Method for producing inserts, well plate with inserts, and insert production kit

Abstract

A technology is provided that enables co-culture of multiple cells without using a porous resin membrane. [Solution] The method for manufacturing an insert includes the steps of: a) preparing a hardened thermoplastic gel; b) after step a), contacting the open end 31P of the insert 31 with the thermoplastic gel to seal the opening of the open end 31P with the thermoplastic gel; c) after step b), introducing unhardened thermosetting gel into the insert; d) after step c), melting the thermoplastic gel by heating and hardening the thermosetting gel in the insert; and e) after step d), removing the melted thermoplastic gel.

Description

[Technical Field]

[0001] The subject matter disclosed herein relates to a method for producing an insert, a well plate with an insert, and an insert production kit. [Background technology]

[0002] Co-culture models of multiple cells are used as cell culture models that mimic organ functions, such as for basic research on pharmaceuticals and toxicity evaluation. As an example of a culture vessel that can be used for co-culture of multiple cells, a culture vessel using a well plate and an insert is commonly available and commercially available. For example, Patent Document 1 reports a method for producing a co-culture model consisting of multiple cells by culturing cells above and below an insert. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2007-166915 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, the bottom of the inserts used in conventional culture systems generally uses a porous resin membrane such as PET. Because the porous resin membrane is a non-biological material, there are concerns that it may affect the biological reproducibility of the model, such as substance permeation and cell-cell interactions. For this reason, there is a demand for a culture system that can co-culture multiple cells without using a porous resin membrane.

[0005] An object of the present invention is to provide a technique that enables co-culture of multiple cells without using a porous resin membrane. [Means for solving the problem]

[0006] In order to solve the above problems, a first aspect is a method for producing an insert to be inserted into a well plate, comprising: a) a step of preparing a hardened thermoplastic gel; b) after step a), a step of contacting the open end of the insert with the thermoplastic gel to seal the opening of the open end with the thermoplastic gel; c) after step b), a step of introducing unhardened thermosetting gel into the insert; d) after step c), a step of melting the thermoplastic gel by heating and hardening the thermosetting gel in the insert; and e) after step d), a step of removing the melted thermoplastic gel.

[0007] A second aspect is a method for producing an insert according to the first aspect, wherein step a) includes: a1) introducing uncured thermoplastic gel into a well plate; and a2) after step a1), curing the thermoplastic gel in the well plate; and step b) is contacting the open end of the insert with the cured thermoplastic gel in the well plate.

[0008] A third aspect is the method for producing an insert according to the second aspect, wherein the step e) further comprises the step of e1) discharging the thermoplastic gel from within the well plate.

[0009] A fourth aspect is a method for manufacturing an insert according to the first aspect, wherein step a) includes: a4) inserting the lower end of a molding jig into the uncured thermoplastic gel; a5) curing the uncured thermoplastic gel while the molding jig is inserted; and a6) after step a5), lifting up the molding jig, wherein the opening end of the insert is provided at the lower end of the insert; and step b) includes inserting the lower end of the insert into a recess in the thermoplastic gel formed by steps a4) to a6) and bringing the opening end of the insert into contact with the inner surface of the recess.

[0010] A fifth aspect is the method for producing an insert according to any one of the first to fourth aspects, wherein the solid component of the thermosetting gel is a biologically derived substance.

[0011] A sixth aspect is the method for producing an insert according to the fifth aspect, wherein the solid component of the thermosetting gel is collagen.

[0012] A seventh aspect is the method for producing an insert according to any one of the first to fifth aspects, wherein the solid component of the thermoplastic gel is a biologically derived substance.

[0013] An eighth aspect is the method for making an insert according to the seventh aspect, wherein the solid component of the thermoplastic gel is gelatin.

[0014] A ninth aspect is the method for producing an insert according to the first aspect, wherein the step c) is a step of introducing a thermosetting gel mixed with cells into the insert.

[0015] A tenth aspect is a well plate with an insert, comprising a well plate having a cylindrical well with a bottom, a cylindrical insert having an open end that can be inserted into the well, and a thermosetting gel that seals the opening at the open end of the insert.

[0016] An eleventh aspect is an insert production kit for use in any of the insert production methods of the first to ninth aspects, comprising a well plate having a cylindrical well with a bottom, a cylindrical insert that can be inserted into the well and has an open end at its lower end, and a molding jig that can be inserted into the well and has an outer width that is the same as or larger than the outer width of the lower end of the insert. [Effects of the Invention]

[0017] According to the first to fifth aspects, a hardened thermosetting gel can be formed at the open end of the insert, which makes it possible to prepare a co-culture model of multiple cells without using a porous resin membrane.

[0018] According to the second embodiment of the method for producing an insert, an insert with a hardened gel can be produced using a well plate.

[0019] According to the fourth aspect of the insert manufacturing method, the lower end of the insert is inserted into a recess in the hardened thermoplastic gel and the open end is brought into contact with the inner surface of the recess, thereby allowing the opening of the open end to be properly sealed with the thermoplastic gel.

[0020] According to the insert production method of the fifth aspect, the use of a thermosetting gel whose solid component is derived from a living body can improve the biological reproducibility of the co-culture model.

[0021] According to the insert manufacturing method of the sixth aspect, the thermosetting gel can be cured at a relatively low temperature.

[0022] According to the insert production method of the seventh aspect, by using a thermoplastic gel whose solid component is derived from a living body, even if the thermoplastic gel remains, it is possible to reduce the influence on cells.

[0023] According to the eighth embodiment of the method for producing an insert, the thermoplastic gel can be melted at a relatively low temperature.

[0024] According to the ninth aspect of the insert production method, by introducing a thermosetting gel containing cells, cells can be cultured within the hardened gel, and by culturing cells on top of the gel, under the gel, or both, more complex co-culture models can be produced.

[0025] According to the insert-equipped well plate of the tenth aspect, a co-culture model can be prepared without using a porous resin membrane.

[0026] According to the insert production kit of the eleventh aspect, an insert can be produced in which the opening at the open end is sealed with a hardened gel, thereby making it possible to produce a co-culture model without using a porous resin membrane. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a perspective view showing a configuration of an insert manufacturing kit according to an embodiment. [Figure 2] FIG. 10 is a partial cross-sectional view showing a well plate to which an insert coupler is attached. [Figure 3] FIG. 10 is a partial cross-sectional view showing a well plate to which a jig connector is attached. [Figure 4] FIG. 10 is a cross-sectional view of an insert with a gel cure. [Figure 5] 1 is a diagram showing the flow of a method for producing an insert with a gel hardening portion (insert production method). FIG. [Figure 6] FIG. 1 is a partial cross-sectional view showing a thermoplastic gel with a mold fixture inserted. [Figure 7] FIG. 10 is a partial cross-sectional view showing the well immediately after the mold jig has been lifted. [Figure 8] FIG. 10 is a partial cross-sectional view showing an insert in contact with a thermoplastic gel. [Figure 9] FIG. 10 is a partial cross-sectional view showing an insert with uncured thermoplastic gel introduced therein. [Figure 10] FIG. 10 is a partial cross-sectional view showing a well into which an insert with a gel hardener is inserted. [Figure 11] FIG. 1 shows a co-culture model that can be created using inserts with gel hardening. [Figure 12] FIG. 1 shows a co-culture model that can be created using inserts with gel hardening. [Figure 13] FIG. 1 shows a co-culture model that can be created using inserts with gel hardening. [Figure 14] FIG. 1 shows a co-culture model that can be created using inserts with gel hardening. DETAILED DESCRIPTION OF THE INVENTION

[0028] Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings. Note that the components described in the embodiment are merely examples and are not intended to limit the scope of the present invention. In the drawings, the dimensions and numbers of each part may be exaggerated or simplified as necessary to facilitate understanding.

[0029] <1. Embodiment> 1 is a perspective view showing the configuration of an insert fabrication kit 1 according to an embodiment. As will be described later, the insert fabrication kit 1 is a kit for fabricating an insert 31 to be attached to a well plate 2, the insert 31 having a hardened gel (hardened gel portion 61) formed thereon. The insert fabrication kit 1 includes the well plate 2, an insert connecting body 3, a jig connecting body 4, and a cover member 5.

[0030] The well plate 2 has a plurality of wells 21 (here, 48 wells) arranged in a matrix. Each well 21 has a cylindrical shape with a bottom that opens upward. The cross section of the well 21 (a cut along a plane perpendicular to the depth direction of the well 21) is rectangular. The cross section of the well 21 may have any shape, such as a circle or a polygon. The well plate 2 may also be a single-well plate having only one well 21. In the following description, the direction perpendicular to the bottom surface 211 will be simply referred to as the "depth direction." In the depth direction, the direction approaching the bottom surface 211 will be referred to as the downward direction, and the direction away from the bottom surface will be referred to as the upward direction.

[0031] The insert assembly 3 has a plurality of inserts 31 arranged in a matrix. The number of inserts 31 in the insert assembly 3 matches the number of wells 21 in the well plate 2. The inserts 31 are components inserted into the wells 21 of the well plate 2 and are components for retaining cells in the wells 21. The depth direction of the wells 21 matches the insertion direction of the inserts 31.

[0032] FIG. 2 is a partial cross-sectional view showing a well plate 2 to which an insert connector 3 is attached. In FIG. 2, only two wells 21 and one insert 31 are shown. As shown in FIG. 2, each well 21 has a bottom surface 211 and a side wall surface 213. The side wall surface 213 extends vertically upward from the bottom surface 211. In planar directions (vertical and horizontal directions) perpendicular to the depth direction, the outer width of the insert 31 is smaller than the inner width of the well 21.

[0033] 2, the insert 31 has a flange 33. The flange 33 is located at the upper end of the insert 31 and is a portion that is hooked onto the top of the well plate 2. Each insert 31 is connected to an adjacent insert 31 via the flange 33.

[0034] The insert 31 has an insert body 35. The insert body 35 is a generally cylindrical portion that extends downward from the flange 33. The insert body 35 has a shape that fits within the well 21. The insert body 35 has, in order from top to bottom, a first segment 351, a second segment 353, and a third segment 355.

[0035] The first segment 351 is a cylindrical (here, cylindrical) portion that opens upward. The second segment 353 is located between the first segment 351 and the third segment 355 in the depth direction and connects the first segment 351 and the second segment 353. The second segment 353 is formed in a hollow cone shape whose inner and outer widths gradually decrease downward. The third segment 355 has an open end 31P that opens downward. The inner and outer widths of the third segment 355 are smaller than those of the first segment 351, respectively. The lower end of the third segment 355 corresponds to the lower end of the insert 31. The interiors of the first segment 351, the second segment 353, and the third segment 355 are vertically connected to one another.

[0036] When the insert connection body 3 is attached to the well plate 2, the flange 33 of the insert 31 is supported on the upper end of the side wall surface 213 of the well 21. In this state, the lower end of the third segment 355 of the insert 31 (the lower end 311 of the insert 31) is positioned above and away from the bottom surface 211 of the well 21.

[0037] As shown in Fig. 1, the jig assembly 4 has a plurality of (four in this example) molding jigs 41 arranged in a row. The molding jigs 41 are formed in the shape of a rod extending linearly in the depth direction. The jig assembly 4 is a member that is attached to the well plate 2. The jig assembly 4 is attached to the well plate 2 with each molding jig 41 inserted into each well 21 of the well plate 2.

[0038] The number of molding jigs 41 included in the jig assembly 4 can be changed as desired. Also, the jig assembly 4 may include only one molding jig 41.

[0039] FIG. 3 is a partial cross-sectional view showing a well plate 2 to which a jig connector 4 is attached. In FIG. 3, only two wells 21 and one molding jig 41 are shown. When the jig connector 4 is attached to the well plate 2, the upper part of the molding jig 41 hooks onto the upper part of the well 21, thereby positioning the molding jig 41 in the depth direction. Note that the molding jig 41 may also be positioned in the vertical and horizontal directions perpendicular to the depth direction. For this reason, grooves or the like may be formed in the well plate 2 at the locations where the molding jig 41 hooks.

[0040] When the jig connecting body 4 is attached to the well plate 2, the lower end 411 of the molding jig 41 is positioned above and away from the bottom surface 211 of the well 21. The outer shape of the lower end 411 of the molding jig 41 is the same as the outer shape of the lower end of the insert 31 (the lower end of the third segment 355). The outer width (outer diameter) of the lower end 411 of the molding jig 41 is smaller than the inner width (inner diameter) of the well 21. In addition, the outer width (outer diameter) of the lower end 411 of the molding jig 41 is the same as or larger than the outer width (outer diameter) of the lower end 311 of the insert 31.

[0041] Each molding jig 41 of the jig connected body 4 is a member used to form a hardened gel portion 61 at the lower end 311 of the insert 31. The procedure for forming the hardened gel portion 61 using the molding jig 41 will be described later.

[0042] The lid member 5 is a member that is detachably attached to the top of the well plate 2. When attached to the well plate 2, the lid member 5 closes the top of each well 21. By closing the top of the wells 21, evaporation of the liquid (culture medium, etc.) in the wells 21, contamination by microorganisms or foreign matter from above the wells 21, cross-contamination between the wells 21, etc. can be reduced.

[0043] FIG. 4 is a cross-sectional view showing an insert 31 with a hardened gel portion 61. The hardened gel portion 61 is formed, for example, by hardening a thermosetting gel. The hardened gel portion 61 is formed so as to close the opening of the open end 31P provided at the lower end 311 of the insert 31. The solid component of the thermosetting gel is preferably a bio-derived substance. By using a bio-derived substance, a hardened gel portion 61 with high biocompatibility can be formed. Therefore, whether cells are cultured on the top surface, inside, or bottom surface of the hardened gel portion 61, bioreproducibility can be improved.

[0044] Furthermore, the lower surface of the insert 31 and the lower surface of the hardened gel portion 61 are formed flush with each other. That is, the lower surface of the insert 31 and the lower surface of the hardened gel portion 61 are smoothly continuous without any steps. This reduces the likelihood of bubbles remaining between the lower surface of the insert 31 and the lower surface of the hardened gel portion 61, even if bubbles are generated in the liquid when a liquid (such as a culture medium) is poured into the well 21. This reduces the impact on cells being cultured using the insert 31.

[0045] In the following description, the solid component of the thermosetting gel is collagen, but the solid component of the thermosetting gel may be other than collagen, such as fibrin or agarose.

[0046] 5 is a diagram showing the flow of a method for producing an insert 31 with a gel-hardened portion 61 (insert production method). The following explanation focuses on one insert 31 and describes the case of forming the gel-hardened portion 61 on one insert 31. However, it is also possible to form the gel-hardened portions 61 on multiple inserts 31 at the same time.

[0047] First, with the insert 31 and mold jig 41 not inserted into the well 21 (i.e., with the insert connector 3 or jig connector 4 not attached to the well plate 2), a liquid thermoplastic gel is introduced into the well 21 (step S1).

[0048] A thermoplastic gel is a polymeric substance that becomes solid when cooled below a predetermined solidification temperature and undergoes a phase transition to a fluid, liquid-like state upon melting when heated above a predetermined melting temperature. The solid component of the thermoplastic gel is preferably a biologically derived substance. By using a biologically derived substance as the solid component of the thermoplastic gel, even if the thermoplastic gel remains in the well 21 during cell culture, the impact on the cells can be reduced. Here, a case where the solid component of the thermoplastic gel is gelatin will be described. Gelatin gel has a solidification (gelling) temperature of 20 to 25°C and a melting temperature of 35 to 40°C. Gelatin gel reversibly changes state within these temperature ranges. In the case of gelatin gel, it is introduced into the well 21 by heating it to, for example, 37°C in step S1. Note that the thermoplastic gel is not limited to gelatin gel, and other gel-like substances may be used. Furthermore, the solid component of the thermoplastic gel does not necessarily have to be biologically derived.

[0049] After the thermoplastic gel is introduced into the well 21, the lower end 411 of the molding jig 41 is inserted into the liquid thermoplastic gel (step S2). Fig. 6 is a partial cross-sectional view showing the thermoplastic gel with the molding jig 41 inserted. As shown in Fig. 6, the insertion of the molding jig 41 forms a depression in the center of the upper part of the thermoplastic gel.

[0050] Then, with the mold jig 41 inserted, the uncured thermoplastic gel is cured (step S3). The thermoplastic gel is cured, for example, by leaving the well plate 2 in an environment below the solidification temperature of the thermoplastic gel (20 to 25°C in the case of gelatin gel). After the thermoplastic gel is cured in step S3, the mold jig 41 is pulled out (step S4). The above steps S1 to S5 correspond to the process of preparing a cured thermoplastic gel.

[0051] 7 is a partial cross-sectional view showing the well 21 immediately after the molding jig 41 has been pulled up. As shown in FIG. 7, a hardened thermoplastic gel 91 is formed on the bottom surface 211 inside the well 21. A recess 93 is formed in the center of the upper part of the thermoplastic gel 91. The shape of the recess 93 corresponds to the outer shape of the lower end 411 of the molding jig 41. The hardened thermoplastic gel 91 is used as a mold for forming a hardened gel portion 61 in the insert 31, as will be described later.

[0052] 5, after the hardened thermoplastic gel 91 is prepared, the insert 31 is inserted into the well 21. Then, the open end 31P of the lower end 311 of the insert 31 comes into contact with the thermoplastic gel 91, so that the opening of the open end 31P is blocked by the thermoplastic gel 91 (step S5).

[0053] Fig. 8 is a partial cross-sectional view showing the insert 31 in contact with the thermoplastic gel 91. As shown in Fig. 8, the lower end 311 of the insert 31 is inserted into the recess 93 of the thermoplastic gel 91 formed by the molding jig 41. Then, the lower end 311 and the open end 31P of the insert 31 come into contact with the bottom surface of the recess 93, thereby closing the opening of the open end 31P of the insert 31.

[0054] Returning to FIG. 5, after the insert 31 comes into contact with the thermoplastic gel 91, a liquid uncured thermosetting gel is introduced into the insert 31 (step S6). FIG. 9 is a partial cross-sectional view showing the insert 31 into which the uncured thermoplastic gel has been introduced. The uncured thermoplastic gel is introduced from an opening at the top of the insert 31. The open end 31P of the insert 31 is blocked by the thermoplastic gel 91. Therefore, the introduced thermoplastic gel is stored in the lower end 311 of the insert 31.

[0055] Returning to FIG. 5, after the uncured thermosetting gel is introduced into the insert 31, the thermoplastic gel 91 is melted by heating, and the thermosetting gel in the insert 31 is cured (step S7). By placing the well plate 2 in an environment at 37°C, the gelatin gel, which is the thermoplastic gel 91, is heated and melted. Furthermore, the collagen gel, which is the thermosetting gel, is heated and cured. As the thermosetting gel hardens, a cured gel portion 61 is formed to close the open end 31P of the lower end 411 of the insert 31.

[0056] Note that the thermosetting gel introduced into the insert 31 in step S6 may contain live cells. For example, a cell suspension gel in which live cells are dispersed in a liquid thermosetting gel may be introduced into the insert 31. This allows a hardened gel portion 61 in which live cells are dispersed and fixed to be formed in the insert 31. In this case, when the thermosetting gel is hardened by heating in step S7, maintaining the thermosetting gel at 40°C or below, more preferably 38°C or below, can reduce the death of live cells.

[0057] Furthermore, the thermosetting gel introduced into the insert 31 in step S6 may contain biochemical substances such as proteins, or drugs, etc. This allows the hardened gel portion 61 to have a specific function.

[0058] Next, the thermoplastic gel melted in step S7 is removed (step S8). Specifically, the melted thermoplastic gel is removed from the well 21. The thermoplastic gel is removed after the insert 31 is removed from the well 21. If possible, the thermoplastic gel may be removed while the insert 31 remains attached to the well 21. This removes the thermoplastic gel adhering to the insert 31 and the hardened gel portion 61. After step S8, the well 21 and the lower end 311 of the insert 31 are washed (step S9). Specifically, a washing liquid (e.g., pure water, physiological saline, or culture medium) is injected into the well 21. The lower end 311 of the insert 31 is then immersed in the injected washing liquid. The washing liquid is then drained from the well 21. The injection and drainage of the washing liquid may be repeated multiple times. By injecting and draining the washing liquid, the thermoplastic gel adhering to the well 21, the insert 31, and the hardened gel portion 61 can be effectively removed.

[0059] 10 is a partial cross-sectional view showing a well 21 into which an insert 31 with a gel hardening portion 61 is inserted. As shown in FIG. 10, by performing the procedure shown in FIG. 5, a gel hardening portion 61 that is smoothly continuous with the underside of the lower end portion 311 of the insert 31 is formed on the insert 31. Furthermore, when the insert connection body 3 is attached to the well plate 2, the underside of the gel hardening portion 61 is positioned above and spaced apart from the bottom surface 211 of the well 21. The well plate 2, insert 31, and gel hardening portion 61 formed on the insert 31 shown in FIG. 10 constitute an insert-equipped well plate.

[0060] According to the insert manufacturing method of this embodiment, a hardened thermosetting gel (gel hardened portion 61) can be formed at the open end 31P of the insert 31. This makes it possible to manufacture a co-culture model of multiple cells without using a resin porous membrane.

[0061] Furthermore, by hardening the thermoplastic gel using the molding jig 41, the recess 93 can be formed to match the height of the lower end 311 of the insert 31. This allows the lower end 311 of the insert 31 to come into contact with the inner bottom surface (inner surface) of the recess 93 without any gaps. Therefore, the hardened gel portion 61 can be formed so as to be smoothly continuous with the lower surface of the lower end 311.

[0062] 11 to 14 are diagrams showing a co-culture model that can be produced using an insert 31 with a hardened gel portion 61. First, in the example shown in FIG. 11, cells C1 are cultured on the upper surface of the hardened gel portion 61, and cells C2 are cultured on the lower surface of the hardened gel portion 61. Furthermore, a liquid such as a culture medium is appropriately injected into the inside and outside of the insert 31. In this way, co-culture between cells C1 and C2 is possible via the hardened gel portion 61.

[0063] 12 to 14 show co-culture models in which cell C3 is cultured within a hardened gel portion 61. In the co-culture model of FIG. 12, cell C1 is cultured on the upper surface of the hardened gel portion 61. In the co-culture model of FIG. 13, cell C2 is cultured on the lower surface of the hardened gel portion 61. In the co-culture model of FIG. 14, cells C1 and C2 are cultured on the upper and lower surfaces of the hardened gel portion 61, respectively.

[0064] As described above, by using the insert 31 with the hardened gel portion 61, it is possible to create a co-culture model consisting of multiple types of cells without using a porous membrane. Furthermore, by using the highly biocompatible hardened gel portion 61, it is possible to create a cell model with a high degree of biomimetic properties.

[0065] <2. Variations> Although the embodiments have been described above, the present invention is not limited to the above and various modifications are possible.

[0066] For example, in the above embodiment, the insert 31 with the gel curing portion 61 is produced in the well 21 by curing a thermoplastic gel in the well 21 of the well plate 2. However, the insert 31 with the gel curing portion 61 may be produced in a container other than the well 21 or on a flat plate. In other words, the thermoplastic gel may be cured in advance on any container or plate, and the insert 31 with the gel curing portion 61 may be produced on the thermoplastic gel.

[0067] Furthermore, it is not essential to form the recesses 93 using the molding jig 41. For example, the upper surface of the thermoplastic gel 91 may be a flat surface without any recesses 93. In this case, the open end 31P of the insert 31 may be brought into contact with the flat surface, thereby closing the open end 31P.

[0068] Furthermore, the open end 31P that is closed by the hardened gel portion 61 does not necessarily have to be formed at the lower end 311 of the insert 31. For example, it may be formed at a location other than the lower end 311, such as the side of the insert 31.

[0069] Although the present invention has been described in detail, the above description is merely illustrative in all respects and does not limit the present invention. It is understood that countless variations not illustrated can be envisioned without departing from the scope of the present invention. The configurations described in the above embodiments and variations can be combined or omitted as appropriate as long as they are not mutually inconsistent. [Explanation of symbols]

[0070] 1: Insert preparation kit 2: Well plate 21: Well 31: Insert 31P: Open end 41: Mold jig 61: Gel hardening part (thermosetting gel) 91: Thermoplastic gel 93: Recess 311: Bottom end 351: First segment 353: Second segment 355: Third segment 411: Bottom end

Claims

1. 1. A method for producing an insert to be inserted into a well plate, comprising: a) providing a hardened thermoplastic gel; b) after step a), contacting the open end of the insert with the thermoplastic gel to close the opening of the open end with the thermoplastic gel; c) after step b), introducing an uncured thermosetting gel into the insert; d) after step c), applying heat to melt the thermoplastic gel and harden the thermosetting gel within the insert; e) after step d), removing the melted thermoplastic gel; A method for making an insert, comprising:

2. 2. The method for manufacturing an insert according to claim 1, The step a) a1) introducing the uncured thermoplastic gel into a well plate; a2) after step a1), hardening the thermoplastic gel in the well plate; Including, The method for making an insert, wherein step b) is a step of contacting the open end of the insert with the hardened thermoplastic gel in the well plate.

3. 3. The method for manufacturing an insert according to claim 2, The step e) e1) discharging the thermoplastic gel from within the well plate; The method for making an insert further comprises:

4. 2. The method for manufacturing an insert according to claim 1, The step a) a4) inserting the lower end of a molding tool into the uncured thermoplastic gel; a5) curing the uncured thermoplastic gel with the molding jig inserted; a6) after step a5), a step of lifting up the molding jig; Including, the open end of the insert is located at a lower end of the insert; The step b) includes a step of inserting the lower end of the insert into the recess of the thermoplastic gel formed by the steps a4) to a6) and bringing the open end of the insert into contact with the inner surface of the recess.

5. 5. The method for manufacturing an insert according to claim 1, The method for producing an insert, wherein the solid component of the thermosetting gel is a biologically derived substance.

6. 6. The method for manufacturing an insert according to claim 5, The method for making an insert, wherein the solid component of the thermosetting gel is collagen.

7. 6. A method for manufacturing an insert according to any one of claims 1 to 5, The method for producing an insert, wherein the solid component of the thermoplastic gel is a biologically derived substance.

8. 8. The method for manufacturing an insert according to claim 7, The method for making an insert, wherein the solid component of the thermoplastic gel is gelatin.

9. 2. The method for manufacturing an insert according to claim 1, The method for producing an insert, wherein the step c) is a step of introducing a thermosetting gel mixed with cells into the insert.

10. A well plate with inserts, a well plate having a cylindrical well with a bottom; an insert insertable into the well and having an open end and formed in a cylindrical shape; a thermosetting gel that seals the opening at the open end of the insert; A well plate with an insert.

11. An insert manufacturing kit used in the insert manufacturing method according to any one of claims 1 to 9, a well plate having a cylindrical well with a bottom; a cylindrical insert that can be inserted into the well and has an open end at its lower end; a molding tool insertable into the well and having an outer width equal to or greater than the outer width of the lower end of the insert; An insert preparation kit comprising:

Images