the second plate portion 17, the first protrusion 41 (first recess 15), and the second protrusion 42 (first recess 16), is preferably 1 to 5 mm, and particularly preferably 1.5 to 4 mm.

[0024] The second recess 15 is a recess for storing a cell treatment solution, specifically, a high-concentration treatment solution containing a high concentration of a cell membrane-permeable cryoprotectant. Similarly, the third recess 16 is a recess for storing a cell treatment solution, specifically, a high-concentration treatment solution containing a high concentration of a cell membrane-permeable cryoprotectant. The concentrations of the cell membrane-permeable cryoprotectant in the treatment solutions stored in the second recess 15 and the third recess 16 may be different.

[0025] Furthermore, the cell pre-freezing treatment working plate 1 is provided with a fourth recess 18 for storing waste liquid, as shown in Figures 1 to 4. Specifically, the recess formed by the side surfaces of the protrusions 41 and 42, the inner surface of the annular side wall, and the bottom 6 serves as the fourth recess 18. As shown in Figure 7, the surface of the working plate portion 2 is provided with a first circular partition 21 for placing a processing liquid, a second circular partition 22 for placing a processing liquid, and a third circular partition 23 for placing a processing liquid.

[0026] The first circular compartment 21 is a circular compartment for placing a treatment liquid not containing a cell membrane-permeable cryoprotectant, the second circular compartment 22 is a circular compartment for placing a treatment liquid containing a low concentration of a cell membrane-permeable cryoprotectant, and the third circular compartment 23 is a circular compartment for placing a treatment liquid containing a low concentration of a cell membrane-permeable cryoprotectant. The size (diameter) of each of the circular compartments 21, 22, 23 is preferably 3 to 8 mm, and more preferably 4 to 7 mm. The area of ​​each of the circular compartments 21, 22, 23 is preferably 7 to 50 mm. 2 is preferable, and particularly, 12 to 40 mm 2 is preferred.

[0027] The circular partitions 21, 22, and 23 are preferably of approximately the same size, but may be different from each other. Also, it is preferable that a predetermined amount of processing liquid can be placed in each of the circular partitions 21, 22, and 23. The amount of processing liquid that can be placed is preferably 10 to 50 μL, and more preferably 15 to 25 μL.

[0028] The first circular partition 21, the second circular partition 22, and the third circular partition 23 are close to each other. The distance between the first circular partition 21 and the second circular partition 22 is preferably 1 to 5 mm, and more preferably 1.5 to 3 mm. The third circular partition 23 is preferably close to the first circular partition 21 and the second circular partition 22 and is spaced apart from them by an equal distance, as shown in FIG. 7. Specifically, as shown in FIG. 7, the third circular partition 23 is preferably located in front of the intermediate portion between the first circular partition 21 and the second circular partition 22. The distance between the first circular partition 21 and the second circular partition 22 and the third circular partition 23 is preferably 1 to 5 mm, and more preferably 1.5 to 3 mm.

[0029] 7 to 9, the outer edge 21a of the first circular partition 21, the outer edge 22a of the second circular partition 22, and the outer edge 23a of the third circular partition 23 are preferably annular grooves. The outer edges 21a, 22a, and 23a may be annular ribs.

[0030] 1 and 7, in the cell pre-freezing treatment working plate 1, the working flat plate portion 2 has a first display portion 26 having an indication of the name and amount of the treatment liquid to be placed in the first circular compartment 21. "BS" in the display portion 26 is an abbreviation for "Basic Solution" and indicates a treatment liquid not containing a cell membrane-permeable cryoprotectant, and is an indication that informs the operator that the treatment liquid to be placed in the first circular compartment 21 is a treatment liquid not containing a cell membrane-permeable cryoprotectant, and "20 μL" next to it is an indication that informs the operator of the amount to be placed.

[0031] As the BS (cell membrane permeable cryoprotectant-free treatment solution), a conventionally used one is preferably used. For example, as the cell membrane permeable cryoprotectant-free treatment solution, a cell culture basal solution (e.g., a cell culture solution containing HEPES [4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid]) is preferably used, which does not contain a cell membrane permeable cryoprotectant, and contains a necessary compound, for example, one or more cell membrane non-permeable cryoprotectants selected from at least one water-soluble cellulose selected from the group consisting of sucrose, trehalose, percoll, polyethylene glycol, polyvinylpyrrolidone, bovine serum albumin, ficoll, hydroxypropyl cellulose, hydroxypropyl methylcellulose, and hydroxyethyl methylcellulose, and an antibiotic such as gentamicin.

[0032] 1 and 7, the cell pre-freezing treatment working plate 1 has a second display section 27 having an indication of the name and amount of the treatment liquid to be placed in the second circular compartment 22. "ES" in the display section 27 is an abbreviation for "Equilibration Solution" and indicates a low-concentration treatment liquid containing a low concentration of a cell membrane-permeable cryoprotectant, and is an indication that informs the operator that the treatment liquid to be placed in the second circular compartment 22 is a low-concentration treatment liquid, and "20 μL" next to it is an indication that informs the operator of the amount to be placed.

[0033] 1 and 7, the cell pre-freezing treatment working plate 1 has a third display section 28 having an indication of the name and amount of the treatment liquid to be placed in the third circular compartment 23. "ES" in the display section 28 is an abbreviation for "Equilibration Solution" and indicates a low-concentration treatment liquid containing a low concentration of a cell membrane-permeable cryoprotectant, and is an indication that informs the operator that the treatment liquid to be placed in the third circular compartment 23 is a low-concentration treatment liquid, and "20 μL" next to it is an indication that informs the operator of the amount to be placed.

[0034] As the ES (low-concentration treatment solution containing a low concentration of a cell membrane-permeable cryoprotectant), a conventionally used one is preferably used. For example, as a low-concentration treatment solution containing a low concentration of a cell membrane-permeable cryoprotectant, a cell culture base solution (e.g., a cell culture solution containing HEPES [4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid]) to which a cell membrane-permeable cryoprotectant such as glycerol, propylene glycol, dimethylsulfoside (DMSO), ethylene glycol, butanediol, etc. is added at a low concentration, and a cell membrane-impermeable cryoprotectant such as sucrose, trehalose, percoll, polyethylene glycol, polyvinylpyrrolidone, bovine serum albumin, ficoll, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, etc. is preferably contained. Furthermore, this ES may also contain an antibiotic such as gentamicin. Furthermore, the content of the cell membrane-permeable cryoprotectant in this ES is preferably 20 to 70% of the content of the cell membrane-permeable cryoprotectant in the "VS" (high-concentration treatment solution containing a high concentration of the cell membrane-permeable cryoprotectant) described below, and particularly preferably 30 to 60%.

[0035] 1, the cell pre-freezing treatment working plate 1 has a fourth display section 29 having an indication of the name and amount of the treatment solution stored in the first well 14. "ES" in the display section 29 is an abbreviation for "Equilibration Solution" and indicates a low-concentration treatment solution that contains a low concentration of a cell membrane-permeable cryoprotectant, and is an indication that informs the operator that the treatment solution stored (injected) in the first well 14 is a low-concentration treatment solution, and "300 μL" next to it informs the operator of the injection amount.

[0036] In addition, the cell pre-freezing treatment work plate may have a fifth display section 43 having an indication of the name and amount of the treatment liquid stored in the second recess 15, and a sixth display section 44 having an indication of the name and amount of the treatment liquid stored in the third recess 16, like plate 1a shown in Figure 10.

[0037] In addition, the cell pre-freezing treatment work plate may have a fourth recess divided by a partition 46 into recess 18a on the second recess side and recess 18b on the third recess side, as in plate 1a shown in Figure 10.

[0038] "VS" in display units 43 and 44 is an abbreviation for "Vitrification Solution," indicating a high-concentration treatment solution containing a high concentration of a cell membrane-permeable cryoprotectant. This display informs the operator that the treatment solution stored (injected) in second recess 15 and third recess 16 is a high-concentration treatment solution, and the "300 μL" next to it informs the operator of the amount to be injected.

[0039] As the VS (high-concentration treatment solution containing a high concentration of a cell membrane-permeable cryoprotectant), a conventionally used one is preferably used. For example, as a high-concentration treatment solution containing a high concentration of a cell membrane-permeable cryoprotectant, a cell culture base solution (e.g., a cell culture solution containing HEPES [4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid]) containing a high concentration of a cell membrane-permeable cryoprotectant such as glycerol, propylene glycol, dimethylsulfoside (DMSO), ethylene glycol, butanediol, etc., and containing a cell membrane-impermeable cryoprotectant such as sucrose, trehalose, percoll, polyethylene glycol, polyvinylpyrrolidone, bovine serum albumin, ficoll, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, etc. may be used. Furthermore, this VS may also contain an antibiotic such as gentamicin. Furthermore, the content of the cell membrane-permeable cryoprotectant in this VS is preferably 1.5 to 3 times, and particularly preferably 1.7 to 2.5 times, the content of the cell membrane-permeable cryoprotectant in the above-mentioned "ES" (low-concentration treatment solution containing a low concentration of the cell membrane-permeable cryoprotectant).

[0040] 1 and 7, the surface of the working flat plate 2 of the cell freezing pretreatment plate 1 has a first arrow 24 extending from the second circular compartment 22 toward the first circular compartment 21, and a second arrow 25 extending from the third circular compartment 23 toward the first arrow 24. The second arrow 25 may extend toward the first circular compartment 21.

[0041] In addition, in the cell pre-freezing treatment working plate 1, as shown in FIG. 1 and FIG. 7, the surface of the working flat plate portion 2 is As shown in Figure 7, The first step order display appears near the first arrow display. 35 (1) and a second step order display near the second arrow display 361, the second flat plate portion 17 is provided with a third step order display (3). Furthermore, in the cell freezing pretreatment operation plate 1 of this embodiment, a first step time display (4) is provided near the first step order display (1). 37 The second step time display is displayed near the second step order display (2). 38 The third step time display is provided near the third step order display (3).

[0042] In the cell freezing pretreatment working plate 1, as shown in Fig. 1 and Fig. 4, the second plate portion 17 having the first recess 14 is located next to (on the right side of) the working plate portion 2, and the second recess 15, the third recess 16, and the fourth recess 18 are located in front of the working plate portion 2 and the second plate portion 17, forming a rectangular working surface. The central portion of the rectangular working surface is provided with a working order display (1, 2, 3, 4) showing the working order of the working plate portion 2, the first recess 14, the second recess 15, and the third recess 16. The working order display 3, 4 may be reversed.

[0043] and, Thin As shown in Figs. 5 and 6, the cell freezing pretreatment working plate preferably includes a lid 5 that simultaneously covers the working flat plate portion 2, the first recess 14, the second recess 15, and the third recess 16. As shown in Fig. 6, the lid 5 of this embodiment covers the entire rectangular working surface described above, and the upper surface portion 12 is exposed when the lid 5 is fitted. Therefore, the lid 5 does not hinder work such as writing characters on the upper surface portion 12. The lid 5 of this embodiment also has a top plate portion 51 and an annular side wall 52 that extends downward from the top plate portion 51. The annular side wall 52 has a plurality of pairs of slits 53 facing each other on the side surface. Furthermore, ribs 54 facing each other are provided on the inner upper surface of the lid 5.

[0044] The cell freezing pretreatment work plate 1 and the lid 5 are made of a synthetic resin material. It is desirable that the cell freezing pretreatment work plate 1 and the lid 5 have high transparency, and a transparent hard synthetic resin is suitable. As the transparent hard synthetic resin, it is preferable to use a styrene-based resin such as polystyrene or SBS, a polycarbonate, an acrylonitrile-based resin, a polyolefin such as polypropylene or polyethylene, a polyester resin such as polyvinyl chloride, PMMA (polymethyl methacrylate), polyethylene terephthalate, or polybutylene terephthalate.

[0045] Next, a cell freezing pretreatment working plate 1b shown in Fig. 11 will be described. The basic configuration of this plate 1b is the same as that of the above-mentioned plate 1. The difference between the two is the arrangement positions of the second recess 15, the third recess 16, and the fourth recess 18 relative to the working flat plate portion 2 and the first recess 14.

[0046] In the cell pre-freezing treatment working plate 1b, the second flat plate portion 17 having the first recess 14 is located next to (on the right side of) the working flat plate portion 2, and the second recess 15, the third recess 16, and the fourth recess 18 are located next to (on the right side of) the second flat plate portion 17, forming a horizontally long rectangular working surface. Therefore, in the plate 1b, the lower annular wall 4a and the annular side wall 3a are also horizontally long rectangular, and the upper surface portion 12 is also horizontally long. In the plate 1b, a narrow flat portion 13 is provided that connects both sides and the lower end of the innermost portion of the annular side wall 3a to the upper end of the lower annular wall 4a. In the plate 1b, a working order display (1, 2, 3, 4) is provided to indicate the working order of the working flat plate portion 2, the first recess 14, the second recess 15, and the third recess 16.

[0047] Next, a cell freezing pretreatment working plate 1c according to an embodiment of the present invention shown in Fig. 12 will be described. The basic configuration of this plate 1c is the same as that of the above-mentioned plate 1. The difference between the two is the configuration of the working plate 2 and the arrangement positions of the working plate 2, the first recess 14, the second recess 15, the third recess 16, and the fourth recess 18.

[0048] In the plate 1c of this embodiment, as shown in Fig. 12, the working plate portion 2a is larger than the working plate portion 2 of the plate 1 described above. The surface of the working plate portion 2a has a first circular partition 21, a second circular partition 22, a third circular partition 23, an enlarged first circular partition 61 surrounding the first circular partition 21, an enlarged second circular partition 62 surrounding the second circular partition 22, and an enlarged third circular partition 63 surrounding the third circular partition 23. The sizes, arrangements, etc. of the first circular partition 21, the second circular partition 22, and the third circular partition 23 are the same as those of the plate 1 described above.

[0049] The expanded first circular partition 61 has a larger diameter than the first circular partition 21, and surrounds the first circular partition 21 not at the center but in a state in which the first circular partition 21 is in close proximity to or in contact with the outer edge of the expanded first circular partition 61. The expanded second circular partition 62 also has a larger diameter than the second circular partition 22, and surrounds the second circular partition 22 not at the center but in a state in which the second circular partition 22 is in close proximity to or in contact with the outer edge of the expanded second circular partition 62. The expanded third circular partition 63 also has a larger diameter than the third circular partition 23, and surrounds the third circular partition 23 not at the center but in a state in which the third circular partition 23 is in close proximity to or in contact with the outer edge of the expanded third circular partition 63.

[0050] The outer edges 21a, 22a, 23a, 61a, 62a, and 63a of the first circular partition 21, the expanded first circular partition 61, the second circular partition 22, the expanded second circular partition 62, the third circular partition 23, and the expanded third circular partition 63 are annular grooves. The outer edges may be annular ribs.

[0051] The enlarged first circular compartment 61 is a circular compartment for placing a treatment liquid not containing a cell membrane permeable cryoprotectant, the enlarged second circular compartment 62 is a circular compartment for placing a treatment liquid containing a low concentration of a cell membrane permeable cryoprotectant, and the enlarged third circular compartment 63 is a circular compartment for placing a treatment liquid containing a low concentration of a cell membrane permeable cryoprotectant. The diameter of each of the enlarged circular compartments 61, 62, and 63 is preferably 1.5 to 3 times the diameter of each of the circular compartments 21, 22, and 23, and more preferably 1.7 to 2.5 times. Similarly, the area of ​​each of the enlarged circular compartments 61, 62, and 63 is preferably 2 to 10 times the area of ​​each of the circular compartments 21, 22, and 23, and more preferably 3 to 7 times.

[0052] As shown in Fig. 12, the cell pre-freezing treatment working plate 1c of this embodiment has a first display section 26a on the working flat plate section 2a, which has a display of the name and amount of the treatment solution to be placed in the first circular compartment 21 and the enlarged first circular compartment 61 (two displays). "BS" in the display section 26a is an abbreviation for "Basic Solution" and indicates a treatment solution not containing a cell membrane-permeable cryoprotectant, and "20μL" written nearby informs the operator of the amount to be placed in the first circular compartment 21, and "100μL" informs the operator of the amount to be placed in the enlarged first circular compartment 61. When multiple cells are to be treated simultaneously, the enlarged first circular compartment 61 is used, and when a single cell is to be treated, the first circular compartment 21 is used.

[0053] Furthermore, as shown in Fig. 12, the cell pre-freezing treatment working plate 1c of this embodiment has a second display section 27a on the working flat plate section 2a, which has a display of the name and amount of the treatment liquid to be placed in the second circular compartment 22 and the enlarged second circular compartment 62 (two displays). "ES" in the display section 27a indicates a low-concentration treatment liquid containing a low concentration of a cell membrane-permeable cryoprotectant, and "20μL" written nearby informs the operator of the amount to be placed in the second circular compartment 22, and "100μL" informs the operator of the amount to be placed in the enlarged second circular compartment 62. When multiple cells are to be treated simultaneously, the enlarged second circular compartment 62 is used, and when a single cell is to be treated, the second circular compartment 22 is used.

[0054] Furthermore, as shown in Fig. 12, the cell pre-freezing treatment working plate 1c of this embodiment has a third display section 28a on the working flat plate section 2a, which has a display of the name and amount of the treatment liquid to be placed in the third circular compartment 23 and the enlarged third circular compartment 63 (two displays). "ES" in the display section 28a indicates a low-concentration treatment liquid containing a low concentration of a cell membrane-permeable cryoprotectant, and "20μL" written nearby informs the operator of the amount to be placed in the third circular compartment 23, and "100μL" informs the operator of the amount to be placed in the enlarged third circular compartment 63. When multiple cells are to be treated simultaneously, the enlarged third circular compartment 63 is used, and when a single cell is to be treated, the third circular compartment 23 is used.

[0055] In the plate 1c of this embodiment, the first recess 14, the second recess 15, the third recess 16, and the fourth recess 18 are disposed on the front side of the working plate portion 2a. The plate 1c of this embodiment also has indications of the names and amounts of the processing liquids stored (poured) in the first recess 14, the second recess 15, and the third recess 16. The working plate portion 2, the first recess 14, the second recess 15, the third recess 16, and the fourth recess 18 are surrounded by the annular side wall 3b, and their upper surfaces are located slightly below the upper end of the annular side wall 3b. In the plate 1c of this embodiment, the lower annular wall 4b is also of a generally rectangular shape, and similarly to the plate 1, it is larger than the annular side wall 3b, and on the upper surface of the lower annular wall 4b in the portion protruding from the annular side wall 3b, it has a horizontally elongated upper surface portion 12 and a narrow flat portion 13 connecting both sides and the lower end of the rear side of the annular side wall 3b to the upper end of the lower annular wall 4b.

[0056] Next, a cell freezing pretreatment working plate 1d of the embodiment shown in Fig. 13 will be described. The basic structure of this plate 1d is the same as that of the above-mentioned plate 1c. The difference between the two is the arrangement positions of the working flat plate portion 2b, the first recess 14, the second recess 15, the third recess 16, and the fourth recess 18.

[0057] In the plate 1d of this embodiment, the first recess 14, the second recess 15, and the third recess 16 are arranged in this order from the back side on the side (specifically, the right side) of the working flat plate portion 2b, and a fourth recess 18 is provided so as to surround the second recess 15 and the third recess 16. The plate 1d of this embodiment also has indications of the names and amounts of the processing liquids stored (poured) in the first recess 14, the second recess 15, and the third recess 16. The working flat plate portion 2b, the first recess 14, the second recess 15, the third recess 16, and the fourth recess 18 are surrounded by the annular side wall 3c, and their upper surfaces are located slightly below the upper end of the annular side wall 3c. In the plate 1d of this embodiment, the lower annular wall 4c is also approximately rectangular, and similarly to the plate 1, the lower annular wall 4c is larger than the annular side wall 3c, and the upper surface of the lower annular wall 4c protruding from the annular side wall 3c has a horizontally elongated upper surface portion 12 and a narrow flat portion 13 connecting both sides and the lower end of the rear side of the annular side wall 3c to the upper end of the lower annular wall 4c.

[0058] Next, a cell pre-freezing treatment working plate 1e of an embodiment shown in FIG. 14 will be described. The basic configuration of this plate 1e is the same as that of the above-mentioned plate 1c. The difference between the two is the arrangement positions of the working flat plate portion 2c, the first recess 14, the second recess 15, the third recess 16, and the fourth recesses 18a, 18b. In the plate 1e of this embodiment, the lower annular wall 4d and the annular side wall 3d are substantially pentagonal in shape with the innermost side being substantially triangular. As in the plate 1, the lower annular wall 4d is larger than the annular side wall 3d, and has a horizontally elongated upper surface portion 12 on the upper surface of the lower annular wall 4d in the portion protruding from the annular side wall 3d, and a narrow flat portion 13 connecting both sides and the innermost lower end of the annular side wall 3b to the upper end of the lower annular wall 4b.

[0059] In the plate 1e of this embodiment, a first recess 14 is arranged at the rear side of the work plate portion 2c, a second recess 15 and a fourth recess 18a are arranged at the front side of one side (left side) of the work plate portion 2c, and a third recess 16 and a fourth recess 18b are arranged at the front side of the other side (right side) of the work plate portion 2c.

[0060] Plate 1e of this embodiment also has indications for the names and amounts of processing liquids stored (poured) in first recess 14, second recess 15, and third recess 16. Working plate portion 2c, first recess 14, second recess 15, third recess 16, and fourth recesses 18a, 18b are surrounded by annular side wall 3d, and their upper surfaces are positioned slightly below the upper end of annular side wall 3d.

[0061] Next, the cell pre-freezing treatment operation using the cell pre-freezing treatment operation plate of the present invention will be described with reference to Figs. First, prepare the cells to be frozen (e.g., embryos), BS (treatment solution not containing a cell membrane-permeable cryoprotectant), ES (low-concentration treatment solution containing a cell membrane-permeable cryoprotectant at a low concentration), and VS (high-concentration treatment solution containing a cell membrane-permeable cryoprotectant at a high concentration). Then, inject a predetermined amount (300 μL) of ES into the first well 14 of the plate 1 according to the markings 29, inject a predetermined amount (300 μL) of VS into the second well 15 and the third well 16, place (drop) an appropriate amount (20 μL) of BS into the first circular section 21 according to the markings 26, place (drop) an appropriate amount (20 μL) of ES into the second circular section 22 according to the markings 27, and place (drop) an appropriate amount (20 μL) of ES into the third circular section 23 according to the markings 28. This completes the preparation for the pre-freezing of the cells.

[0062] First, cells to be frozen are collected using a micropipette or the like and transplanted into the BS placed on the first circular compartment 21. Next, according to the display (1) 35 and the arrow display 24 on the working plate 2 shown in FIG. 4, the second circular compartment 22 and the first circular compartment 21 are connected with the member used for transplantation (e.g., a pipette). This allows the second circular compartment 22 to communicate with the first circular compartment 21. Then, according to the display 37, the BS is left for 3 minutes. This operation causes the ES to gradually mix with the BS into which the cells of the first circular compartment 21 have been transplanted, forming a mixed area in which the concentration of the low-concentration treatment solution in the treatment solution surrounding the cells has increased.

[0063] Next, according to the display (2) 36 and the arrow display 25 of the working plate 2 shown in FIG. 4, the third circular compartment 23 is connected to the above-mentioned mixing section by the member used for transplantation (e.g., a pipette). This allows the second circular compartment 22, the first circular compartment 21, and the third circular compartment 23 to communicate with each other. Then, according to the display 38, it is left for 3 minutes. This operation gradually and further increases the low-concentration treatment solution concentration of the treatment solution surrounding the target cells. In this way, the treatment solution surrounding the cells to be frozen gradually increases the cell membrane-permeable cryoprotectant concentration, so that the equilibration treatment of the target cells can be advanced without damaging the cells. The above operation completes the first equilibration step treatment.

[0064] In particular, in the plate 1 of this embodiment, the surface of the working plate portion 2 is provided with a first circular compartment 21 for placing a processing liquid that does not contain a cell membrane-permeable cryoprotectant, a second circular compartment 22 for placing a low-concentration processing liquid, and a third circular compartment 23 for placing a low-concentration processing liquid, and since the first circular compartment, the second circular compartment, and the third circular compartment are close to each other, preparation for the above-mentioned equilibration and the work thereof can be carried out easily and reliably.

[0065] Then, the cells that have been subjected to the first equilibration step on the working plate 2 as described above are aspirated using a micropipette or the like, placed on the surface of the ES liquid injected into the first well 14, and left for 9 minutes. In the case of embryos, the cells placed on the ES surface can be observed to sink and shrink within 30 seconds, and the embryos that had once significantly shrunk are allowed to fully recover in volume. This completes the second equilibration step.

[0066] Next, the cells in the first well 14 are aspirated using a micropipette or the like and placed on the surface of the second well 15 into which VS has been injected. During this procedure, the amount of ES liquid carried over into the VS liquid in the second well 15 is minimized. After placing the cells, the ES in the micropipette is discarded into the fourth well 18, which is used to store waste treatment liquid. Next, fresh VS is aspirated and discarded from the edge of the second well 15, and the micropipette is removed. Second recess 15Co-washing is performed using VS. Second recess 15 Pipette thoroughly in about three different locations in the VS (eject the embryos from the pipette into the VS vigorously and stir the area around the embryos several times quickly). Second recess 15 This completes the third equilibration step.

[0067] Next, the VS in the micropipette is discarded into the fourth recess 18 for storing waste treatment liquid, and fresh VS injected into the third recess 16 is aspirated. Then, the cells in the second recess 15 are aspirated using the micropipette that has aspirated the fresh VS, and placed into the third recess 16 into which the VS has been injected. Sufficient pipetting is performed (the embryo is forcefully discharged from the pipette into the VS, and the area around the embryo is quickly stirred several times) while changing the location to about two places. It is confirmed that the cells have shrunk into a flat shape due to dehydration. This completes the equilibration.

[0068] Next, cells to be frozen are collected at the tip of a micropipette, and the biological cell holder (not shown) of a biological cell cryopreservation device (not shown) is placed under a microscope together with a small amount of VS. The biological cell holder of the biological cell cryopreservation device with the cells attached is immersed in liquid nitrogen that has been prepared in advance and frozen (vitrified). The biological cell cryopreservation device with the cells attached is then inserted into a cylindrical member (not shown), and the cylindrical storage member containing the biological cell cryopreservation device is stored in a storage container (not shown), and the storage container is then placed in a liquid nitrogen tank for storage. [Industrial Applicability]

[0069] The cell pre-freezing treatment plate of the present invention is as follows. (1) A cell pre-freezing treatment plate, the plate comprising a work flat section, a first recess for accommodating a low-concentration treatment liquid containing a low concentration of a cell membrane-permeable cryoprotectant, a second recess for accommodating a high-concentration treatment liquid containing a high concentration of a cell membrane-permeable cryoprotectant, a third recess for accommodating a high-concentration treatment liquid containing a high concentration of a cell membrane-permeable cryoprotectant, and a fourth recess for accommodating a waste liquid, the surface of the work flat section further comprising a first circular recess for placing a treatment liquid not containing a cell membrane-permeable cryoprotectant. a first circular partition, a second circular partition for placing the low-concentration treatment liquid, and a third circular partition for placing the low-concentration treatment liquid, the first circular partition, the second circular partition, and the third circular partition being adjacent to one another, and the surface of the working flat portion having an enlarged first circular partition surrounding the first circular partition, an enlarged second circular partition surrounding the second circular partition, and an enlarged third circular partition surrounding the third circular partition.

[0070] In this plate, an appropriate amount of cell membrane permeable cryoprotectant-free treatment liquid is placed (dropped) on the first circular compartment, an appropriate amount of low-concentration treatment liquid is placed (dropped) on the second circular compartment, and an appropriate amount of low-concentration treatment liquid is placed (dropped) on the third circular compartment. Then, the target cells to be frozen are transplanted into the cell membrane permeable cryoprotectant-free treatment liquid, and then, by connecting the second circular compartment and the first circular compartment with a member used for transplantation (e.g., a pipette), the low-concentration treatment liquid gradually mixes with the non-containing treatment liquid, and the concentration of the low-concentration treatment liquid in the treatment liquid surrounding the target cells increases. Furthermore, by connecting the third circular compartment to a mixing section of the non-containing liquid and the low-concentration treatment liquid with a member used for transplantation (e.g., a pipette), the low-concentration treatment liquid gradually mixes with the mixing section, and the concentration of the low-concentration treatment liquid in the treatment liquid surrounding the target cells gradually increases. By doing so, the concentration of the cell membrane-permeable cryoprotectant in the treatment solution surrounding the cells to be frozen gradually increases, so that the equilibration treatment of the cells to be frozen can be performed using an excellent cell membrane-permeable cryoprotectant.

[0071] In particular, the surface of the working plate is provided with a first circular partition for placing a processing liquid that does not contain a cell membrane-permeable cryoprotectant, a second circular partition for placing a low-concentration processing liquid, and a third circular partition for placing a low-concentration processing liquid, and since the first, second and third circular partitions are close to each other, preparation for the above-mentioned equilibration and further, good equilibration processing can be carried out easily and reliably. Furthermore, when multiple cells are treated simultaneously, the enlarged circular compartment is used, and when a single cell is treated, a small circular compartment located within the enlarged circular compartment is used. Therefore, it is possible to use either one depending on the number of cells to be treated.

[0072] The above-mentioned cell pre-freezing treatment working plate can be embodied as follows. (2) The working flat plate has a first display section having a name and amount of the processing liquid to be placed in the first circular compartment, a second display section having a name and amount of the processing liquid to be placed in the second circular compartment, and a third display section having a name and amount of the processing liquid to be placed in the third circular compartment, the cell freezing pretreatment working plate described in (1) above. (3) The cell pre-freezing treatment work plate according to (1) or (2) above, further comprising a fourth display section having a name of the treatment liquid stored in the first recess and an indication of the amount of the treatment liquid stored therein. (4) A cell freezing pretreatment work plate as described in any of (1) to (3) above, wherein the surface of the work flat portion has a first arrow marking extending from the second circular compartment toward the first circular compartment, and a second arrow marking extending from the third circular compartment toward the first arrow marking or the first circular compartment. (5) The cell pre-freezing treatment working plate according to any one of (1) to (4) above, further comprising a lid that simultaneously covers the working plate portion, the first recess, the second recess and the third recess. (6) The cell pre-freezing treatment work plate according to any one of (1) to (5) above, comprising a bottom, an annular side wall extending upward from the bottom, and a protrusion protruding upward from the bottom, the protrusion being provided with the second recess or the third recess, and the fourth recess being formed by the side of the protrusion, the inner surface of the annular side wall, and the bottom. (7) The cell pre-freezing treatment work plate according to any of (1) to (6) above, comprising a bottom, an annular side wall extending upward from the bottom, a first protrusion protruding upward from the bottom, and a second protrusion protruding upward from the bottom, wherein the first protrusion comprises the second recess, the second protrusion comprises the third recess, and the side of the protrusion, the inner surface of the annular side wall, and the bottom form the fourth recess. (8) The cell freezing pretreatment work plate according to any one of (1) to (7) above, further comprising a second flat plate portion, the first recess being formed in the second flat plate portion. (9) A cell freezing pretreatment work plate as described in any of (1) to (8) above, wherein the work flat section has a first display section having an indication of the name and amount of processing liquid to be placed in the first circular partition section and the enlarged first circular partition section, a second display section having an indication of the name and amount of processing liquid to be placed in the second circular partition section and the enlarged second circular partition section, and a third display section having an indication of the name and amount of processing liquid to be placed in the third circular partition section and the enlarged third circular partition section.

[0073] The cell pre-freezing treatment plate of the present invention is as follows: (10) A cell pre-freezing treatment working plate comprising a working flat portion, a first recess for accommodating a processing liquid, a second recess for accommodating a processing liquid, a third recess for accommodating a processing liquid, and a fourth recess for accommodating waste processing liquid, the surface of the working flat portion comprising a first circular partition for placing a processing liquid, a second circular partition for placing a processing liquid, and a third circular partition for placing a processing liquid, the first circular partition, the second circular partition, and the third circular partition being adjacent to one another, the surface of the working flat portion comprising an enlarged first circular partition surrounding the first circular partition, an enlarged second circular partition surrounding the second circular partition, and an enlarged third circular partition surrounding the third circular partition.

[0074] The above-mentioned cell pre-freezing treatment working plate can be embodied as follows. (11) The working flat section has a first display section having an indication of the name and amount of the processing liquid to be placed in the first circular compartment, a second display section having an indication of the name and amount of the processing liquid to be placed in the second circular compartment, and a third display section having an indication of the name and amount of the processing liquid to be placed in the third circular compartment, and the cell freezing pretreatment working plate has a fourth display section having an indication of the name and amount of the processing liquid to be stored in the first recess. (12) A cell freezing pretreatment work plate as described in (10) or (11) above, wherein the work flat section has a first display section having an indication of the name and amount of processing liquid to be placed in the first circular partition section and the enlarged first circular partition section, a second display section having an indication of the name and amount of processing liquid to be placed in the second circular partition section and the enlarged second circular partition section, and a third display section having an indication of the name and amount of processing liquid to be placed in the third circular partition section and the enlarged third circular partition section. (13) A cell freezing pretreatment work plate as described in any of (10) to (12) above, wherein the surface of the work flat portion has a first arrow marking extending from the second circular compartment toward the first circular compartment, and a second arrow marking extending from the third circular compartment toward the first arrow marking or the first circular compartment.

Claims

1. A cell pre-freezing processing plate, the plate comprising a processing flat portion, a first recess for accommodating a low-concentration processing liquid containing a low concentration of a cell membrane-permeable cryoprotectant, a second recess for accommodating a high-concentration processing liquid containing a high concentration of a cell membrane-permeable cryoprotectant, a third recess for accommodating a high-concentration processing liquid containing a high concentration of a cell membrane-permeable cryoprotectant, and a fourth recess for accommodating a waste liquid, the surface of the processing flat portion further comprising a first circular partition for placing a processing liquid not containing a cell membrane-permeable cryoprotectant, a second circular partition for placing the low-concentration processing liquid, and a third circular partition for placing the low-concentration processing liquid, the first circular partition, the second circular partition, and the third circular partition being adjacent to each other, the surface of the processing flat portion further comprising an enlarged first circular partition surrounding the first circular partition, and a second circular partition for placing the low-concentration processing liquid. the outer edges of the expanded first circular partition, the expanded second circular partition, and the expanded third circular partition are annular grooves; the outer edges of the first circular partition, the second circular partition, and the expanded third circular partition are annular grooves; the expanded first circular partition surrounds the first circular partition in a state of being in contact with the inner side and outer edge of the expanded first circular partition; the expanded second circular partition surrounds the second circular partition in a state of being in contact with the inner side and outer edge of the expanded second circular partition; and the expanded third circular partition surrounds the third circular partition in a state of being in contact with the inner side and outer edge of the expanded third circular partition.

2. 2. The cell freezing pretreatment work plate according to claim 1, wherein the work flat portion has a first display portion having a name and amount of the processing liquid to be placed in the first circular partition portion, a second display portion having a name and amount of the processing liquid to be placed in the second circular partition portion, and a third display portion having a name and amount of the processing liquid to be placed in the third circular partition portion.

3. 3. The cell freezing pretreatment work plate according to claim 1, further comprising a fourth display section having a name of a treatment liquid stored in the first recess and an indication of a storage amount of the treatment liquid.

4. 4. The cell freezing pretreatment work plate according to claim 1, wherein the surface of the work flat portion has a first arrow mark extending from the second circular partition toward the first circular partition, and a second arrow mark extending from the third circular partition toward the first arrow mark or the first circular partition.

5. 5. The cell freezing pretreatment working plate according to claim 1, further comprising a lid for simultaneously covering the working flat plate portion, the first recess, the second recess and the third recess.

6. The cell freezing pretreatment working plate according to any one of claims 1 to 5, comprising a bottom, an annular side wall extending upward from the bottom, and a protrusion protruding upward from the bottom, the protrusion being provided with the second recess or the third recess, and the fourth recess being formed by a side surface of the protrusion, an inner surface of the annular side wall, and the bottom.

7. 7. The cell pre-freezing treatment working plate according to claim 1, further comprising: a bottom; an annular side wall extending upward from the bottom; a first protrusion protruding upward from the bottom; and a second protrusion protruding upward from the bottom, wherein the first protrusion comprises the second recess, the second protrusion comprises the third recess, and the side surface of the protrusion, an inner surface of the annular side wall, and the bottom form the fourth recess.

8. 8. The cell freezing pretreatment working plate according to claim 1, further comprising a second flat plate portion, the first recess being formed in the second flat plate portion.

9. 9. A cell freezing pretreatment work plate as described in any one of claims 1 to 8, wherein the work flat plate portion has a first display portion having a name and amount of processing liquid to be placed in the first circular partition portion and the enlarged first circular partition portion, a second display portion having a name and amount of processing liquid to be placed in the second circular partition portion and the enlarged second circular partition portion, and a third display portion having a name and amount of processing liquid to be placed in the third circular partition portion and the enlarged third circular partition portion.

10. A cell pre-freezing processing working plate, the plate comprising a working flat portion, a first recess for accommodating a processing liquid, a second recess for accommodating a processing liquid, a third recess for accommodating a processing liquid, and a fourth recess for accommodating a waste processing liquid, the surface of the working flat portion further comprising a first circular partition for placing a processing liquid, a second circular partition for placing a processing liquid, and a third circular partition for placing a processing liquid, the first circular partition, the second circular partition, and the third circular partition being adjacent to each other, the surface of the working flat portion further comprising an enlarged first circular partition surrounding the first circular partition, an enlarged second circular partition surrounding the second circular partition, and an enlarged third circular partition surrounding the third circular partition, a first circular partition portion and a second circular partition portion, the outer edges of which are annular grooves, the first circular partition portion and the second circular partition portion and the second circular partition portion are annular grooves, the first circular partition portion surrounds the first circular partition portion in a state of being in contact with the inner side and outer edge of the first circular partition portion, the second circular partition portion surrounds the second circular partition portion in a state of being in contact with the inner side and outer edge of the second circular partition portion, and the third circular partition portion surrounds the third circular partition portion in a state of being in contact with the inner side and outer edge of the third circular partition portion.

11. The cell freezing pretreatment work plate according to claim 10, wherein the work flat portion has a first display portion having an indication of the name and amount of the processing liquid to be placed in the first circular partition portion, a second display portion having an indication of the name and amount of the processing liquid to be placed in the second circular partition portion, and a third display portion having an indication of the name and amount of the processing liquid to be placed in the third circular partition portion, and the cell freezing pretreatment work plate has a fourth display portion having an indication of the name and amount of the processing liquid to be stored in the first recess.

12. The cell freezing pretreatment work plate of claim 10 or 11, wherein the work flat plate has a first display section having a name and amount of processing liquid to be placed in the first circular partition section and the enlarged first circular partition section, a second display section having a name and amount of processing liquid to be placed in the second circular partition section and the enlarged second circular partition section, and a third display section having a name and amount of processing liquid to be placed in the third circular partition section and the enlarged third circular partition section.

13. 13. A cell freezing pretreatment work plate as described in any one of claims 10 to 12, wherein the surface of the work flat portion has a first arrow mark extending from the second circular partition toward the first circular partition, and a second arrow mark extending from the third circular partition toward the first arrow mark or the first circular partition.

14. 14. The cell freezing pretreatment work plate according to any one of claims 1 to 13, wherein a diameter of the expanded first circular partition is 1.5 to 3 times a diameter of the first circular partition, a diameter of the expanded second circular partition is 1.5 to 3 times a diameter of the second circular partition, and a diameter of the expanded third circular partition is 1.5 to 3 times a diameter of the third circular partition.

Images