Pressing tool and cell culture method

Abstract

To provide a technique capable of molding a gel-like medium into a desired shape in the case of culturing cells by using the gel-like medium.SOLUTION: A pressing tool 2 is used together with a culture vessel 1 whose upper part is open and which has an a cup-shaped culture chamber 10. The pressing tool 2 has a fixing part 22 and a press surface 20. The fixing part 22 extends in a horizontal direction in the upper part. The press surface 20 is arranged below the fixing part 22, and faces a bottom part 11 of the culture chamber 10 each other with a space. In the case where the pressing tool 2 is attached to the culture vessel 1, the fixing part 22 is mounted on an edge part of an opening of the culture chamber 10, and the press surface 20 faces the bottom surface 11 of the culture chamber 10 each other with a space. This makes it possible to mold the gel-like medium into a desired shape. Therefore, in the case of performing three-dimensional culture of cells by the gel-like medium, observation or imaging can be easily performed.SELECTED DRAWING: Figure 9

Description

[Technical field]

[0001] The present invention relates to a pressing tool used when culturing cells, and a cell culture method using the pressing tool. [Background technology]

[0002] Conventionally, image analysis of drug sensitivity of cells cultured in a container has been performed. In such research, there is a demand for culturing 3D cell aggregates, because the conditions are closer to those in vivo than those of cells cultured in a 2D manner.

[0003] In particular, it is known that in the evaluation of anticancer drugs against cancer cells, the need for three-dimensional culture is high because the tumor microenvironment cannot be reproduced in two-dimensional culture. In three-dimensional culture of cancer cells, particularly in spheroid culture, three-dimensional culture is performed using a gel-like medium containing various gel components such as agarose gel and hydrogel. In this way, three-dimensional culture using a gel-like medium is described, for example, in Patent Document 1.

[0004] In particular, Corning's gel-like medium, Matrigel, contains basement membrane components isolated from EHS mouse tumor cells, and cancer cells embedded in Matrigel have been shown to exhibit site-specific behavior. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2022-181076 A Summary of the Invention [Problem to be solved by the invention]

[0006] When performing 3D cell culture using gelatinous medium, the shape of the gelatinous medium can make observation difficult. For example, when performing fluorescent staining to visualize the localization of a protein specific to the cell, excitation light is irradiated onto the fluorescent substance and the emitted fluorescence is detected. In this case, if the gel is thick, the excitation light is attenuated and scattered, resulting in weaker detectable fluorescence.

[0007] Furthermore, when the gel is thick, the cells are distributed in the Z direction (height direction) regardless of whether bright-field imaging or fluorescent imaging is used, and if images are to be acquired by focusing on each position in the Z direction, a problem occurs in that it takes a long time to capture the images.

[0008] The present invention has been made in consideration of the above circumstances, and aims to provide a technique that enables a gelatinous medium to be molded into a desired shape when cells are cultured using the gelatinous medium. [Means for solving the problem]

[0009] In order to solve the above problems, the first invention of the present application is a pressing jig to be used with a culture vessel having a cup-shaped culture chamber that is open at the top, the pressing jig having a fixing part extending horizontally at the top and a pressing surface that is positioned below the fixing part and extends horizontally, and when the pressing jig is attached to the culture vessel, the fixing part is placed on the edge of the opening of the culture chamber of the culture vessel, and the pressing surface faces the bottom surface of the culture chamber with a gap between them.

[0010] The second invention of the present application is a pressing jig of the first invention, further comprising a body portion having a cylindrically extending outer circumferential surface and having the pressing surface at its lower end, and the fixing portion extends horizontally from an upper end of the body portion.

[0011] A third invention of the present application is the pressing jig of the second invention, further comprising legs extending downward from the outer periphery of the lower end of the body.

[0012] A fourth invention of the present application is a pressing jig of the second invention, wherein the body portion has a through hole extending in the vertical direction between an upper opening provided on the upper end surface and a lower opening provided in the center of the pressing surface.

[0013] A fifth invention of the present application is a pressing jig according to any one of the first to fourth inventions, wherein the culture vessel is a well plate having a plurality of the culture chambers and has a plurality of the pressing surfaces.

[0014] The sixth invention of the present application is a cell culture method using a pressing jig of any one of the first to third inventions, comprising the steps of: a) dropping a suspension of cells and liquefied gel into the culture chamber; b) fixing the pressing jig to the culture vessel and bringing the pressing surface into contact with the suspension; c) solidifying the liquefied gel to obtain a gel containing the cells; d) removing the pressing jig from the culture vessel; and e) dropping liquid medium into the culture chamber and culturing the cells.

[0015] The seventh invention of the present application is a cell culture method using a pressing jig of any one of the first to third inventions, comprising the steps of A) dropping a liquefied gel into the culture chamber, B) fixing the pressing jig to the culture vessel and bringing the pressing surface into contact with the liquefied gel, C) solidifying the liquefied gel to obtain a gel that does not contain cells, D) removing the pressing jig from the culture vessel, and E) dropping a suspension of cells and liquid medium into the culture chamber and culturing the cells.

[0016] The eighth invention of the present application is a cell culture method using the pressing jig of the fourth invention, comprising the steps of p) fixing the pressing jig to the culture vessel, q) injecting a suspension of cells and liquefied gel below the pressing surface through the through-hole, r) solidifying the liquefied gel to obtain a gel containing the cells, s) removing the pressing jig from the culture vessel, and t) dropping liquid medium into the culture chamber and culturing the cells.

[0017] The ninth invention of the present application is a cell culture method using the pressing jig of the fourth invention, comprising the steps of P) fixing the pressing jig to the culture vessel, Q) injecting a liquefied gel below the pressing surface through the through-hole, R) solidifying the liquefied gel to obtain a gel that does not contain cells, S) removing the pressing jig from the culture vessel, and T) dropping a suspension of cells and liquid medium into the culture chamber and culturing the cells. Effect of the Invention

[0018] According to the first to ninth aspects of the present application, the gelatinous medium can be molded into a desired shape, which makes it easier to observe and capture images when three-dimensionally culturing cells using the gelatinous medium. [Brief description of the drawings]

[0019] [Figure 1] FIG. 2 is a perspective view of a well plate. [Diagram 2] FIG. 2 is a longitudinal sectional view of a well plate. [Diagram 3] FIG. 1 is a partial cross-sectional view of a well plate showing a conventional cell culture method. [Figure 4] FIG. 1 is a partial cross-sectional view of a well plate showing a conventional cell culture method. [Diagram 5] FIG. 2 is a perspective view of a pressing jig according to the first embodiment. [Figure 6] FIG. 4 is a view showing a state in which the pressing jig according to the first embodiment is inserted into a well. [Figure 7] 4 is a flowchart showing a flow of a first cell culture method using a pressing jig according to the first embodiment. [Figure 8] FIG. 2 is a diagram showing the cell / gel suspension dropping step in the first cell culture method. [Figure 9] FIG. 13 is a diagram showing a pressing tool placement step in the first cell culture method. [Figure 10] FIG. 2 is a diagram showing the cell culture step of the first cell culture method. [Figure 11]13 is a flowchart showing a flow of a second cell culture method using the pressing jig according to the first embodiment. [Figure 12] FIG. 13 is a diagram showing the liquefied gel dropping step of the second cell culture method. [Figure 13] FIG. 13 is a diagram showing a pressing tool placement step in the second cell culture method. [Figure 14] FIG. 13 is a diagram showing the cell / medium suspension dropping step in the second cell culture method. [Figure 15] FIG. 13 is a diagram showing the cell culture step of the second cell culture method. [Figure 16] FIG. 11 is a perspective view of a pressing jig according to a second embodiment. [Figure 17] FIG. 11 is a view showing a state in which a pressing jig according to a second embodiment is inserted into a well. [Figure 18] 13 is a flowchart showing the flow of a third cell culture method using a pressing jig according to the second embodiment. [Figure 19] 13 is a flowchart showing the flow of a fourth cell culture method using a pressing jig according to a second embodiment. [Figure 20] FIG. 13 is a perspective view of a pressing jig according to a first modified example. [Figure 21] FIG. 13 is a view showing a state in which a pressing jig according to a first modified example is inserted into a well. [Figure 22] FIG. 11 is a perspective view of a pressing jig according to a second modified example. [Figure 23] FIG. 13 is a vertical cross-sectional view of a pressing jig and a well plate according to a third modified example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

[0021] <1. Well plate configuration> FIG. 1 is a perspective view of a well plate 1, which is an example of a culture vessel. FIG. 2 is a longitudinal sectional view of the well plate 1. The well plate 1 is a generally plate-shaped vessel having a plurality of wells (recesses) 10. The well plate 1 is made of, for example, a transparent resin that transmits light. As shown in FIG. 1, the plurality of wells 10 are regularly arranged on the upper surface of the well plate 1. In this embodiment, the shape of the wells 10 when viewed from above is circular. However, the shape of the wells 10 when viewed from above may be other shapes, such as rectangular.

[0022] A biological sample to be observed, together with a liquid medium or a gel medium, is held in each well 10. In this way, the biological sample, such as cells, is cultured in each well 10.

[0023] 3 and 4 are partial cross-sectional views of a well plate 1 showing a state of conventional three-dimensional cell culture. In FIG. 3 and FIG. 4, the state near one well 10 is shown. As shown in FIG. 3 and FIG. 4, the well 10 has a bottom surface 11 and a side surface 12. The bottom surface 11 is a flat surface extending perpendicularly to the up-down direction. The side surface 12 is a cylindrical surface extending upward from the periphery of the bottom surface 11. Note that the side surface 12 may be tapered so that the diameter slightly increases toward the top in order to facilitate release from a mold when molding the well plate 1.

[0024] A description will be given of an example of a conventional procedure for three-dimensionally culturing cells 9 using a gelatinous medium in such a well plate 1. In the following, the liquefied gelatinous medium will be referred to as a "liquefied gel", and the suspension of cells and the liquefied gel will be referred to as a "cell-gel suspension".

[0025] When three-dimensionally culturing cells using a gelatinous medium, first, a cell / gel suspension is dropped onto the bottom surface 11 of a well 10. The liquefied gel is then solidified to obtain a gelatinous medium 91. Then, liquid medium 92 is dropped into the well 10 to fill the well 10 with the liquid medium 92. Then, cells 9 are cultured in this state.

[0026] The gelatinous medium 91 is, for example, a gelatinous medium containing various gel components such as agarose gel and hydrogel. In this embodiment, a gelatinous medium "Matrigel" manufactured by Corning is used as the gelatinous medium 91. Matrigel becomes solid at -20°C, liquefies at about 0 to 4°C, and solidifies and becomes gelatinous at 37°C. Therefore, the temperature of the liquefied gel is about 0 to 4°C, and the liquefied gel is solidified and becomes gelatinous by heating it in an incubator.

[0027] For example, DMEM (Dulbecco's Modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium), physiological saline, etc. are used as the liquid medium 92. The cell 9 may be a single cell, or may be a cell mass (spheroid) formed by a plurality of cells.

[0028] When cells 9 are cultured according to the above procedure, the liquefied gel has fluidity but is viscous, and therefore does not spread evenly parallel to the bottom surface 11. Furthermore, when the liquefied gel comes into contact with the side surface 12, it forms a meniscus. For this reason, it is difficult to maintain a constant thickness of the liquefied gel.

[0029] 2. First embodiment <2-1. Composition of pressing jig> Next, a pressing jig 2 according to a first embodiment of the present invention will be described. Fig. 5 is a perspective view of the pressing jig 2 according to the first embodiment. Fig. 6 is a partial cross-sectional view of the well plate 1 and the pressing jig 2 when the pressing jig 2 is in use.

[0030] The pressing jig 2 is used together with a culture vessel having a cup-shaped culture chamber (well 10) with an open top, such as the well plate 1. The pressing jig 2 of this embodiment is used for one well 10 of the well plate 1.

[0031] The pressing jig 2 has a body 21 and a fixing part 22. The pressing jig 2 is integrally molded from a resin or elastic material such as acrylic resin or PDMS (polydimethylsiloxane). The material of the pressing jig 2 is preferably a material such as PDMS that has excellent releasability and heat resistance of 120°C or more. If the pressing jig 2 is made of a heat-resistant material, it can be autoclaved. In addition, when an elastic material such as PDMS is used, it is preferable that the hardness measured by a type A durometer specified in JIS K6253 is 50 degrees or more from the viewpoint of accuracy.

[0032] The body 21 is a cylindrical portion extending vertically. The diameter of the body 21 is smaller than the diameter of the well 10. Therefore, the body 21 can be inserted into the well 10. The body 21 has a pressing surface 20 at its lower end. In other words, the pressing surface 20 is the circular lower end surface of the body 21. The pressing surface 20 is positioned below the fixing portion 22 and extends horizontally.

[0033] In this embodiment, the cross section of the well 10 viewed from the top and bottom is circular, and the pressing surface 20 is also circular, but the present invention is not limited to this. The shape of the pressing surface 20 can be changed appropriately depending on the shape of the well 10.

[0034] The fixing portion 22 is a portion that extends horizontally in the upper portion of the pressing jig 2. Specifically, the fixing portion 22 is a flange that extends horizontally from the upper end portion of the body portion 21 in an annular shape.

[0035] With the pressing jig 2 attached to the well plate 1, the fixing part 22 is placed on the edge of the upper opening of the well 10. The length of the body part 21 is slightly smaller than the depth of the well 10. As a result, with the pressing jig 2 attached to the well plate 1, the pressing surface 20 faces the bottom of the well 10 with a gap therebetween in the vertical direction.

[0036] <2-2. First cell culture method> Next, the first cell culture method using the pressing jig 2 according to the first embodiment will be described with reference to Figs. 7 to 10. Fig. 7 is a flow chart showing the flow of the first cell culture method using the pressing jig 2. Fig. 8 is a diagram showing the cell / gel suspension dropping step (step S101) of the first cell culture method. Fig. 9 is a diagram showing the pressing jig placement step (step S102) of the first cell culture method. Fig. 10 is a diagram showing the cell culture step (step S105) of the first cell culture method.

[0037] In this first cell culture method, first, an appropriate amount of cell / gel suspension 82, in which cells 9 are mixed with liquefied gel medium 81, is dropped into well 10 (step S101: cell / gel suspension dropping step). At this time, the cells 9 mixed with liquefied gel medium 81 may contain cell clumps. At this time, the temperature of liquefied gel medium 81 is 0 to 4° C. As a result, the cell / gel suspension 82 is placed in a dome shape on the bottom surface 11 of well 10, as shown in FIG. 8.

[0038] Next, pressing jig 2 is fixed to well plate 1, and pressing surface 20 is brought into contact with cell / gel suspension 82 (step S102: pressing jig placement step), whereby the upper surface of cell / gel suspension 82 is molded flat by pressing surface 20, as shown in FIG.

[0039] Next, with the pressing jig 2 fixed to the well plate 1, the liquefied gel 81 is solidified to obtain a gelled medium 91 containing the cells 9 (step S103: gel solidification step). Specifically, the well plate 1 is placed in an incubator and heated to about 37°C. Thereafter, the pressing jig 2 is removed from the well plate 1 (step S104: pressing jig removal step). As a result, a gelled medium 91 having a certain thickness and containing the cells 9 is formed on the bottom surface 11 of the well 10.

[0040] 10, liquid medium 92 is dropped into well 10, and cells 9 encapsulated in gelatinous medium 91 are cultured (step S105: cell culture step). In this way, cells 9 are cultured three-dimensionally inside gelatinous medium 91 that has been molded flat.

[0041] In this way, by using the pressing jig 2, the liquefied gel 81 can be molded into a desired shape and solidified. This allows the gelatinous medium 91 to have a constant thickness. As a result, the distribution range of the cultured cells 9 in the Z direction can be set within a predetermined range. This makes it easier to image and observe the cultured cells 9.

[0042] <2-3.Second cell culture method> Next, the second cell culture method using the pressing jig 2 according to the first embodiment will be described with reference to Figs. 11 to 15. Fig. 11 is a flow chart showing the flow of the second cell culture method using the pressing jig 2. Fig. 12 is a diagram showing the liquefied gel dropping step (step S201) of the second cell culture method. Fig. 13 is a diagram showing the pressing jig placement step (step S202) of the second cell culture method. Fig. 14 is a diagram showing the cell / liquid medium suspension dropping step (step S205) of the second cell culture method. Fig. 15 is a diagram showing the cell culture step (step S206) of the second cell culture method.

[0043] In this second cell culture method, first, an appropriate amount of liquefied gel medium 81 is dropped into the well 10 (step S201: liquefied gel dropping step). At this time, the temperature of the liquefied gel medium 81 is 0 to 4° C. As a result, the liquefied gel medium 81 is placed in a dome shape on the bottom surface 11 of the well 10, as shown in FIG.

[0044] Next, the pressing jig 2 is fixed to the well plate 1, and the pressing surface 20 is brought into contact with the liquefied gel medium 81 (step S202: pressing jig placement step). As a result, the upper surface of the liquefied gel medium 81 is molded flat by the pressing surface 20, as shown in FIG.

[0045] Next, with the pressing jig 2 fixed to the well plate 1, the liquefied gel 81 is solidified to obtain a gelatinous medium 91 (step S203: gel solidification step). Specifically, the well plate 1 is placed in an incubator and heated to about 37°C. Thereafter, the pressing jig 2 is removed from the well plate 1 (step S104: pressing jig removal step). As a result, a gelatinous medium 91 having a certain thickness is formed on the bottom surface 11 of the well 10.

[0046] Thereafter, as shown in Fig. 14, a cell / liquid medium suspension 83 in which cells 9 are mixed with liquid medium 92 is dropped into well 10 (step S205: cell / liquid medium suspension dropping step). By leaving the mixture to stand as is, cells 9 in cell / liquid medium suspension 83 sink and are placed on the upper surface of gelatinous medium 91, as shown in Fig. 15. By culturing cells 9 in this state, cells 9 are cultured three-dimensionally near the upper surface of gelatinous medium 91 that has been molded flat (step S206: standing / cell culturing step).

[0047] In this way, by using the pressing jig 2, the liquefied gel 81 can be molded into a desired shape and solidified. This allows the gelatinous medium 91 to have a constant thickness. As a result, the distribution range of the cultured cells 9 in the Z direction can be set within a predetermined range. This makes it easier to image and observe the cultured cells 9.

[0048] 3. Second embodiment <3-1. Composition of pressing jig> Next, a pressing jig 2A according to a second embodiment of the present invention will be described. Fig. 16 is a perspective view of the pressing jig 2A according to the second embodiment. Fig. 17 is a partial cross-sectional view of the well plate 1 and the pressing jig 2A when the pressing jig 2A is in use. The pressing jig 2A according to the second embodiment will be described below, mainly focusing on the differences from the pressing jig 2 according to the first embodiment. Other points are the same as those of the pressing jig 2 according to the first embodiment.

[0049] The pressing jig 2A has a body 21A and a fixing part 22A. The body 21A is a cylindrical part extending vertically. The body 21A has a pressing surface 20A at its lower end. That is, the pressing surface 20 is the circular lower end surface of the body 21A. The body 21A has an upper opening 211A provided on its upper end surface and a lower opening 212A provided in the center of the pressing surface 20A, which is the lower end surface. The body 21A also has a through hole 210A that passes through the body 21A in the vertical direction between the upper opening 211A and the lower opening 212A.

[0050] This pressing jig 2A has a through hole 210A, which makes it possible to drip a liquid or the like onto the bottom surface 11 of the well 10 with the pressing jig 2A attached to the well plate 1. Therefore, after the pressing jig 2A is attached to the well plate 1, a liquefied gel medium 81 or a cell / gel suspension 82 can be dripped onto the bottom surface 11 of the well 10.

[0051] <3-2. Third cell culture method> Next, a third cell culture method using the pressing jig 2A according to the second embodiment will be described with reference to Fig. 11. Fig. 18 is a flow chart showing the flow of the third cell culture method using the pressing jig 2A.

[0052] In this third cell culture method, first, the pressing jig 2A is fixed to the well plate 1 (step S301: pressing jig placement step). At this time, nothing is placed in the space between the bottom surface 11 of the well 10 and the pressing surface 20A of the pressing jig 2A.

[0053] Next, a cell / gel suspension 82, in which cells 9 are mixed with liquefied gel medium 81, is injected into the space between bottom surface 11 of well 10 and pressing surface 20A of pressing jig 2A through through-hole 210A using a micropipette or the like (step S302: cell / gel suspension injection step). This causes cell / gel suspension 82 to fill the space between bottom surface 11 of well 10 and pressing surface 20A of pressing jig 2A. That is, the upper surface of cell / gel suspension 82 is molded flat along pressing surface 20A of pressing jig 2A. At this time, the temperature of cell / gel suspension 82 is 0 to 4°C.

[0054] Next, with the pressing tool 2A fixed to the well plate 1, the liquefied gel 81 is solidified to obtain a gelled medium 91 containing the cells 9 (step S303: gel solidification step). Specifically, the well plate 1 is placed in an incubator and heated to about 37°C. Thereafter, the pressing tool 2A is removed from the well plate 1 (step S304: pressing tool removal step). As a result, a gelled medium 91 having a certain thickness and containing the cells 9 is formed on the bottom surface 11 of the well 10.

[0055] Thereafter, liquid medium 92 is dropped into well 10, and cells 9 encapsulated in gelatinous medium 91 are cultured (step S305: cell culture step). In this manner, cells 9 are cultured three-dimensionally inside gelatinous medium 91 that has been molded flat.

[0056] In this manner, by using the pressing jig 2A of this embodiment, the pressing jig 2A can be fixed to the well plate 1 first, and then the cell / gel suspension 82 can be injected into the wells 10.

[0057] Also in this embodiment, the liquefied gel 81 can be molded into a desired shape and solidified. This allows the gelatinous medium 91 to have a constant thickness. As a result, the distribution range of the cultured cells 9 in the Z direction can be set within a predetermined range. This makes it easier to image and observe the cultured cells 9.

[0058] <3-3. Fourth cell culture method> Next, a fourth cell culture method using the pressing jig 2A according to the second embodiment will be described with reference to Fig. 19. Fig. 19 is a flow chart showing the flow of the fourth cell culture method using the pressing jig 2A.

[0059] In this fourth cell culture method, first, the pressing jig 2A is fixed to the well plate 1 (step S401: pressing jig placement step). At this time, nothing is placed in the space between the bottom surface 11 of the well 10 and the pressing surface 20A of the pressing jig 2A.

[0060] Next, the liquefied gel medium 81 is injected into the space between the bottom surface 11 of the well 10 and the pressing surface 20A of the pressing jig 2A through the through-hole 210A using a micropipette or the like (step S402: gel injection step). As a result, the liquefied gel medium 81 fills the space between the bottom surface 11 of the well 10 and the pressing surface 20A of the pressing jig 2A. That is, the upper surface of the liquefied gel medium 81 is molded flat along the pressing surface 20A of the pressing jig 2A. At this time, the temperature of the liquefied gel medium 81 is 0 to 4°C.

[0061] Next, with the pressing jig 2A fixed to the well plate 1, the liquefied gel 81 is solidified to obtain a gelatinous medium 91 (step S403: gel solidification step). Specifically, the well plate 1 is placed in an incubator and heated to about 37°C. Thereafter, the pressing jig 2A is removed from the well plate 1 (step SA04: pressing jig removal step). As a result, a gelatinous medium 91 having a certain thickness is formed on the bottom surface 11 of the well 10.

[0062] Thereafter, cell / liquid medium suspension 83, in which cells 9 are mixed with liquid medium 92, is dropped into well 10 (step S405: cell / liquid medium suspension dropping step). Then, by leaving the mixture to stand, cells 9 in cell / liquid medium suspension 83 sink and are placed on the upper surface of gelatinous medium 91. By culturing cells 9 in this state, cells 9 are cultured three-dimensionally near the upper surface of gelatinous medium 91 that has been molded flat (step S406: standing / cell culturing step).

[0063] In this way, by using the pressing jig 2A, the liquefied gel 81 can be molded into a desired shape and solidified. This allows the gelatinous medium 91 to have a constant thickness. As a result, the distribution range of the cultured cells 9 in the Z direction can be set within a predetermined range. This makes it easier to image and observe the cultured cells 9.

[0064] <4. Modifications> Although one embodiment of the present invention has been described above, the present invention is not limited to the above embodiment.

[0065] <4-1. First modified example> Fig. 20 is a perspective view of a pressing jig 2B according to the first modified example, and Fig. 21 is a partial cross-sectional view showing a state in which the pressing jig 2B according to the first modified example is in use.

[0066] 20 and 21, pressing jig 2B has leg portions 23B in addition to body portion 21B and fixed portion 22B. The configurations of body portion 21B and fixed portion 22B are similar to those of pressing jig 2 according to the first embodiment, and therefore description thereof will be omitted.

[0067] The legs 23B extend downward from the outer periphery of the lower end of the body 21 B. In the pressing jig 2B according to the first modification, the legs 23B are cylindrical portions extending downward from the outer periphery of the pressing surface 20B.

[0068] In the pressing jig 2 according to the first embodiment, the distance between the bottom surface 11 of the well 10 and the pressing surface 20 may become smaller than expected due to manufacturing errors in the well plate 1 and the pressing jig 2. In that case, when the pressing jig 2 is inserted into the well 10, the liquefied gelled medium 81 or cell / gel suspension 82 that has been dropped onto the bottom surface 11 may come into contact with the side surface 12 and may then flow upward along the side surface 12. In that case, it may not be possible to keep the distribution range of the gelled medium 91 in the Z direction within a predetermined range.

[0069] In the pressing jig 2B according to the first modification, the presence of the legs 23B makes it possible to prevent the distance between the bottom surface 11 of the well 10 and the pressing surface 20B from becoming smaller than the length of the legs 23B. As a result, the gelatinous medium 91 can be prevented from contacting the side surface 12.

[0070] <4-2. Second modified example> 22 is a perspective view of a pressing jig 2C according to the second modification. This pressing jig 2C also has legs 23C, similar to the pressing jig 2C according to the first modification. However, the legs 23C of this pressing jig 2C extend downward from several points spaced apart in the circumferential direction on the outer periphery of the lower end of the body 21C. In this way, the legs 23C do not have to be connected in a cylindrical shape.

[0071] <4-3.Third modified example> 23 is a perspective view of a pressing jig 2D according to a third modified example. This pressing jig 2D has a plurality of body parts 21D to be inserted into the plurality of wells 10, respectively, and a single fixing part 22D. The fixing part 22D spreads in a plate shape along the upper surface of the well plate 1 and is connected to the upper end parts of the plurality of body parts 21D. Each body part 21D has a pressing surface 20D at its lower end. That is, this pressing jig 2D has a plurality of pressing surfaces 20D that are arranged at intervals from the bottom surfaces 11 of the plurality of wells 10 at the same time. In this way, the pressing jig 2D may be one that can be used simultaneously for a plurality of wells 10.

[0072] <4-4. Other variations> In the above embodiment, the well plate 1 having a plurality of wells (culture chambers) 10 has been described. However, the culture vessel of the present invention may be a petri dish or a beaker having one recess.

[0073] Furthermore, the elements appearing in the above-described embodiments and modifications may be combined as appropriate to the extent that no contradiction arises. [Explanation of symbols]

[0074] 1 Well plate (culture vessel) 2,2A,2B,2C,2D Pressing jig 9 cells 10 wells (culture chamber) 11 Bottom 12 Side 20,20A,20B,20D Pressing surface 21, 21A, 21B, 21D Torso 22,22A,22B,22D Fixed part 23B, 23C: Legs 81 Liquefied Gel 82 Cell-gel suspension 83 Cell / liquid medium suspension 91 Gel-like medium 92 Liquid medium 210A through hole 211A Top opening 212A Bottom opening

Claims

1. A pressing tool used with a culture vessel having a cup-shaped culture chamber with an open top, a fixing portion extending horizontally at an upper portion; A pressing surface that is disposed below the fixing portion and extends horizontally; having A pressing jig, in a state where the pressing jig is attached to the culture vessel, the fixing part is placed on the edge part of the opening of the culture chamber of the culture vessel, and the pressing surface faces the bottom surface of the culture chamber with a gap therebetween.

2. The pressing jig according to claim 1, A body portion having an outer peripheral surface extending in a cylindrical shape and having the pressing surface at a lower end portion. and The fixing portion is a pressing jig extending horizontally from an upper end of the body portion.

3. The pressing jig according to claim 2, A leg portion extending downward from the outer periphery of the lower end of the body portion The pressing jig further comprises:

4. The pressing jig according to claim 2, The body portion is A through hole that penetrates in the vertical direction between an upper opening provided on the upper end surface and a lower opening provided in the center of the pressing surface. A pressing jig having the following structure.

5. The pressing jig according to any one of claims 1 to 4, the culture vessel is a well plate having a plurality of the culture chambers, A pressing jig having a plurality of the pressing surfaces.

6. A cell culture method using the pressing tool according to any one of claims 1 to 3, a) dropping a suspension of cells and liquefied gel into the culture chamber; b) fixing the pressing tool to the culture vessel and bringing the pressing surface into contact with the suspension; c) solidifying the liquefied gel to obtain a gel encapsulating the cells; d) removing the pressing tool from the culture vessel; e) dropping a liquid medium into the culture chamber and culturing the cells; A cell culture method comprising the steps of:

7. A cell culture method using the pressing tool according to any one of claims 1 to 3, A) dropping a liquefied gel into the culture chamber; B) fixing the pressing tool to the culture vessel and bringing the pressing surface into contact with the liquefied gel; C) solidifying the liquefied gel to obtain a cell-free gel; D) removing the pressing tool from the culture vessel; E) dropping a suspension of cells and liquid medium into the culture chamber and culturing the cells; A cell culture method comprising the steps of:

8. A cell culture method using the pressing jig according to claim 4, p) fixing the pressing tool to the culture vessel; q) injecting a suspension of cells and liquefied gel below the pressing surface through the through-hole; r) solidifying the liquefied gel to obtain a gel containing the cells; s) removing the pressing tool from the culture vessel; t) dropping a liquid medium into the culture chamber and culturing the cells; A cell culture method comprising the steps of:

9. A cell culture method using the pressing jig according to claim 4, P) fixing the pressing tool to the culture vessel; Q) injecting liquefied gel below the pressing surface through the through hole; R) solidifying the liquefied gel to obtain a cell-free gel; S) removing the pressing tool from the culture vessel; T) dropping a suspension of cells and a liquid medium into the culture chamber and culturing the cells; A cell culture method comprising the steps of:

Images