Insert member, culture container, component for insert member, insert member manufacturing method, and seeding method

Abstract

This insert member is provided with an insert body. The insert body is inserted into a container for co-culturing different types of cells. The insert body is provided with a cylindrical part having a tubular shape and a closing part. The cylindrical part has an opening at the bottom. Different types of cells can be seeded on the closing part. The closing part closes the opening. The biocompatibility of the closing part is higher than that of a porous resin. The biocompatibility indicates the degree of biological environment recreatability.

Description

Insert member, culture vessel, parts of insert member, method for manufacturing insert member, and seeding method

[0001] The present invention relates to an insert member, a culture vessel, a component of an insert member, a method for manufacturing an insert member, and a seeding method.

[0002] Patent Document 1 describes a method for creating an in vitro blood-brain barrier model. The in vitro blood-brain barrier model is obtained by co-culturing primary cultured brain capillary endothelial cells, primary cultured brain pericytes, and primary cultured astrocytes in a three-dimensional culture device containing a porous filter.

[0003] Specifically, a culture medium is placed in a container, and a filter is immersed in the culture medium using a hanger. Primary cultured brain capillary endothelial cells are seeded on the surface of the porous filter, primary cultured brain pericytes are seeded on the backside of the filter, and primary cultured astrocytes are seeded below the filter, reproducing the in vivo blood-brain barrier. Furthermore, the porosity of the filter allows crosstalk between the three cell types, making it possible to reproduce the complex mechanisms of maintenance and regulation of the blood-brain barrier in vivo.

[0004] Japanese Patent Application Laid-Open No. 2007-166915

[0005] However, the filter described in Patent Document 1 is a porous resin membrane such as polyester. The porous resin membrane is a non-biological material. Therefore, when co-culture is performed using a porous resin membrane, for example, the reproducibility of substance permeation or cell-cell interactions may be insufficient. In other words, in this case, the porous resin membrane may affect the bioreproducibility of the cell model obtained by co-culture.

[0006] One object of the present invention is to provide an insert member, a culture vessel, parts of an insert member, a method for manufacturing an insert member, and a seeding method that can improve the biological reproducibility of a cell model obtained by co-culture.

[0007] According to one aspect of the present invention, an insert member includes an insert body. The insert body is inserted into a container for co-culturing different types of cells. The insert body includes a cylindrical tube portion and a closing portion. The tube portion has an opening at the bottom. The different types of cells can be seeded in the closing portion. The closing portion closes the opening. The biocompatibility of the closing portion is higher than the biocompatibility of a porous resin. The biocompatibility indicates the degree of reproducibility of a biological environment.

[0008] In one aspect of the present invention, the blocking portion is preferably made of gel.

[0009] In one aspect of the present invention, the blocking portion preferably contains a biological substance.

[0010] In one aspect of the present invention, the occlusion portion preferably contains cells.

[0011] In one aspect of the present invention, it is preferable that the temperature of the blocking portion is lower than room temperature.

[0012] In one aspect of the present invention, it is preferable that the insert body further includes a support portion that supports the closing portion.

[0013] In one aspect of the present invention, the cylindrical portion preferably has a communication portion that communicates the inside and the outside of the cylindrical portion.

[0014] According to another aspect of the present invention, a culture vessel includes the insert member described above and a vessel into which the insert member is inserted.

[0015] According to yet another aspect of the present invention, a component of an insert member has an insert body to be inserted into a container for co-culturing different types of cells. The component includes a cylindrical tube portion and a cover member. The tube portion has an opening at its bottom. The cover member covers the opening and is detachable from the tube portion.

[0016] According to yet another aspect of the present invention, a method for manufacturing an insert member includes the steps of: preparing an insert member component having an insert body to be inserted into a container for co-culturing different types of cells, the component comprising a cylindrical tube portion having an opening at the bottom and a cover member covering the opening; placing a substance in a state before it changes to a gel on the upper surface of the cover member; changing the state of the substance to a gel to form a blocking portion that covers the opening; and peeling the cover member from the tube portion. Different types of cells can be seeded in the blocking portion. The biocompatibility of the blocking portion is higher than that of a porous resin. The biocompatibility indicates the degree of reproducibility of the biological environment.

[0017] According to yet another aspect of the present invention, a seeding method includes the steps of: preparing an insert member including an insert body to be inserted into a container for co-culturing different types of cells, the insert body having a cylindrical portion with an opening at the bottom and a closing portion that closes the opening; and seeding the cells into the closing portion. The biocompatibility of the closing portion is higher than that of a porous resin. The biocompatibility indicates the degree of reproducibility of a biological environment.

[0018] In one aspect of the present invention, the cells preferably include first cells. The seeding step preferably includes seeding the first cells in a first region between the second region and the third region of the occlusion part. The second region preferably refers to a region including the upper surface of the occlusion part. The third region preferably refers to a region including the lower surface of the occlusion part.

[0019] In one aspect of the present invention, the cells preferably include second cells. The seeding step preferably includes seeding the second cells in a second region including an upper surface of the closure portion.

[0020] In one aspect of the present invention, the cells preferably include third cells, and the seeding step preferably includes the step of seeding the third cells in a third region including the underside of the closure portion before the step of inserting the insert body into the container body of the container.

[0021] According to at least one aspect of the present invention, the bioreproducibility of a cell model obtained by co-culture can be improved.

[0022] 5A-5D are perspective views showing a culture vessel according to an embodiment of the present invention. (a) is a plan view showing an insert member of the culture vessel according to the embodiment. (b) is a plan view showing a vessel of the culture vessel according to the embodiment. (a) is a cross-sectional view taken along line IIIA-IIIA in FIG. 2A. (b) is a cross-sectional view taken along line IIIB-IIIB in FIG. 2B. (a)-(d) are cross-sectional views showing first to fourth examples of co-culture using the culture vessel according to the embodiment. (a) is a perspective view showing components of the insert member of the culture vessel according to the embodiment. (b) is a cross-sectional view taken along line VB-VB in FIG. 5A. (a) is a cross-sectional view showing a first example of a method for manufacturing an insert member according to the embodiment. (b) is a cross-sectional view showing a second example of a method for manufacturing an insert member according to the embodiment. (b) is a cross-sectional view showing an example of a seeding method using the culture vessel according to the embodiment. (a)-(d) are cross-sectional views showing first to fourth examples of an insert member in which different types of cells have been seeded. (a) is a cross-sectional view showing an insert member of a culture vessel according to a first modification of the embodiment. 14(a) is a cross-sectional view showing a state in which co-culture is performed using a culture vessel according to a first modified example. (a) is a cross-sectional view showing an insert member of a culture vessel according to a second modified example of this embodiment. (b) is a plan view showing an insert member of an insert vessel according to a second modified example with the closure removed in the culture vessel according to the second modified example. (a) is a cross-sectional view showing an insert member of an insert vessel according to a third modified example of this embodiment. (b) is a plan view showing an insert member of an insert vessel according to a third modified example of this embodiment with the closure removed in the culture vessel according to the third modified example. (a) is a cross-sectional view showing an insert member of an insert vessel according to a fourth modified example of this embodiment. (b) is a plan view showing an insert member of an insert vessel according to the fourth modified example of this embodiment with the closure removed in the culture vessel according to the fourth modified example. (a) is a perspective view showing an insert member of an insert vessel according to a fifth modified example of this embodiment. (b) is a cross-sectional view taken along line XIVB-XIVB in FIG. 14(a).

[0023] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the drawings, the same or corresponding parts are designated by the same reference numerals, and description thereof will not be repeated. Furthermore, in this specification, to facilitate understanding of the invention, mutually orthogonal X-axis, Y-axis, and Z-axis may be described. Typically, the X-axis and Y-axis are parallel to the horizontal direction, and the Z-axis is parallel to the vertical direction.

[0024] A culture vessel 1 according to an embodiment of the present invention will be described with reference to Figures 1 to 9. Figure 1 is a perspective view showing the culture vessel 1 according to this embodiment. The culture vessel 1 is a vessel for co-culturing different types of cells. For example, the cells are animal cells. The animal cells are, for example, human, mouse, or rat cells.

[0025] As shown in Fig. 1, the culture vessel 1 includes an insert member 100 and a vessel 200. The insert member 100 is inserted into the vessel 200. The vessel 200 is a vessel in which different types of cells are co-cultured with the insert member 100 inserted into the vessel 200. The vessel 200 is, for example, a well plate or a dish. The well plate has a size conforming to, for example, the ANSI / SBS standard.

[0026] The container 200 includes a container base 201. The container base 201 has, for example, a substantially flat plate shape. In the example of Fig. 1, the container base 201 has a substantially rectangular flat plate shape.

[0027] The container 200 further includes at least one container body 203. In the example of Fig. 1, the container 200 includes a plurality of container bodies 203. The number of the container bodies 203 is not particularly limited. The number of the container bodies 203 is, for example, 6, 12, 24, or 96. The plurality of container bodies 203 are arranged on the container base portion 201.

[0028] The container body 203 is recessed relative to the container base 201. In other words, the container body 203 protrudes downward from the container base 201. The container body 203 is hollow and has a space 211. The container body 203 also has an opening 209. The container body 203 has, for example, a substantially cylindrical shape with a bottom. In the example of FIG. 1 , the container body 203 has a substantially cylindrical shape with a bottom.

[0029] Specifically, the container body 203 includes a bottom portion 205 and a tubular portion 207. The bottom portion 205 has a generally flat plate shape. In the example of FIG. 1 , the bottom portion 205 has a generally disc shape. The tubular portion 207 extends from the bottom portion 205 to the container base portion 201. The tubular portion 207 has a generally tubular shape. In the example of FIG. 1 , the tubular portion 207 has a generally cylindrical shape.

[0030] The container 200 is made of, for example, synthetic resin or glass. In this case, the synthetic resin is, for example, polystyrene, polyethylene, polyethylene terephthalate, or polycarbonate.

[0031] The container 200 may be, for example, an integrally molded product. Alternatively, the container base 201 and the container body 203 may be separate components, and the container body 203 may be joined to the container base 201.

[0032] The insert member 100 includes an insert base portion 101. The insert base portion 101 has, for example, a substantially flat plate shape. In the example of Fig. 1, the insert base portion 101 has a substantially rectangular flat plate shape.

[0033] The insert member 100 further includes at least one insert body 103. In the example of FIG. 1 , the insert member 100 includes a plurality of insert bodies 103. The number of insert bodies 103 is not particularly limited. The number of insert bodies 103 is, for example, 6, 12, 24, or 96. In the example of FIG. 1 , the number of insert bodies 103 is the same as the number of container bodies 203. The plurality of insert bodies 103 are provided corresponding to the plurality of container bodies 203, respectively. The insert bodies 103 are inserted into the corresponding container bodies 203. In other words, the insert bodies 103 are inserted into the container 200. The plurality of insert bodies 103 are arranged on the insert base portion 101.

[0034] The insert body 103 is recessed relative to the insert base portion 101. In other words, the insert body 103 protrudes downward from the insert base portion 101. Therefore, the insert body 103 can be easily inserted into the container body 203. The insert body 103 is hollow and has a space 111. The insert body 103 also has a first opening 109. The first opening 109 is defined by the upper end of the cylindrical portion 107.

[0035] Specifically, the insert body 103 includes a tubular portion 107. The tubular portion 107 has a substantially tubular shape. In the example of FIG. 1 , the tubular portion 107 has a substantially cylindrical shape. The tubular portion 107 extends downward from the insert base portion 101. The tubular portion 107 has a bottom portion 105.

[0036] The insert base portion 101 and the cylindrical portion 107 are made of a material such as synthetic resin or glass. In this case, the synthetic resin is, for example, polystyrene, polyethylene, polyethylene terephthalate, or polycarbonate.

[0037] The insert base portion 101 and the cylindrical portion 107 may be a single member, for example, an integrally molded product. Alternatively, the insert base portion 101 and the cylindrical portion 107 may be separate components, and the cylindrical portion 107 may be joined to the insert base portion 101.

[0038] FIG. 2(a) is a plan view showing the insert member 100. FIG. 3(a) is a cross-sectional view taken along line IIIA-IIIA in FIG. 2(a). As shown in FIG. 3(a), the cylindrical portion 107 has a second opening 115 in the bottom portion 105. The second opening 115 faces the first opening 109 in a first direction D1. The first direction D1 is substantially parallel to the direction in which the insert body 103 is inserted into the container body 203 (FIG. 1). In other words, the first direction D1 is substantially parallel to the direction in which the cylindrical portion 107 extends. In this embodiment, the first direction D1 is substantially parallel to the vertical direction.

[0039] The second opening 115 corresponds to an example of the "opening" of the present invention.

[0040] The insert body 103 further includes a closing portion 113. The closing portion 113 closes the second opening 115. The closing portion 113 has a generally flat plate shape. In the example of FIG. 2( a), the closing portion 113 has a generally circular plate shape. As shown in FIG. 3( a), the closing portion 113 faces the first opening 109 in the first direction D1. In other words, the closing portion 113 forms the bottom of the insert body 103. The first direction D1 is generally perpendicular to the closing portion 113.

[0041] Different types of cells can be seeded into the occluding portion 113. The biocompatibility of the occluding portion 113 is higher than that of the porous resin. Biocompatibility indicates the degree of reproducibility of the biological environment. The biological environment refers to the biological environment of an animal. In this case, the animal is typically a human, but is not particularly limited and may be, for example, another mammal. The biological environment refers to the internal state or environment of a living organism. Specifically, the biological environment refers to the state or environment inside a living organism in which cells can survive. The porous resin is, for example, porous polyester. Biocompatibility can also be defined as follows: That is, biocompatibility refers to the property of having affinity for the biological tissues or organs of an animal and not causing a foreign body reaction or rejection reaction to the biological tissues or organs of an animal. High biocompatibility indicates high affinity for the biological tissues or organs of an animal.

[0042] The occluding portion 113 is formed of, for example, a gel. The gel is, for example, a protein gel, a fibrin gel, or a polysaccharide gel. The protein gel is, for example, a gel containing collagen (e.g., gelatin gel). The fibrin gel is a gel in which fibrin forms a mesh and water molecules are fixed within the mesh. The polysaccharide gel is, for example, a gel containing agarose. Other examples of the occluding portion 113 include, but are not limited to, chitosan gel, Matrigel (MATRIGEL: registered trademark), Vitrigel (VITRIGEL: registered trademark), or dextran gel. The gel is also, for example, a hydrogel. The gel may be, for example, an extracellular matrix gel. The extracellular matrix contains two types of polymers: fibrous proteins and proteoglycans. The fibrous protein is, for example, collagen.

[0043] The blocking portion 113 includes a first region 135, a second region 129, and a third region 131. The first region 135 indicates the region inside the blocking portion 113. Specifically, the first region 135 indicates the region between the second region 129 and the third region 131. The second region 129 indicates the region including the upper surface 125 of the blocking portion 113. In other words, the second region 129 indicates the region closer to the upper surface 125 than the first region 135. The third region 131 indicates the region including the lower surface 127 of the blocking portion 113. In other words, the third region 131 indicates the region closer to the lower surface 127 than the first region 135. Note that in FIG. 3A , for ease of understanding, the boundary between the second region 129 and the first region 135 is indicated by a dashed line, and the boundary between the third region 131 and the first region 135 is also indicated by a dashed line, but in reality, no boundary lines exist.

[0044] Fig. 2(b) is a plan view showing the container 200. Fig. 3(b) is a cross-sectional view taken along line IIIB-IIIB in Fig. 2(b). As shown in Fig. 3(b), the container body 203 opens upward. The opening 215 is defined by the upper end of the cylindrical portion 207. The opening 215 faces the bottom portion 205 in the first direction D1.

[0045] As shown in Figures 3(a) and 3(b), the outer width W1 of the insert body 103 is smaller than the inner width W3 of the container body 203. Therefore, when the insert body 103 is inserted into the container body 203, the cylindrical portion 107 of the insert body 103 is spaced apart from the cylindrical portion 207 of the container body 203 in the second direction D2. The second direction D2 is approximately perpendicular to the first direction D1. The outer width W1 indicates the length of the outer portion of the cylindrical portion 107 in the second direction D2. In the example of Figure 3(a), the outer width W1 indicates the outer diameter of the cylindrical portion 107. The inner width W3 indicates the length of the inner portion of the cylindrical portion 207 in the second direction D2. In the example of Figure 3(b), the inner width W3 indicates the inner diameter of the cylindrical portion 207.

[0046] Furthermore, the length W2 of the insert body 103 is smaller than the length W4 of the space 211 in the container body 203. Therefore, when the insert body 103 is inserted into the container body 203, the closing portion 113 of the insert body 103 is spaced apart in the first direction D1 from the bottom 205 of the container body 203. The length W2 indicates the length of the tubular portion 207 in the first direction D1 relative to the lower surface of the insert base portion 101. The length W4 indicates the length of the space 211 in the container body 203 in the first direction D1.

[0047] Next, an example of co-culture using the culture vessel 1 will be described with reference to Figure 4. Figures 4(a) to 4(d) show a state in which the insert body 103 is inserted into the vessel body 203. In other words, Figures 4(a) to 4(d) show a state in which co-culture is performed using the culture vessel 1. In this case, the type of the first cell 20 is different from the type of the second cell 121 and the type of the third cell 123. Hereinafter, overlapping explanations of the first to fourth examples will be omitted as appropriate.

[0048] 4(a) is a cross-sectional view showing a first example of co-culture using the culture vessel 1. As shown in FIG. 4(a), second cells 121 are seeded in the second region 129 of the closure portion 113. Furthermore, third cells 123 are seeded in the third region 131. Furthermore, a culture medium 317 is introduced into the vessel body 203, and a culture medium 319 is introduced into the insert body 103. The culture media 317 and 319 are, for example, liquid culture media. The culture media 317 and 319 are, for example, the same.

[0049] The culture medium 317 comes into contact with the lower surface 127 of the closing portion 113 and enters the inside of the closing portion 113 from below. The culture medium 319 comes into contact with the upper surface 125 of the closing portion 113 and enters the inside of the closing portion 113 from above.

[0050] Culture medium 317 provides nutrients to at least third cells 123 and may also provide nutrients to second cells 121. Culture medium 319 provides nutrients to at least second cells 121 and may also provide nutrients to third cells 123.

[0051] In the first example, the second cell 121 and the third cell 123 are co-cultured to create a cell model consisting of the second cell 121 and the third cell 123 .

[0052] Fig. 4(b) is a cross-sectional view showing a second example of co-culture using the culture vessel 1. As shown in Fig. 4(b), second cells 121 are seeded in the second region 129 of the closure portion 113. Furthermore, first cells 133 are seeded in the first region 135.

[0053] Culture medium 317 provides nutrients to at least first cell 133 and may also provide nutrients to second cell 121. Culture medium 319 provides nutrients to at least second cell 121 and may also provide nutrients to first cell 133. Otherwise, culture media 317, 319 of the second example are similar to culture media 317, 319 of the first example.

[0054] In the second example, the second cell 121 and the first cell 133 are co-cultured to create a cell model consisting of the second cell 121 and the first cell 133 .

[0055] Fig. 4(c) is a cross-sectional view showing a third example of co-culture using the culture vessel 1. As shown in Fig. 4(c), first cells 133 are seeded in the first region 135 of the closure portion 113. Furthermore, third cells 123 are seeded in the third region 131.

[0056] Culture medium 317 provides nutrients to at least third cells 123 and may also provide nutrients to first cells 133. Culture medium 319 provides nutrients to at least first cells 133 and may also provide nutrients to third cells 123. Otherwise, culture media 317, 319 of the third example are similar to culture media 317, 319 of the first example.

[0057] In the third example, the first cell 133 and the third cell 123 are co-cultured to create a cell model consisting of the first cell 133 and the third cell 123 .

[0058] Fig. 4(d) is a cross-sectional view showing a fourth example of co-culture using the culture vessel 1. As shown in Fig. 4(d), first cells 133 are seeded in the first region 135 of the closure portion 113. Second cells 121 are seeded in the second region 129. Third cells 123 are seeded in the third region 131.

[0059] Culture medium 317 provides nutrients to at least third cell 123, and may also provide nutrients to first cell 133 and second cell 121. Culture medium 319 provides nutrients to at least second cell 121, and may also provide nutrients to first cell 133 and third cell 123. Otherwise, culture media 317, 319 of the fourth example are similar to culture media 317, 319 of the first example.

[0060] In the fourth example, the first cell 133, the second cell 121, and the third cell 123 are co-cultured to create a cell model consisting of the first cell 133, the second cell 121, and the third cell 123.

[0061] The first cell 133, the second cell 121, and the third cell 123 are examples of the "cell" of the present invention.

[0062] As described above with reference to FIG. 3 , according to this embodiment, the biocompatibility of the closure portion 113 of the insert body 103 is higher than that of a porous resin. Therefore, when different types of cells are seeded in the closure portion 113 and co-cultured ( FIG. 4 ), the reproducibility of substance permeation or cell-cell interactions can be improved compared to when a porous resin is used. As a result, the bioreproducibility of the cell model obtained by co-culture can be improved compared to when a porous resin is used. In other words, a cell model with a higher degree of biomimetic properties can be created by co-culture compared to when a porous resin is used.

[0063] 3, in this embodiment, the occluding portion 113 is preferably made of gel. This preferred example further enhances the biocompatibility of the occluding portion 113 compared to when a porous resin is used. As a result, the bioreproducibility of the cell model obtained by co-culture can be further improved.

[0064] Furthermore, in this embodiment, by forming the blocking portion 113 from a gel, cells (first cells 133) can be seeded and co-cultured not only in the second region 129 and the third region 131 but also in the first region 135 inside the blocking portion 113. As a result, a cell model with a complex structure can be created by co-culture.

[0065] Furthermore, it is more preferable that the blocking portion 113 contains a biological material. According to this preferable example, the biocompatibility of the blocking portion 113 can be further improved compared to when a non-biological material is used. As a result, the bioreproducibility of the cell model obtained by co-culture can be further improved.

[0066] A biological substance is a substance derived from humans or other animals. In other words, a biological substance is a substance obtained from humans or other animals. A substance obtained from humans or other animals may be a substance obtained directly from humans or other animals (hereinafter referred to as a "direct substance"), or may be a substance obtained from a direct substance. Furthermore, a direct substance may be a substance obtained by purifying a substance obtained from humans or other animals. An example of a biological substance is collagen or fibrin.

[0067] Non-biological substances are substances that are not derived from humans or other animals. Examples of non-biological substances include resins.

[0068] Next, a method for manufacturing the insert member 100 (hereinafter referred to as an "insert member manufacturing method") will be described with reference to FIGS.

[0069] Fig. 5(a) is a perspective view showing a part 1000 of the insert member 100. Fig. 5(b) is a cross-sectional view taken along line VB-VB in Fig. 5(a). As shown in Figs. 5(a) and 5(b), the part 1000 of the insert member 100 includes an insert base portion 101, at least one cylindrical portion 107, and at least one cover member 119. In the example of Fig. 5(a), the part 1000 includes the insert base portion 101, a plurality of cylindrical portions 107, and a plurality of cover members 119. A plurality of cover members 119 are arranged corresponding to the plurality of cylindrical portions 107, respectively.

[0070] The cover member 119 is affixed to the bottom 105 of the tubular portion 107. Specifically, the cover member 119 is affixed to the lower end surface 117 of the tubular portion 107. For example, the cover member 119 is affixed to the lower end surface 117 with an adhesive or double-sided tape. The lower end surface 117 is generally annular. In the example of FIG. 5 , the lower end surface 117 is generally annular. The cover member 119 is removable from the tubular portion 107. The cover member 119 covers the second opening 115 from below the tubular portion 107. In other words, the cover member 119 blocks the second opening 115 from below the tubular portion 107.

[0071] The cover member 119 is made of, for example, a resin. The resin is, for example, polyester. The cover member 119 is, for example, a film or a sheet. The cover member 119 has, for example, a substantially circular shape.

[0072] 6 is a cross-sectional view showing a first example of a method for manufacturing an insert member according to this embodiment. In this first example, the closure portion 113 is made of fibrin gel. As shown in FIG. 6, the first example of the method for manufacturing an insert member includes steps S1 to S5.

[0073] First, in step S1, a substance 311 is prepared. The substance 311 is a raw material of the blocking portion 113. The substance 311 is in a state before it changes into the gel 114. The substance 311 is, for example, a liquid.

[0074] In the first example, in step S1, a cooled substance 311 is prepared. The cooled state indicates that the temperature of the substance 311 is lower than room temperature. The substance 311 is a mixed liquid composed of multiple components. For example, the substance 311 is a mixed liquid containing a substrate and an enzyme. The room temperature is, for example, about 20°C.

[0075] Specifically, a fibrinogen solution 303 is prepared in a container 301 under a cooled environment. Fibrinogen is a protein. Fibrinogen is a substrate. Also under a cooled environment, a thrombin solution 307 is prepared in a container 305. Thrombin is an enzyme. Then, under a cooled environment, the fibrinogen solution 303 and the thrombin solution 307 are mixed in a container 309 to create a mixed liquid substance 311. In other words, a cooled substance 311 is created. The reason for mixing under a cooled environment is to suppress the enzyme reaction. The cooled environment is an environment at a temperature lower than room temperature. The cooled environment is, for example, an environment of approximately 4°C. The cooled state is, for example, a state of approximately 4°C.

[0076] The container 309 may be the container 301, the container 305, or a container separate from the containers 301 and 305. The containers 301, 305, and 309 are, for example, test tubes.

[0077] Next, in step S2, a part 1000 is prepared.

[0078] Next, in step S3, the substance 311 is introduced into the interior of each of the cylindrical portions 107 of the component 1000, thereby disposing the substance 311 on the upper surface 120 of the cover member 119.

[0079] Next, in step S4 , the state of the substance 311 is changed to a gel 114 to form a blocking portion 113 that covers the second opening 115 .

[0080] In the first example, in step S4, the cooled substance 311 is heated to change the state of the substance 311 into a gel 114.

[0081] Specifically, substance 311, which is a mixed liquid of fibrinogen solution 303 and thrombin solution 307, is heated from a cooled state, and an enzyme reaction causes substance 311 to change into gel 114. Gel 114 is a fibrin gel. For example, substance 311 is heated from a cooled state to room temperature and changes into gel 114.

[0082] Next, in step S5, the cover member 119 is peeled off from the cylindrical portion 107. As a result, the insert member 100 (insert body 103) is completed, and the insert member manufacturing method is completed.

[0083] As explained above with reference to Figure 6, according to the first example of the insert member manufacturing method of this embodiment, the insert member 100 having the blocking portion 113 can be easily manufactured by introducing the pre-gelled substance 311 into the tubular portion 107 (step S3), gelling the substance 311 (step S4), and peeling off the cover member 119 (step S5).

[0084] In particular, in step S1, by preparing substance 311, which is a liquid mixture obtained by mixing fibrinogen solution 303 and thrombin solution 307, it is possible to create occlusion portion 113 made of fibrin gel.

[0085] The part 1000 of the insert member 100 described with reference to FIG. 5 is suitable for carrying out the method for manufacturing the insert member shown in FIG.

[0086] 7 is a cross-sectional view showing a second example of the method for manufacturing an insert member according to this embodiment. In the second example, the closure portion 113 is made of a protein gel (e.g., a gel containing collagen). The following mainly describes the differences between the second example and the first example.

[0087] As shown in FIG. 7, the second example of the method for manufacturing an insert member includes steps S11 to S15.

[0088] First, in step S11, a substance 315 is prepared. In a second example, a cooled substance 315 is prepared in step S11. Specifically, the substance 315, which is a collagen solution, is prepared in a container 313 in a cooled environment. The cooled environment is, for example, an environment of about 4°C. The cooled state is, for example, a state of about 4°C. The container 313 is, for example, a test tube.

[0089] Next, in step S12, a part 1000 is prepared.

[0090] Next, in step S13 , the substance 315 is introduced into the interior of each of the cylindrical portions 107 of the component 1000 , thereby disposing the substance 315 on the upper surface 120 of the cover member 119 .

[0091] Next, in step S14, the state of the substance 311 is changed to a gel 114. As a result, the blocking portion 113 is formed.

[0092] In the second example, in step S14, the substance 315, which is a collagen solution, is heated from a cooled state to the body temperature, thereby changing the state of the substance 315 into a gel 114. The gel 114 is a gelatin gel. The body temperature is body temperature (e.g., approximately 37°C).

[0093] Next, in step S15, the cover member 119 is peeled off from the cylindrical portion 107. As a result, the insert member 100 (insert body 103) is completed, and the insert member manufacturing method is completed.

[0094] As described above with reference to Figure 7, according to the second example of the insert member manufacturing method of this embodiment, the insert member 100 having the blocking portion 113 can be easily manufactured, similar to the first example.

[0095] In particular, in step S11, by preparing substance 315, which is a collagen solution, it is possible to create occlusion section 113 made of a gel containing collagen. The gel containing collagen is, for example, an extracellular matrix gel.

[0096] The part 1000 of the insert member 100 described with reference to FIG. 5 is suitable for carrying out the method for manufacturing the insert member shown in FIG.

[0097] Next, a method for seeding cells using the culture vessel 1 will be described with reference to Fig. 8. Fig. 8 is a cross-sectional view showing an example of the seeding method according to this embodiment. As shown in Fig. 8, the seeding method includes steps S21 to S27.

[0098] First, in step S21, the insert member 100 is prepared.

[0099] Next, in step S22, third cells 123 are seeded in the closure portion 113. That is, the third cells 123 are seeded in the closure portion 113 before step S24 of inserting the insert body 103 into the container body 203. Specifically, the third cells 123 are seeded in the third region 131 of the closure portion 113.

[0100] Next, in step S23, the culture medium 317 is introduced into the container 200. Specifically, the culture medium 317 is introduced into the container body 203.

[0101] Next, in step S24, the insert member 100 in step S22 is inserted into the container 200 in step S23. Specifically, the insert body 103 in step S22 is inserted into the container body 203 in step S23.

[0102] Next, in step S25, first cells 133 are seeded in occluded portion 113. Specifically, first cells 133 are seeded in first region 135 of occluded portion 113.

[0103] Next, in step S26, second cells 121 are seeded in occluded portion 113. Specifically, second cells 121 are seeded in second region 129 of occluded portion 113.

[0104] Next, in step S27, the culture medium 319 is introduced into the insert body 103. Then, the seeding method is completed.

[0105] As described above with reference to FIG. 8 , the seeding method according to this embodiment allows different types of cells (first cell 133, second cell 121, and third cell 123) to be seeded into the highly biocompatible occluding portion 113. Therefore, compared to when a porous resin is used, the reproducibility of substance permeation or cell-cell interactions can be improved when performing co-culture. As a result, compared to when a porous resin is used, the bioreproducibility of the cell model obtained by co-culture can be improved.

[0106] Furthermore, according to the seeding method of this embodiment, cells (first cells 133) can be seeded and co-cultured not only in the second region 129 and the third region 131 but also in the first region 135 inside the occluding portion 113. As a result, a cell model with a complex structure can be created by co-culture. Specifically, the co-culture shown in FIG. 4( d ) is performed, and a cell model with a complex structure can be created.

[0107] Continuing with reference to FIG. 8, a modified seeding method will be described.

[0108] Step S22 (seeding of the third cells 123) may be performed at any timing as long as it is performed before step S24 (insertion of the insert body 103 into the container body 203). Hereinafter, the timing of performing step S22 includes not only the timing shown in FIG. 8 but also "any timing."

[0109] Furthermore, step S25 (seeding the first cells 133) may be performed at any timing as long as it is performed before step S26 (seeding the second cells 121). For example, step S25 may be performed before step S24 and after or before step S22. Hereinafter, the timing of performing step S25 includes not only the timing shown in FIG. 8 but also "any timing."

[0110] Furthermore, step S26 (seeding the second cells 121) may be performed at any timing as long as it is performed after step S25 (seeding the first cells 133). For example, step S26 may be performed before step S24 and after or before step S22. Hereinafter, the timing of performing step S26 includes not only the timing shown in FIG. 8 but also "any timing."

[0111] Furthermore, step S27 (introduction of culture medium 319 into insert body 103) may be performed at any timing as long as seeding of cells (first cell 133, second cell 121, and third cell 123) into closure portion 113 is complete. For example, step S27 may be performed before step S24. Hereinafter, the timing of performing step S27 includes not only the timing shown in FIG. 8 but also "any timing."

[0112] Furthermore, the timing of execution of step S23 (introduction of the culture medium 317 into the container body 203) is not particularly limited. For example, step S23 may be executed at any timing before step S22, or may be executed in parallel with step S21 or step S22. For example, step S23 may be executed at any timing after step S24 (insertion of the insert body 103 into the container body 203).

[0113] Continuing with reference to FIG. 8, a modified seeding method will be further described.

[0114] Any one of step S22 (seeding of the third cells 123), step S25 (seeding of the first cells 133), and step S26 (seeding of the second cells 121) may not be performed. In this case, step S23 (introduction of the culture medium 317 into the container body 203) and step S27 (introduction of the culture medium 319 into the insert body 103) are the same as in the above-described modified example.

[0115] For example, when step S22 is not performed, the co-culture shown in FIG. 4(b) is performed. According to this example, co-culture of the second cell 121 and the first cell 133 can be performed. In this seeding method, the execution timing of steps S25 and S26 is not limited to the timing shown in FIG. 8. Specifically, step S25 may be performed at "any timing" as long as it is performed before step S26. Furthermore, step S26 may be performed at "any timing" as long as it is performed after step S25. These points are the same as in the above-described modified example. Furthermore, as shown in FIG. 4(b), in this example, co-culture of the second cell 121 and the first cell 133 can be performed.

[0116] For example, when step S25 is not performed, the co-culture shown in FIG. 4( a) is performed. According to this example, co-culture of the second cell 121 and the third cell 123 can be performed. In this seeding method, the timing of performing step S26 is not limited to the timing shown in FIG. 8. That is, in this case, for example, step S26 may be performed before step S24 and after or before step S22. This is the same as in the above-described modified example. Furthermore, the timing of performing step S22 is also the same as in the above-described modified example.

[0117] For example, if step S26 is not performed, the co-culture shown in FIG. 4(c) is performed. According to this example, co-culture of the first cell 133 and the third cell 123 can be performed. In this seeding method, the timing of performing step S25 is not limited to the timing shown in FIG. 8. That is, in this case, for example, step S25 may be performed before step S24 and after or before step S22. This is the same as in the above-described modified example. Furthermore, the timing of performing step S22 is also the same as in the above-described modified example.

[0118] Next, the insert member 100 will be described with reference to FIG. 9 . The insert member 100 may be distributed with cells seeded in the closing portion 113. In this case, the insert member 100 is cooled to a temperature T1 that is lower than room temperature. In other words, the temperature of the closing portion 113 is a temperature T1 (hereinafter referred to as the "predetermined temperature T1") that is lower than room temperature. In further terms, the temperature of the closing portion 113 is maintained at the predetermined temperature T1. The predetermined temperature T1 is a temperature at which cells will not die. Therefore, even if the temperature of the closing portion 113 rises from the predetermined temperature T1 to the temperature during co-culture, the cells seeded in the closing portion 113 will survive.

[0119] 9(a) to 9(d) are cross-sectional views showing first to fourth examples of the insert member 100 in which different types of cells have been seeded. In Fig. 9(a) to 9(d), the temperature of the closure portion 113 of the insert member 100 is a predetermined temperature T1.

[0120] 9( a), the occluding portion 113 of the insert body 103 includes a second cell 121 and a third cell 123. Specifically, a second region 129 of the occluding portion 113 includes the second cell 121. A third region 131 of the occluding portion 113 includes the third cell 123.

[0121] 9(b), the occluding portion 113 of the insert body 103 includes a first cell 133 and a second cell 121. Specifically, a first region 135 of the occluding portion 113 includes the first cell 133. A second region 129 includes the second cell 121.

[0122] 9( c), the occluding portion 113 of the insert body 103 includes a first cell 133 and a third cell 123. Specifically, a first region 135 of the occluding portion 113 includes the first cell 133. A third region 131 includes the third cell 123.

[0123] 9( d ), the occluding portion 113 of the insert body 103 includes a first cell 133, a second cell 121, and a third cell 123. Specifically, a first region 135 of the occluding portion 113 includes the first cell 133. A second region 129 includes the second cell 121. A third region 131 includes the third cell 123.

[0124] 9(a) to 9(d), in the insert member 100 according to this embodiment, the closure portion 113 contains cells (two or more types of cells selected from the first cell 133, the second cell 121, and the third cell 123). In this way, by preseeding the cells in the closure portion 113, the purchaser of the insert member 100 can reduce the need for cell seeding.

[0125] Furthermore, the temperature of the closing portion 113 is a temperature T1 that is lower than room temperature. Therefore, the insert member 100 can be distributed while keeping the cells alive. For example, the insert member 100 is distributed while being stored in a refrigerator. Furthermore, for example, the insert member 100 may be distributed while being stored in a freezer. In this case, the closing portion 113 and the cells contained in the closing portion 113 (two or more types of cells among the first cell 133, the second cell 121, and the third cell 123) are maintained in a frozen state.

[0126] (First Modification) A first modification of this embodiment will be described with reference to Figure 10. The first modification differs from the embodiment described above with reference to Figures 1 to 9 mainly in that the inside and outside of the cylindrical portion 107 of the insert body 103 are connected to each other. Below, the differences between the first modification and the embodiment will be mainly described.

[0127] Fig. 10(a) is a cross-sectional view showing an insert member 100 according to a first modified example of this embodiment. Fig. 10(b) is a cross-sectional view showing a state in which co-culture is performed using the culture vessel 1 according to the first modified example.

[0128] As shown in Fig. 10(a) , the cylindrical portion 107 of the insert body 103 has at least one communication portion 141. In the example of Fig. 10(a) , the cylindrical portion 107 has a plurality of communication portions 141. The communication portions 141 communicate between the inside and outside of the cylindrical portion 107. Specifically, the communication portions 141 are holes that penetrate the cylindrical portion 107. The number and shape of the communication portions 141 are not particularly limited. In the example of Fig. 10(a) , the heights of the plurality of communication portions 141 relative to the bottom portion 105 are approximately equal.

[0129] According to the first modified example, the provision of the communication portion 141 allows communication between the interior and exterior of the tubular portion 107. Therefore, as shown in FIG. 10( b), when the insert body 103 is inserted into the container body 203, the interior of the tubular portion 107 of the insert body 103 is connected to the interior of the container body 203. Additionally, the culture medium 319 inside the tubular portion 107 and the culture medium 317 inside the container body 203 are connected via the communication portion 141. Therefore, material transfer and cell-cell interaction are possible between the interior of the tubular portion 107 of the insert body 103 and the interior of the container body 203 via the communication portion 141 and the culture media 317, 319. As a result, the bioreproducibility of the cell model obtained by co-culture can be further improved.

[0130] (Second Modification) A second modification of this embodiment will be described with reference to Figure 11. The second modification is different from the embodiment described above with reference to Figures 1 to 9 mainly in that the insert body 103 includes a support portion 143 that supports the closing portion 113. Below, the differences between the second modification and the embodiment described above will be mainly described.

[0131] Fig. 11(a) is a cross-sectional view showing an insert member 100 according to a second modified example of the present embodiment. Fig. 11(b) is a plan view showing the insert member 100 according to the second modified example with the blocking portion 113 removed.

[0132] 11( a), the insert body 103 includes a support portion 143. The support portion 143 supports the closing portion 113. Therefore, according to the second modification, the closing portion 113 can be prevented from falling off from the tubular portion 107 and from being deformed by gravity. In particular, when the closing portion 113 is made of gel, the support portion 143 is effective in preventing the closing portion 113 from falling off and being deformed.

[0133] Specifically, the support portion 143 protrudes from a lower end portion, including a lower end surface 117, of the tubular portion 107 toward the inside of the tubular portion 107. The support portion 143 supports the lower end of the blocking portion 113. The second opening 115 is defined by the support portion 143. In the example of FIG. 11( a), the support portion 143 forms part of the bottom portion 105 of the tubular portion 107. Furthermore, as shown in FIG. 11( b), the support portion 143 has a substantially ring shape. For example, the support portion 143 has a substantially circular ring shape.

[0134] (Third Modification) A third modification of this embodiment will be described with reference to Fig. 12. The third modification is mainly different from the second modification described with reference to Fig. 11 in that the support portion 143 of the insert body 103 is positioned higher than the lower end surface 117 of the tubular portion 107. Below, the differences between the third modification and the second modification will be mainly described.

[0135] Fig. 12(a) is a cross-sectional view showing an insert member 100 according to a third modified example of the present embodiment. Fig. 12(b) is a plan view showing the insert member 100 according to the third modified example with the blocking portion 113 removed.

[0136] 12( a), the insert body 103 includes a support portion 143. The support portion 143 supports the closing portion 113. Therefore, according to the third modification, similar to the second modification, it is possible to prevent the closing portion 113 from falling off and also to prevent deformation of the closing portion 113.

[0137] Specifically, the support portion 143 is located above the lower end surface 117 of the tubular portion 107 and protrudes from a position on the lower end side of the tubular portion 107 toward the inside of the tubular portion 107. In other words, the support portion 143 supports the closing portion 113 from inside the closing portion 113. Therefore, in the third modified example, the area of ​​the third region 131 of the closing portion 113 can be made larger than that of the closing portion 113 of the insert body 103 shown in FIG. 11 . Furthermore, as shown in FIG. 12( b ), the support portion 143 has a generally ring shape. For example, the support portion 143 has a generally circular ring shape.

[0138] (Fourth Modification) A fourth modification of this embodiment will be described with reference to Fig. 13. The fourth modification is mainly different from the second modification described with reference to Fig. 11 in that the support portion 145 of the insert body 103 has a mesh-like shape. Below, the differences between the fourth modification and the second modification will be mainly described.

[0139] Fig. 13(a) is a cross-sectional view showing an insert member 100 according to a fourth modified example of the present embodiment. Fig. 13(b) is a plan view showing the insert member 100 according to the fourth modified example with the blocking portion 113 removed.

[0140] 13A, the insert body 103 includes a support portion 145. The support portion 145 supports the closing portion 113. Therefore, according to the fourth modification, similar to the second modification, it is possible to prevent the closing portion 113 from falling off and also to prevent deformation of the closing portion 113.

[0141] Specifically, the support portion 145 is positioned above the lower end surface 117 of the tubular portion 107, at a position toward the lower end of the tubular portion 107. The support portion 145 is also positioned inside the blocking portion 113. The support portion 145 supports the blocking portion 113 from within the blocking portion 113. Therefore, in the fourth modified example, the area of ​​the third region 131 can be increased compared to the blocking portion 113 of the insert body 103 shown in FIG. 11 . Furthermore, when the second cells 121 seeded in the second region 129 and the third cells 123 seeded in the third region 131 are co-cultured (e.g., FIG. 4( a)), the influence of the support portion 145 on the co-culture can be reduced. Furthermore, the first cells 133 (e.g., FIG. 4( d)) can also be seeded in the first region 135.

[0142] 13(b), the support portion 145 has a mesh-like structure. Therefore, material transfer and cell-cell interaction are possible inside the blocking portion 113. As a result, when different types of cells are seeded in the blocking portion 113 and co-cultured, the bioreproducibility of the cell model can be improved. In particular, in the example of FIG. 13(b), the support portion 145 has a substantially circular mesh-like structure.

[0143] The support portion 145 may be attached to the lower end surface 117 of the cylindrical portion 107 .

[0144] (Fifth Modification) A fifth modification of this embodiment will be described with reference to Figure 14. The fifth modification differs from the embodiment described above with reference to Figures 1 to 9 mainly in that the insert body 103 has a tapered shape. Below, the differences between the fifth modification and the embodiment will be mainly described.

[0145] Fig. 14(a) is a perspective view showing an insert member 100 according to a fifth modified example of this embodiment, and Fig. 14(b) is a cross-sectional view taken along line XIVB-XIVB in Fig. 14(a).

[0146] 14(a) and 14(b), the insert body 103 has a tapered shape. Specifically, the cylindrical portion 1070 of the insert body 103 has a tapered shape. In other words, the cylindrical portion 1070 has a hollow inverted truncated cone shape.

[0147] Furthermore, the blocking portion 113 has an inverted truncated cone shape. Therefore, the sizes of the regions into which different types of cells are seeded can be varied along the first direction D1. For example, in a plan view, the area of ​​the second region 129 is the largest, and the area of ​​the third region 131 is the smallest. In addition, in a plan view, the area of ​​the first region 135 is smaller than the area of ​​the second region 129 and larger than the area of ​​the third region 131. Note that a plan view refers to viewing the object from the vertical direction.

[0148] The container body 203 (FIG. 1) may have a tapered shape.

[0149] (Sixth Modification) A sixth modification of this embodiment will be described with reference to Figure 15. The sixth modification differs from the embodiment described above with reference to Figures 1 to 9 mainly in that the cylindrical portion 1071 of the insert body 103 and the cylindrical portion 2071 of the container body 203 each have a substantially rectangular cylindrical shape. Below, the differences between the sixth modification and the embodiment will be mainly described.

[0150] Fig. 15 is a perspective view showing a culture vessel 1 according to a sixth modified example of this embodiment. As shown in Fig. 15, the tubular portion 1071 of the insert body 103 has a generally angular tubular shape. In the example of Fig. 15, the tubular portion 1071 has a generally square tubular shape. Therefore, the closure portion 113 has a generally square, flat plate shape. Furthermore, the tubular portion 2071 of the vessel body 203 has a generally square tubular shape with a bottom. In the example of Fig. 15, the tubular portion 2071 has a generally square tubular shape with a bottom.

[0151] The embodiments of the present invention have been described above with reference to the drawings. However, the present invention is not limited to the above embodiments and can be implemented in various forms without departing from the spirit of the present invention. Furthermore, the components disclosed in the above embodiments can be modified as appropriate. For example, some of the components shown in one embodiment may be added to the components of another embodiment, or some of the components shown in one embodiment may be deleted from the embodiment.

[0152] Furthermore, the drawings mainly show each component in a schematic manner to facilitate understanding of the invention, and the thickness, length, number, spacing, etc. of each component shown in the drawings may differ from the actual ones due to the convenience of creating the drawings. Furthermore, the configuration of each component shown in the above embodiment is merely an example and is not particularly limited, and it goes without saying that various modifications are possible within a range that does not substantially deviate from the effects of the present invention.

[0153] (1) In the embodiment and modified examples described with reference to FIGS. 1 to 15 , the insert member 100 may be formed from a single insert body 103. In this case, the insert member 100 may not have an insert base portion 101. A single container 200 may be formed from a single container body 203. In this case, the container 200 may not have a container base portion 201.

[0154] (2) As shown in Figures 4(d), 8, 9(d), and 10(b), the occluding portion 113 has one first region 135. However, the occluding portion 113 may have multiple first regions 135 arranged in the first direction D1 between the second region 129 and the third region 131. In this case, different cells are seeded in each of the multiple first regions 135. Therefore, by increasing the number of first regions 135, a more complex cell model can be created.

[0155] In particular, the thickness of the blocking portion 113 is not limited and can be set to any value depending on the number of regions in which different cells are to be seeded. The thickness indicates the length of the blocking portion 113 in the first direction D1.

[0156] INDUSTRIAL APPLICABILITY The present invention relates to an insert member, a culture vessel, a component of an insert member, a method for manufacturing an insert member, and a seeding method, and has industrial applicability.

Claims

1. An insert member comprising an insert body to be inserted into a container for co-culturing different types of cells, the insert body comprising: a cylindrical tube portion having an opening at the bottom; and a blocking portion that blocks the opening and into which the different types of cells can be seeded, the blocking portion having a higher biocompatibility than the biocompatibility of a porous resin, and the biocompatibility indicating the degree of reproducibility of a biological environment.

2. The insert member according to claim 1, wherein the blocking portion is made of gel.

3. An insert member according to claim 1 or claim 2, wherein the closure portion contains a biological substance.

4. An insert member according to claim 1 or claim 2, wherein the occlusion portion contains cells.

5. The insert member according to claim 4, wherein the temperature of the closing portion is lower than room temperature.

6. An insert member according to claim 1 or claim 2, wherein the insert body further comprises a support portion that supports the closing portion.

7. An insert member according to claim 1 or claim 2, wherein the cylindrical portion has a communication portion that connects the inside and outside of the cylindrical portion.

8. A culture vessel comprising: an insert member according to claim 1 or 2; and a vessel into which the insert member is inserted.

9. A component of an insert member having an insert body to be inserted into a container for co-culturing different types of cells, the component comprising: a cylindrical tube portion having an opening at the bottom; and a cover member that covers the opening and can be peeled off from the tube portion.

10. A method for manufacturing an insert member, the method comprising the steps of: preparing an insert member component having an insert body to be inserted into a container for co-culturing different types of cells, the component comprising a cylindrical tube portion having an opening at the bottom and a cover member covering the opening; placing a substance in a state before it changes to a gel on the upper surface of the cover member; changing the state of the substance to a gel to form a blocking portion that covers the opening; and peeling the cover member off the tube portion, wherein different types of cells can be seeded in the blocking portion, the biocompatibility of the blocking portion is higher than the biocompatibility of a porous resin, and the biocompatibility indicates the degree of reproducibility of the biological environment.

11. A seeding method comprising the steps of: preparing an insert member having an insert body to be inserted into a container for co-culturing different types of cells, the insert body having a cylindrical tubular portion with an opening at the bottom and a blocking portion that closes the opening; and seeding the cells into the blocking portion, wherein the biocompatibility of the blocking portion is higher than the biocompatibility of a porous resin, and the biocompatibility indicates the degree of reproducibility of the biological environment.

12. The seeding method of claim 11, wherein the cells include first cells, the seeding step includes seeding the first cells in a first region between a second region and a third region of the occluding portion, the second region indicating a region including an upper surface of the occluding portion, and the third region indicating a region including a lower surface of the occluding portion.

13. A seeding method according to claim 11 or claim 12, wherein the cells include second cells, and the seeding step includes a step of seeding the second cells in a second region including the upper surface of the occluding portion.

14. A seeding method according to claim 11 or claim 12, wherein the cells include third cells, and the seeding step includes a step of seeding the third cells in a third region including the underside of the closure portion prior to the step of inserting the insert body into the container body of the container.

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