Cell culture device

Abstract

To provide a cell culture device that allows for easy determination of influence of VOCs on cells, and simulation of the concentration gradient of VOCs in a living body.SOLUTION: Disclosed is a cell culture device capable of culturing cells with exposure to a chemical substance, the device comprising a flow channel-forming part, a first culture medium layer formed above the flow channel-forming part, a second culture medium layer formed above the first culture medium layer, a vessel to contain the flow channel-forming part and the first and second culture medium layers, where cells are cultured in the first culture medium layer where the cells form organoids, the flow channel-forming part comprises a substrate and a gel layer formed with a hydrogel as a forming material on the upper surface of the substrate, where in the interface between the substrate and the gel layer, there are an attaching area where the substrate and the gel layer are attached and a non-attaching area where the substrate and the gel layer are not attached, and in the non-attaching area, the gel layer is separated from the substrate to form a flow channel enclosed by the gel layer and the substrate.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a cell culture device.

Background Art

[0002] Volatile Organic Compounds (VOCs) are known to be associated with various diseases such as reduced lung function, dementia, and cancer from an epidemiological perspective. For example, it has been reported that acetaldehyde, one of the VOCs, is strongly associated with carcinogenesis such as esophageal cancer. Therefore, biological verification of the effects of VOCs on the human body is underway.

[0003] However, until now, a basic experimental system for verifying the effects of VOCs on cells has not been constructed, and detailed verification has not been possible.

[0004] For example, when attempting to verify the effects by exposing cells cultured on a plastic plate to VOCs, it is difficult to control the exposure amount of VOCs to the cells due to the volatility of VOCs. Therefore, a device has been proposed that exposes cells to a humid atmosphere containing formaldehyde at a specified concentration to confirm the effects on the cells (see, for example, Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, the device described in Non-Patent Document 1 is large-scale and not easily experimentable. Also, VOCs spread into the body through the blood and are exposed to the living body from blood vessels. Therefore, a concentration gradient of VOCs in the living body occurs according to the distance from blood vessels. However, known methods cannot form such a concentration gradient of VOCs.

[0007] Furthermore, in recent years, it has been desired to verify the effects of VOCs on organoids. In this specification, "organoid" means a three-dimensional cell tissue body self-organized by densely accumulating cells in a controlled space.

[0008] Normally, organoids are formed by self-organization through three-dimensional culture from tissue cells or stem cells (ES cells, iPS cells) and are buried in a culture medium. Therefore, even if the atmosphere in which the organoids are cultured is filled with VOCs with controlled concentration using, for example, the device described in Non-Patent Document 1, it is difficult to expose the organoids to VOCs.

[0009] The present invention has been made in view of such circumstances, and an object thereof is to provide a cell culture device capable of easily confirming the effects of VOCs on cells and simulating the concentration gradient of VOCs in the living body.

Means for Solving the Problems

[0010] In order to solve the above problems, one aspect of the present invention is a cell culture device capable of culturing cells while exposing them to a chemical substance, comprising a flow path forming part, a first culture medium layer laminated above the flow path forming part, a second culture medium layer laminated above the first culture medium layer, and a container for accommodating the flow path forming part, the first culture medium layer, and the second culture medium layer. Cells are cultured inside the first culture medium layer, and the cells form organoids. The flow path forming part has a base material and a gel layer provided on the upper surface of the base material with a hydrogel as a forming material. At the interface between the base material and the gel layer, an adhesion region where the base material and the gel layer adhere to each other and a non - adhesion region where the base material and the gel layer do not adhere to each other are formed. In the non - adhesion region, the gel layer is separated from the base material, and a flow path surrounded by the gel layer and the base material is formed. A cell culture device is provided.

Advantages of the Invention

[0011] According to the present invention, it is possible to easily confirm the influence of VOCs on cells, and to provide a cell culture device capable of mimicking the concentration gradient of VOCs in vivo. In such a cell culture device, the influence of the concentration gradient of VOCs can be confirmed.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] [First Embodiment] Hereinafter, the cell culture device according to the first embodiment will be described with reference to FIGS. 1 to 8. In all the following drawings, for ease of viewing, the dimensions and ratios of each component are appropriately different.

[0014] In the following description, an xyz orthogonal coordinate system is set, and the positional relationship of each member will be described with reference to this xyz orthogonal coordinate system. Here, a predetermined direction in the horizontal plane is defined as the x-axis direction, a direction orthogonal to the x-axis direction in the horizontal plane is defined as the y-axis direction, and a direction orthogonal to each of the x-axis direction and the y-axis direction (i.e., the vertical direction) is defined as the z-axis direction.

[0015] Also, “up” is defined as the +z direction which is above in the vertical direction, and “down” is defined as the -z direction which is below in the vertical direction.

[0016] In the following description, “plan view” means looking at the object downward (-z direction) from above in the vertical direction (+z side).

[0017] 《Cell Culture Device》 FIG. 1 is an exploded perspective view of the cell culture device 1 of the present embodiment. FIG. 2 is a cross-sectional view taken along the line II-II of FIG. 1. As shown in FIGS. 1 and 2, the cell culture device 1 includes a flow path forming portion 10, a first culture medium layer 20, a second culture medium layer 30, and a container 50. The cell culture device 1 has a plurality (three in the figure) of flow paths 10x partially formed of hydrogel.

[0018] In the cell culture device 1, the flow path forming portion 10 is disposed at the bottom of the container 50, and the first culture medium layer 20 and the second culture medium layer 30 are laminated in order above the flow path forming portion 10. An organoid O is disposed inside the first culture medium layer 20.

[0019] The cell culture device 1 is an instrument used to confirm the influence of VOCs on the organoid O. Hereinafter, each component will be described in order.

[0020] [Flow path forming portion] FIG. 3 is a schematic perspective view of the flow path forming portion 10. FIG. 4 is a cross-sectional view taken along the line IV-IV of FIG. 3. The flow path forming portion 10 includes a base material 11 and a gel layer 15.

[0021] (Base material) The base material 11 supports the gel layer 15. The rigidity modulus of the base material 11 is different from that of the gel layer 15. For example, the rigidity modulus of the base material 11 is higher than that of the gel layer 15.

[0022] As the material for forming the base material 11, various materials can be selected regardless of whether they are organic materials or inorganic materials as long as the effects of the invention are not impaired. The base material 11 may or may not have light transmissibility.

[0023] Examples of the organic material that forms the base material 11 include polymer materials and elastomers. Examples of the polymer materials include thermoplastic resins such as polyvinyl chloride, polystyrene, ABS resin, and polylactic acid, and thermosetting resins such as polyimide and phenolic resin. Examples of the elastomers include polysilicon and synthetic rubber.

[0024] The base material 11 may be subjected to various processes on at least one of the surface and the inside by known microfabrication techniques. For example, the base material 11 may have irregularities or grooves on the surface.

[0025] (Gel layer) The gel layer 15 is formed of a hydrogel and provided on the upper surface 11a of the base material 11. In the flow path forming portion 10, the gel layer 15 is formed in a rectangular shape having the same width as the base material 11 in the y-axis direction and a shorter dimension than the base material 11 in the x-axis direction. The planar shape of the gel layer 15 is not particularly limited, and various shapes according to the usage form can be selected.

[0026] Examples of the polymer material constituting the hydrogel include water-soluble polymers such as polyacrylamide and polyvinyl alcohol, polysaccharides such as chitosan and alginic acid, and proteins such as collagen and albumin. These materials have a three-dimensional network structure and swell with a solvent contained in most of the volume. The representative of the solvent in which the polymer material constituting the hydrogel swells is water.

[0027] In addition, a stimulus-responsive gel capable of adjusting the degree of swelling in response to an external stimulus may be used as the forming material of the gel layer 15.

[0028] For example, as gels that respond to heat (heat-responsive gels), gels made of poly(N-isopropylacrylamide) or poly(methyl vinyl ether) can be mentioned. A photothermal conversion material (such as a metal nanomaterial, a carbon nanomaterial, or a conductive polymer) may be dispersed in the heat-responsive gel, and the stimulus response may be triggered by heat generation due to light irradiation.

[0029] Examples of gels that respond to pH (pH-responsive gels) include gels composed of polyelectrolytes synthesized from anionic or cationic monomers.

[0030] Examples of gels that respond to light (light-responsive gels) include gels composed of polymers having a spiropyran or azobenzene backbone. For the light-responsive gel, a mechanism in which the degree of swelling changes by light stimulation may be introduced using an inclusion complex of azobenzene and cyclodextrin as a crosslinking point.

[0031] In addition, examples of the material for forming the gel layer 15 include a molecularly imprinted gel in which a specific molecule bound to a hydrogel backbone is used as a crosslinking point of the gel network. For example, examples of protein-responsive gels include a biomolecule-crosslinked gel in which a biomolecule complex is used as a crosslinking point of the gel network, and an antigen-responsive gel in which an antigen-antibody complex is introduced into the network as a crosslinking point of the gel.

[0032] Also, the material for forming the gel layer 15 may be a hydrogel that responds to multiple stimuli by mixing a plurality of these polymer materials. Further, as the material for forming the gel layer 15, tough hydrogels such as double network gels, slide ring gels, Tetra-PEG gels, and nanoclay gels can also be used.

[0033] Regarding the synthesis method of the polymer material constituting the hydrogel, various known methods can be adopted. For example, when the polymer material constituting the hydrogel is an acrylic polymer material, an acrylic group may be crosslinked during the polymerization of an acrylic monomer to form a three-dimensional network structure.

[0034] The type of polymerization reaction when polymerizing the acrylic monomer is not particularly limited, and examples include radical polymerization using a water-soluble photoinitiator. Examples of the water-soluble photoinitiator include 2-oxoglutaric acid, 4'-(2-hydroxyethoxy)-2-hydroxy-2-methylpropiophenone (trade name: Irgacure 2959), lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (abbreviation: LAP), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] (trade name: VA-086), and the like.

[0035] During radical polymerization, a deoxidizer may be added to the reaction system to prevent polymerization inhibition by oxygen. Examples of the deoxidizer include a combination of glucose and glucose oxidase. Further, radical polymerization may be carried out under an inert gas atmosphere such as nitrogen or argon.

[0036] When the polymer material constituting the hydrogel is a polysaccharide or a protein, a three-dimensional network structure may be formed by physical bonding of the polysaccharide or the protein, or the polysaccharide or the protein may be crosslinked using a crosslinking agent to form a three-dimensional network structure. Examples of the crosslinking agent include glutaraldehyde.

[0037] The thickness of the gel layer 15 is not particularly limited, but it is preferably a thickness that exhibits a structural strength such that it is not crushed by its own weight. For example, when a hydrogel containing polyacrylamide is used as the forming material of the gel layer 15, the thickness of the gel layer 15 is preferably 50 μm to 1000 μm, and more preferably 120 μm to 200 μm.

[0038] The strength of the gel layer 15 can be improved by increasing the crosslinking of the polymer material constituting the hydrogel by chemical crosslinking or physical crosslinking, or by increasing the concentration of the polymer material constituting the hydrogel.

[0039] For example, when preparing a hydrogel containing polyacrylamide by polymerizing a monomer (precursor) of acrylamide, the monomer concentration is preferably 0.8 mol / L to 8 mol / L, more preferably 2 mol / L to 4 mol / L.

[0040] Also, when using methylenebisacrylamide as a chemical crosslinking agent in the polymerization of acrylamide monomers, the crosslinking agent concentration is preferably 0.01 mol% to 2.0 mol% with respect to the monomer, more preferably 0.03 mol% to 1 mol%.

[0041] The hydrogel can contain various additives. The type of additive is not particularly limited as long as it does not inhibit hydrogel formation. Examples of additives include biomolecules that improve biocompatibility, silver nanoparticles for expressing antibacterial properties, and surfactants. By adding these additives to the hydrogel, arbitrary functions can be imparted to the hydrogel.

[0042] (Interface structure) In the flow path forming portion 10, an adhesion region 10a where the base material 11 and the gel layer 15 are adhered and a non - adhesion region 10b where the base material 11 and the gel layer 15 are not adhered are formed at the interface between the base material 11 and the gel layer 15.

[0043] The non - adhesion regions 10b are formed in three strip - like portions extending in the x - axis direction. The shape of the non - adhesion region 10b is an example, and various shapes according to the design can be adopted.

[0044] The portion of the gel layer 15 overlapping with the non - adhesion region 10b is lifted upward away from the base material 11, forming a flow path 10x. The width of the flow path 10x can be controlled by adjusting the width of the non - adhesion region 10b. The height of the flow path 10x can be controlled by adjusting the thickness of the gel layer 15 and the width of the non - adhesion region 10b.

[0045] [First culture medium layer] The first culture medium layer 20 is in contact with the flow path forming portion 10 and is laminated above the flow path forming portion 10.

[0046] The first culture layer 20 is an extracellular matrix (ECM) used as a scaffold for cell growth. As the material of the first culture layer 20, a known three-dimensional culture medium capable of culturing organoids can be adopted. For example, it is composed of water, various polysaccharides, proteins, glycoproteins, etc. The first culture layer 20 can be adjusted using known extracellular matrix-producing cells. Also, as the material of the first culture layer 20, commercially available extracellular matrices such as Matrigel (registered trademark, Corning) can be used.

[0047] [Second culture layer] The second culture layer 30 is in contact with the first culture layer 20 and is laminated above the first culture layer 20. The second culture layer 30 is a solid medium used for cell culture, and a known medium can be used.

[0048] As the second culture layer 30, for example, in addition to general cell media (DMEM, RPMI), the medium KSFM for esophageal squamous epithelial cells can be mentioned.

[0049] [Organoid] Inside the first culture layer 20, an organoid O is cultured. The organoid O may be cultured in the first culture layer 20, or may be separately cultured and then placed inside the first culture layer 20.

[0050] The cell culture device 1 is an instrument used to confirm the influence of VOCs on the organoid O. As the organoid O, organoids of various cells for which the influence of VOCs is to be confirmed can be adopted.

[0051] Specifically, as the cells constituting the organoid O, epithelial cells, hepatocytes, mucosal cells, nerve cells, etc. can be used.

[0052] In addition, as cells other than organoids, stromal cells such as fibroblasts may be included.

[0053] [Other configurations] The cell culture device 1 has pipes 41 and 42 connected to the flow path 10x. The cell culture device 1 can introduce various fluids into the flow path 10x via the pipes 41 and 42. The pipes 41 and 42 are respectively fixed with an adhesive 49 at the openings on both sides of the flow path 10x.

[0054] The pipes 41 and 42 are bent pipes that extend in the x-axis direction and bend to extend in the z-axis direction. The configuration of the pipes 41 and 42 is an example, and various configurations can be adopted.

[0055] The adhesive 49 fixes the pipes 41 and 42 between the base material 11 and the first culture medium layer 20. The adhesive 49 covers the periphery of the pipes 41 and 42 on both sides of the flow path 10x. Further, a part of the adhesive 49 penetrates into the inside of the flow path 10x and is densely filled in the space between the pipes 41 and 42 and the flow path 10x.

[0056] The adhesive 49 preferably has water resistance and adhesiveness to the base material 11 and the first culture medium layer 20. Examples of the adhesive 49 include cyanoacrylate adhesives, silicone adhesives, epoxy adhesives, and the like.

[0057] [Container] The container 50 has a container body 51 and a lid member 52. The container body 51 is open at the top and houses a laminate of the flow path forming portion 10, the first culture medium layer 20, and the second culture medium layer 30 in the internal space S.

[0058] The lid member 52 closes the opening 51x of the container body 51 from above the container body 51. A through hole through which the pipes 41 and 42 can be inserted may be formed in the lid member 52.

[0059] The container 50 preferably has light transparency. The container 50 can be formed of a material known as a material for a cell culture container. For example, the material of the container 50 can include glass, polystyrene, and the like.

[0060] After forming the flow path forming portion 10, the cell culture device 1 can be formed by housing the flow path forming portion 10 in the container body 51, connecting the pipes 41 and 42 as appropriate, and then laminating the first culture medium layer 20 and the second culture medium layer 30 in the container.

[0061] FIG. 5 is a schematic perspective view of the flow path forming portion 10A before forming the flow path 10x, and FIG. 6 is a cross-sectional view taken along the line VI-VI of FIG. 5. FIG. 5 corresponds to FIG. 3, and FIG. 6 corresponds to FIG. 3. The flow path forming portion 10 can be obtained by forming the gel layer 12 shown in FIGS. 5 and 6 and then swelling the gel layer 12.

[0062] The adhesive region 10a and the non-adhesive region 10b of the flow path forming portion 10A can be manufactured by patterning and forming a hydrogel using a known photolithography technique.

[0063] Specifically, first, using a known photolithography technique, a layer 121 of a silane coupling agent having an adhesive functional group is formed on the substrate 11 in accordance with the shape of the adhesive region 10a. The "adhesive functional group" refers to a functional group that can be polymerized with the monomer (precursor) of the polymer material constituting the above-described hydrogel.

[0064] For example, when an acrylic monomer is used as the monomer, examples of the adhesive functional group include (meth)acrylic groups. In this case, for example, 3-(methacryloyloxy)propyltrimethoxysilane can be used as the silane coupling agent.

[0065] The formed surface treatment layer is a layer formed by the chemical (covalent) bonding of the silane coupling agent to the substrate surface. The adhesive functional group is introduced into the upper surface 11a of the substrate 11 by the surface treatment layer.

[0066] Next, the monomer of the polymer material constituting the hydrogel is polymerized on top of the formed layer 121 to form the gel layer 12 before swelling. At this time, the functional group of the monomer and the adhesive functional group of the silane coupling agent are polymerized, so that the monomer overlapping the layer 121 adheres to the upper surface 11a of the base material 11. On the other hand, in the region of the base material 11 where the layer 121 is not formed, the monomers polymerize with each other without bonding to the base material 11.

[0067] As described above, it is possible to form the gel layer 12 having the adhesive region 10a where the gel layer 12 overlaps the layer 121 and adheres to the base material 11 and the non-adhesive region 10b where the gel layer 12 is not adhered to the base material 11.

[0068] Next, the obtained gel layer 12 is swollen. At this time, in the gel layer 12, the portion that overlaps the adhesive region 10a in a planar manner is restricted from deforming during the swelling of the gel layer 12. On the other hand, in the gel layer 12, the portion that overlaps the non-adhesive region 10b in a planar manner can freely increase in volume in a direction away from the base material 11 when the volume increases due to swelling.

[0069] As a result, in the laminate 1B, the portion that overlaps the non-adhesive region 10b in a planar manner greatly swells and deforms in a direction away from the base material 11 in order to relieve the increase in internal pressure due to the volume increase. As a result, as shown in FIGS. 3 and 4, a flow path 10x surrounded by the gel layer 12 and the base material 11 is formed in the flow path forming portion 10.

[0070] The shape of the flow path 10x can be controlled by controlling the pattern shapes of the adhesive region 10a and the non-adhesive region 10b.

[0071] In addition, the shape of the flow path 10x can be controlled by adjusting the type of the gel layer 12, the ratio of the rigidity modulus of the base material 11 to the rigidity modulus of the gel layer 12, the thickness of the gel layer 12, etc. The rigidity modulus of the gel layer 12 and the swelling ratio of the gel layer 12 can be controlled by changing the type of the monomer of the polymer material constituting the gel layer 12, the type and amount of the crosslinking agent used, etc.

[0072] (Function and effect) Figures 7 and 8 are explanatory diagrams for explaining a method for confirming the influence of VOCs on the organoid O using the cell culture device 1.

[0073] The cell culture device 1 can simulate the structure of the skin and the structure of digestive organs such as the esophagus and intestine, for example, by regarding the flow path 10x as a circulatory system tubular tissue such as blood vessels or lymph, and regarding the first culture medium layer 20 as stromal tissue.

[0074] In such a cell culture device 1, a solution containing VOCs (VOCs solution) is caused to flow inside the flow path 10x. As a result, the VOCs solution diffuses into the side wall (gel layer 15) of the flow path 10x made of the hydrogel forming material and the first culture medium layer 20. Thereby, the VOCs contained in the VOCs solution diffuse into the first culture medium layer 20. In FIG. 7, the VOCs are indicated by reference numeral A.

[0075] Examples of the VOCs include formaldehyde, benzene, toluene, and trichloroethylene.

[0076] Formaldehyde is used in building materials such as processed wood products, and is also included in household goods such as glue and adhesives, and is associated with cancers of the nose and throat and lung cancer.

[0077] Benzene is contained in gasoline, tobacco smoke, industrial exhaust gas, etc., and is associated with blood cancer and lung cancer.

[0078] Toluene is contained in paints, thinners, and adhesives, affects growth and reproduction, and is associated with kidney and liver disorders.

[0079] Trichloroethylene is used as a solvent, is contained in some industrial and household products, and is associated with kidney cancer.

[0080] When more time elapses after the start of the liquid feeding of the VOCs solution, as shown in FIG. 7, the VOCs reach the organoid O disposed in the first culture layer 20 and affect the organoid O. For example, if the VOCs is acetaldehyde, it is considered that the organoid O will undergo changes such as changes in the activity of ALDH2 (acetaldehyde dehydrogenase 2) or death.

[0081] The amount of VOCs reaching the organoid O (the exposure amount of VOCs to the organoid O) is affected by the flow rate and flow volume of the VOCs solution and the concentration of VOCs in the VOCs solution. Also, the amount of VOCs reaching the organoid O varies depending on the configuration of the cell culture device 1, such as the material of the first culture layer 20, the depth position of the organoid O in the first culture layer 20, the material of the gel layer 15, and the thickness of the gel layer 15. Therefore, for the cell culture device 1 to be used, the exposure amount of VOCs to the organoid O can be grasped by experimentally confirming the amount of VOCs reaching the arrangement position of the organoid O, either beforehand or afterwards.

[0082] When confirming the influence of VOCs exposure on organoids using a conventional cell culture container, it is conceivable to culture the organoids with VOCs filled in the gas phase of the cell culture container. However, usually, the organoids are buried in the culture medium. Therefore, for example, even if the concentration-controlled VOCs are filled into the atmosphere in which the organoids are cultured using the device described in Non-Patent Document 1, it has been difficult to expose the organoids to VOCs. Also, it has been extremely difficult to control and grasp the exposure amount of VOCs to the organoids, and the influence by VOCs could not be evaluated.

[0083] On the other hand, when using the cell culture device 1 of the present embodiment, it is possible to expose the organoids buried in the culture medium to VOCs, and furthermore, it is possible to control the exposure amount. Therefore, it becomes possible to easily confirm the influence of VOCs on the organoids.

[0084] The cell culture device 1 shown in Fig. 7 has three flow paths 10x (10xa, 10xb, 10xc). For example, by flowing a liquid containing no VOCs through the flow path 10xa, a VOCs solution through the flow path 10xb, and a VOCs solution with a higher concentration than that in the flow path 10xb through the flow path 10xc, the amount of VOCs exposure to the organoids arranged above the flow path 10x can be varied.

[0085] Therefore, by comparing the organoids O (Oa, Ob, Oc) respectively adjacent to the three flow paths 10x, the influence of the amount of VOCs exposure on the organoid O can be easily confirmed.

[0086] Fig. 8 is a schematic plan view showing a cell culture device 2 according to a modified example. Fig. 8 omits the illustration of the second culture medium layer and shows the first culture medium layer 20 and the flow path 16x located below the first culture medium layer 20.

[0087] The flow path 16x of the cell culture device 2 has four main flow paths 161x extending in the x-axis direction while bending, and connection paths 162x connecting the main flow paths 161x to each other in the y-axis direction at the bending positions of the main flow paths 161x. Due to the connection paths 162x, the flow path 16x branches within the path and forms an annular path within the path.

[0088] In the example shown in Fig. 8, a plurality of annular paths that are hexagonal in plan view are formed. Adjacent annular paths share a common flow path at the positions of the sides of the overlapping hexagons and are arranged in a honeycomb shape.

[0089] The cell culture device can also have a configuration with a plurality of annular paths. The plurality of annular paths may be laid out in plan view by sharing a part of the flow path between adjacent annular paths. In such a cell culture device, the portions where no annular paths are formed are neatly laid out in a tile-like manner.

[0090] In the first culture layer 20, the organoid O includes a first organoid O1 and a second organoid O2 whose distance from the flow path 16x is different from that of the first organoid O1. Specifically, the first organoid O1 lies in a plane overlapping the flow path 16x, and the second organoid O2 is spaced apart from the flow path 16x in a plan view (separation distance L).

[0091] When a VOCs solution is flowed through the flow path 16x of such a cell culture device 2, due to the difference in the distance between the flow path 16x and the organoid O, for example, the difference between the first organoid O1 and the second organoid O2, a plurality of organoids O with different VOCs exposure states are formed. Therefore, by analyzing the cells for each organoid, accurate analysis can be performed without mixing cells with different VOCs exposure amounts, as compared to the case where cells are cultured on the entire surface of the interface between the first culture layer 20 and the second culture layer 30 and the same operation is performed.

[0092] According to the cell culture device configured as described above, it becomes possible to easily confirm the influence of VOCs on cells.

[0093] Note that although the cell culture device of the present embodiment has a plurality of flow paths, it is not limited thereto. Even a cell culture device having only one flow path can achieve the effects of the present invention.

[0094] [Second Embodiment] FIG. 9 is an explanatory diagram of a cell culture device 3 according to the second embodiment and is an exploded perspective view of a flow path forming portion included in the cell culture device 3. As shown in FIG. 9, the cell culture device 3 includes a flow path forming portion 60 and a base 70. In the following description, the illustration of the above-described first culture layer and second culture layer is omitted.

[0095] The flow path forming portion 60 has a flow path partially formed of hydrogel. The flow path forming portion 60 and the base 70 are adhered to each other.

[0096] [Flow Path Forming Portion] The flow path forming portion 60 includes a base material 61 and a gel layer 62.

[0097] (Base material) The base material 61 has a first through-hole 611 and a second through-hole 612 that penetrate in the thickness direction (z-axis direction) of the base material 61. In FIG. 9, the base material 61 is shown as having three first through-holes 611 and three second through-holes 612. The number of the first through-holes 611 and the second through-holes 612 is not limited to this as long as the number of the first through-holes 611 and the second through-holes 612 is the same, and they may be one each, or may be a plurality other than three.

[0098] In FIG. 9, the first through-holes 611 are arranged at equal intervals in the y direction on the +x side of the base material 61. The second through-holes 612 are arranged at equal intervals in the y direction on the -x side of the base material 61.

[0099] (Gel layer) The gel layer 62 is formed of a hydrogel and is provided on the upper surface 61a of the base material 61. The gel layer 62 is provided so as to overlap with a set of the first through-holes 611 and the second through-holes 612 arranged in the x-axis direction, and the upper surface 61a of the base material 61 is exposed around the gel layer 62. That is, in the flow path forming portion 60, the gel layer 62 has a strip shape extending in the x-axis direction and three gel layers 62 are formed.

[0100] (Interface structure) FIG. 10 is a cross-sectional view taken along the line α-α in FIG. 9 as viewed in the arrow direction. FIG. 11 is a plan view of the gel layer 62 and the vicinity of the gel layer 62.

[0101] As shown in FIG. 10, in the flow path forming portion 60, an adhesion region 60a where the base material 61 and the gel layer 62 adhere and a non-adhesion region 60b where the base material 61 and the gel layer 62 do not adhere are formed at the interface between the base material 61 and the gel layer 62.

[0102] Also, as shown in FIG. 11, the non-adhesion region 60b is formed in a strip shape extending in the x-axis direction and is surrounded in a closed ring shape by the adhesion region 60a in plan view. The shape of the non-adhesion region 60b is an example, and various shapes according to the design can be adopted.

[0103] Further, the first through hole 611 and the second through hole 612 each open into the non - adhesion region 60b.

[0104] Such an adhesion region 60a and a non - adhesion region 60b can be formed by forming a layer 621 of a silane coupling agent according to the shape of the adhesion region 60a, then overlapping it with the formed layer 621, and polymerizing the monomers of the polymer material constituting the hydrogel. Thereby, the monomers overlapping the layer 621 adhere to the upper surface 61a of the base material 61, and in the region where the layer 621 is not formed, the monomers polymerize with each other without bonding to the base material 61.

[0105] In the above - described manner, the gel layer 62 (flow - path forming portion 60) having the adhesion region 60a and the non - adhesion region 60b can be formed. The shape of the gel layer 62 can be controlled by arranging spacers that suppress the outflow of monomers around when arranging the monomers. In FIG. 10, the periphery of the gel layer 62 coincides with the periphery of the layer 621.

[0106] [Base] The base 70 supports the flow - path forming portion 60 on the support surface 70a. The base 70 has a base body 71 having a support surface 70a, a pipe connection portion 72 connected to the side surface of the base body 71 on the +x side of the base body 71, and a pipe connection portion 73 connected to the side surface of the base body 71 on the -x side of the base body 71.

[0107] The base body 71 has a substantially rectangular - parallelepiped shape. The planar shape of the base body 71 is the same as the planar shape of the flow - path forming portion 60 (base material 61), and when the flow - path forming portion 60 is overlapped, the contours of the two overlap.

[0108] The pipe connection portion 72 has a substantially rectangular - parallelepiped shape, and the +x - side corner has a rounded shape. Similarly, the pipe connection portion 73 has a substantially rectangular - parallelepiped shape, and the -x - side corner has a rounded shape. The upper surfaces 72a, 73a of both are located on the +z side with respect to the upper surface (support surface 70a) of the base body 71. That is, the upper surfaces 72a, 73a of the pipe connection portions 72, 73 are higher than the support surface 70a.

[0109] (First flow path, second flow path) The base 70 has three first flow paths 701, which are the same number as the non - adhesion regions 60b formed in the flow - path forming portion 60, and three second flow paths 702, which are also the same number as the non - adhesion regions 60b.

[0110] One end of the first flow path 701, which is the first inner end portion 701x, opens to the support surface 70a, and the other end of the first flow path 701, which is the first outer end portion 701y, opens to the upper surface 72a of the pipe connection portion 72. The first flow path 701 is provided so as to penetrate the inside of the base body 71 and the pipe connection portion 72, that is, the inside of the base 70.

[0111] The first inner end portion 701x opens in a region X that overlaps the non - adhesion region 60b in plan view. In plan view, the first inner end portion 701x and the first through - hole 611 of the flow - path forming portion 60 overlap and communicate with each other.

[0112] Similarly, one end of the second flow path 702, which is the second inner end portion 702x, opens to the support surface 70a, and the other end of the second flow path 702, which is the second outer end portion 702y, opens to the upper surface 73a of the pipe connection portion 73. The second flow path 702 is provided so as to penetrate the inside of the base body 71 and the pipe connection portion 73, that is, the inside of the base 70.

[0113] The second inner end portion 702x opens in a region X that overlaps the non - adhesion region 60b in plan view. In plan view, the second inner end portion 702x and the second through - hole 612 of the flow - path forming portion 60 overlap and communicate with each other.

[0114] The base 70 can be manufactured using a known 3D printing technology.

[0115] In FIGS. 12 to 14, the state in which the gel layer 62 shown in FIG. 9 is swollen is shown. Hereinafter, the swollen gel layer is denoted by reference numeral 65.

[0116] FIG. 12 is a perspective view of the flow path forming portion 60 and the base 70 having the swollen gel layer 65. FIG. 13 is a cross-sectional view taken along the line β-β of FIG. 12, and is a view corresponding to FIG. 10. FIG. 14 is a cross-sectional view taken along the line γ-γ of FIG. 12.

[0117] As shown in FIGS. 12 to 14, the gel layer 65 is not fixed to the base material 61 in the non-adhesive region 60b. Further, the gel layer 65 is fixed to the base material 61 in the adhesive region 60a. Therefore, in the gel layer 65, the portion that overlaps the non-adhesive region 60b in a planar manner can freely increase in volume in the extending direction of the non-adhesive region 60b or in the direction away from the base material 61 when the volume increases due to swelling. On the other hand, the portion that overlaps the non-adhesive region 60b in a planar manner is restricted from increasing in volume in a direction intersecting the extending direction of the non-adhesive region 60b.

[0118] As a result, in the gel layer 65, the portion that overlaps the non-adhesive region 60b in a planar manner greatly bulges and deforms in the direction away from the base material 61 in order to relieve the increase in internal pressure due to the volume increase. Thereby, a partial flow path 60x surrounded by the gel layer 65 and the base material 61 is formed in the flow path forming portion 60.

[0119] The partial flow path 60x (the space surrounded by the gel layer 62 and the base material in the non-adhesive region 60b) formed in this way, the first flow path 701, and the second flow path 702 communicate with each other to form the flow path 3x of the cell culture device 3.

[0120] As shown in FIG. 12, in the cell culture device 3, a pipe 43 can be connected to the first outer end portion 701y that opens to the pipe connection portion 72 via a connector 45, and a pipe 44 can be connected to the second outer end portion 702y that opens to the pipe connection portion 73 via a connector 47 and used.

[0121] Even in the cell culture device having the above-described configuration, according to the cell culture device, the influence of VOCs on cells can be easily confirmed.

[0122] The preferred embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to such examples. The various shapes, combinations, etc. of the constituent members shown in the above examples are merely examples, and various modifications can be made based on design, specifications, etc. without departing from the gist of the present invention.

Explanation of Reference Signs

[0123] 1, 2, 3... cell culture device, 10, 10A, 60... flow path forming part, 10a, 60a... adhesion region, 10b, 60b... non-adhesion region, 10x, 10xa, 10xb, 10xc, 16x... flow path, 11, 61... base material, 11a, 61a... upper surface, 12, 15, 62, 65... gel layer, 20... first culture medium layer, 30... second culture medium layer, 50... container, O... organoid

Claims

1. A cell culture device capable of culturing cells while exposing them to a chemical substance, comprising a flow path forming part, a first culture medium layer laminated above the flow path forming part, a second culture medium layer laminated above the first culture medium layer, and a container for accommodating the flow path forming part, the first culture medium layer, and the second culture medium layer, wherein the cells are cultured inside the first culture medium layer, the cells form organoids, the flow path forming part includes a base material, and a gel layer provided on the upper surface of the base material with hydrogel as a forming material, at the interface between the base material and the gel layer, there are an adhesion region where the base material and the gel layer adhere to each other, and a non - adhesion region where the base material and the gel layer do not adhere to each other, and in the non - adhesion region, the gel layer is separated from the base material, forming a flow path surrounded by the gel layer and the base material. A cell culture device.

2. The cell culture device according to claim 1, wherein the flow path branches within the path.

3. The cell culture device according to claim 2, wherein the flow path has an annular path within the path.

4. The flow path has a plurality of annular paths, and in the cell culture device according to claim 3, the plurality of annular paths share a part of the flow path with adjacent annular paths and are laid flat in a plan view.

5. The cell culture device according to any one of claims 1 to 4, having a plurality of the flow paths.

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