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 layer formed above the flow channel forming part, a second layer formed above the first layer, a vessel to contain the flow channel-forming part, the first and second layers, where the first layer comprises an extracellular matrix layer and a plurality of fibroblasts dispersed in the extracellular matrix layer, the second layer contains a plurality of epithelial cells, where the flow channel-forming part comprises a substrate and a gel layer formed on the upper surface of the substrate with a hydrogel as a forming material, where the interface between the substrate and the gel layer comprises an attaching area where the substrate and the gel layer are attached and a non-attaching area where the gel layer is separated from the substrate to form a flow channel enclosed by the gel layer and the substrate and the flow channel comprises a main channel and a loop structured part branching off from the main channel.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 decreased 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 it cannot be easily experimented. 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, with known methods, such a concentration gradient of VOCs cannot be formed.

[0007] The present invention has been made in view of such circumstances, and an object thereof is to provide a cell culture device that can easily confirm the influence of VOCs on cells and can simulate the concentration gradient of VOCs in the living body.

Means for Solving the Problems

[0008] Acetaldehyde, which is a kind of VOCs, is a metabolite during alcohol metabolism and is known as a causative substance of esophageal cancer. However, so far, no basic medical elucidation has been made on the behavior of acetaldehyde in the living body and its influence on living organs. In particular, the realization of a verifiable experimental system has been desired for the influence of VOCs dissolved in the blood after drinking on living organs.

[0009] Regarding the influence of acetaldehyde on cells, the relationship between drinking and esophageal cancer has been studied. It is known that during the process of esophageal epithelial carcinogenesis, abnormalities (morphological changes) are observed in the loop-shaped capillaries within the epithelial papilla. The inventors considered that such changes in the vascular structure are involved in the influence exerted on the epithelium by acetaldehyde in the blood, and completed the present invention for the realization of a verifiable experimental system.

[0010] 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 channel forming part, a first layer laminated above the flow channel forming part, a second layer laminated above the first layer, and a container accommodating the flow channel forming part, the first layer, and the second layer. The first layer includes an extracellular matrix layer and a plurality of fibroblasts dispersed inside the extracellular matrix layer. The second layer includes a plurality of epithelial cells. The flow channel 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 and a non - adhesion region where the base material and the gel layer do not adhere are formed. In the non - adhesion region, the gel layer is separated from the base material, forming a flow channel surrounded by the gel layer and the base material. The flow channel has a main flow channel and a loop structure part branching from the main flow channel, and provides a cell culture device.

Advantages of the Invention

[0011] According to the present invention, it is possible to easily confirm the influence of VOCs on cells and 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

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Mode for Carrying Out the Invention

[0013] [First Embodiment] Hereinafter, with reference to FIGS. 1 to 8, the cell culture device according to the first embodiment will be described. In all the following drawings, for ease of viewing the drawings, 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 layer 20, a second 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 layer 20 and the second layer 30 are sequentially laminated above the flow path forming portion 10. Cells (fibroblasts C1, epithelial cells C2) are disposed inside the first layer 20 and the second layer 30, respectively.

[0019] The cell culture device 1 is an instrument used to confirm the influence of VOCs on cells. 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 ratio of the base material 11 is different from that of the gel layer 15. For example, the rigidity ratio 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 materials 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] Further, as the forming material of the gel layer 15, a stimulus-responsive gel capable of adjusting the degree of swelling in response to an external stimulus may be used.

[0028] For example, as a gel that responds to heat (heat-responsive gel), 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 by 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 molecular imprint gel in which a specific molecule bonded to a hydrogel backbone is used as a crosslinking point of the gel network. For example, as a protein-responsive gel, there are mentioned a biomolecule crosslinked gel having a biomolecule complex 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 when polymerizing 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. Examples include radical polymerization using a water-soluble photoinitiator. Examples of water-soluble photoinitiators 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 deoxidizers include a combination of glucose and glucose oxidase. Also, 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 crosslinking agents 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, and 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, and 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) At the interface between the base material 11 and the gel layer 15, an adhesion region 10a where the base material 11 and the gel layer 15 adhere to each other and a non - adhesion region 10b where the base material 11 and the gel layer 15 do not adhere to each other are formed in the flow path forming portion 10.

[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] (Flow path) 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] The flow path 10x has a main flow path 101 extending in the x-axis direction and a loop structure portion 102 branching from the main flow path 101. The loop structure portion 102 has an annular path 102a formed in a closed loop shape in plan view and a connection path 102b connecting the annular path 102a to the main flow path 101. The flow path 10x shown in FIG. 3 has four loop structure portions 102.

[0046] In the loop structure portion 102, the end of the flow path (connection path 102b) extending from the main flow path 101 is a dead-end annular path 102a. Also, the loop structure portion 102 has a smaller inner diameter of the flow path compared to the main flow path 101. Therefore, when the fluid flowing through the flow path 10x flows into the loop structure portion 102, it is considered to be likely to stay inside the loop structure portion 102.

[0047] In FIG. 3, the flow path 10x is assumed to have four loop structure portions 102, but it is not limited to this, and the number of loop structure portions 102 may be five or more, or may be three or less.

[0048] A plurality of vascular endothelial cells C3 may be arranged on the inner wall of the flow path 10x. The flow path 10x having the vascular endothelial cells C3 can be considered as a model closer to the structure of blood vessels in the living body than a flow path without the vascular endothelial cells C3. By configuring such a flow path 10x, the cell culture device 1 can construct an experimental system closer to the in-vivo environment.

[0049] [Layer 1] As shown in FIGS. 1 and 2, the first layer 20 is in contact with the flow path forming portion 10 and is laminated above the flow path forming portion 10.

[0050] The first layer 20 includes an extracellular matrix (ECM) layer 21 and a plurality of fibroblasts C1 dispersed inside the ECM layer 21.

[0051] The material of the ECM layer 21 may be any material that forms an extracellular matrix capable of encapsulating and culturing fibroblasts. Examples of the material of the ECM layer 21 include Matrigel, GelMA (gelatin methacrylate), and type I collagen.

[0052] The ECM layer 21 may contain a chemical probe that reacts with VOCs to emit light. For example, by including a chemical probe in the ECM layer 21 that reacts with acetaldehyde to emit light, it becomes possible to visualize the exposure amount of acetaldehyde to the ECM layer 21.

[0053] Examples of the fibroblast C1 include human dermal fibroblasts (NHDF), human cardiac fibroblasts (NHCF), and human gingival fibroblasts (HGF). In addition, as the fibroblast C1, fibroblasts isolated from mouse or human esophagus such as human fetal esophageal fibroblasts (FEF3), mouse fibroblasts (3T3), normal diploid fibroblasts derived from fetal lung (WI-38), normal human dermal fibroblasts (NHLF), etc. can also be employed.

[0054] [Layer 2] The second layer 30 is in contact with the first layer 20 and laminated above the first layer 20. The second layer 30 includes a plurality of epithelial cells C2 and may include a medium (not shown) for culturing the epithelial cells C2. The second layer 30 is a model mimicking the epithelial layer of a living organ.

[0055] Examples of the epithelial cells C2 include tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, and corneal epithelial cells, etc. The epithelial cells C2 may be stratified squamous epithelial cells.

[0056] Examples of the medium that the second layer 30 may contain include a medium for esophageal squamous epithelial cells (KSFM), Dulbecco's modified Eagle's medium (DMEM), and Eagle's minimum essential medium (EMEM).

[0057] The first layer 20 and the second layer 30 as described above can be considered as a model simulating the stromal layer of a living organ. For example, when using the cell culture device 1 as a model simulating the skin, it is conceivable to capture the first layer 20 as a model of the dermis and the second layer 30 as a model of the epidermis and conduct experiments. Also, when using the cell culture device 1 as a model simulating the digestive organ, it is conceivable to capture the first layer 20 as the lamina propria mucosae and the second layer 30 as the mucosal epithelium and conduct experiments.

[0058] [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 through 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.

[0059] The pipes 41 and 42 are bent pipes that extend in the x-axis direction and then 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.

[0060] The adhesive 49 fixes the pipes 41 and 42 between the base material 11 and the first layer 20. The adhesive 49 covers the peripheries 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.

[0061] The adhesive 49 desirably has water resistance and adhesiveness to the base material 11 and the first layer 20. Examples of the adhesive 49 include cyanoacrylate adhesives, silicone adhesives, and epoxy adhesives.

[0062] [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 the laminated body of the flow path forming portion 10, the first layer 20, and the second layer 30 in the internal space S.

[0063] 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.

[0064] The container 50 preferably has light transmissivity. The container 50 can be formed of a material known as a material for a cell culture container. For example, examples of the material of the container 50 include glass and polystyrene.

[0065] The cell culture device 1 can be formed by forming the flow path forming portion 10, housing the flow path forming portion 10 in the container body 51, appropriately connecting the pipes 41 and 42, and then laminating the first layer 20 and the second layer 30 in the container.

[0066] 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 is obtained by forming the gel layer 12 shown in FIGS. 5 and 6 and then swelling the gel layer 12.

[0067] 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.

[0068] 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.

[0069] For example, when an acrylic monomer is used as the monomer, examples of the adhesive functional group include a (meth)acrylic group. In this case, for example, 3-(methacryloyloxy)propyltrimethoxysilane can be used as the silane coupling agent.

[0070] The formed surface treatment layer is a layer formed by the chemical (covalent) bonding of a silane coupling agent to the substrate surface. The surface treatment layer introduces adhesive functional groups to the upper surface 11a of the base material 11.

[0071] Next, a 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 groups of the monomer and the adhesive functional groups 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.

[0072] In the above manner, a gel layer 12 can be formed having an adhesive region 10a where the gel layer 12 overlaps the layer 121 and adheres to the base material 11, and a non-adhesive region 10b where the gel layer 12 is not adhered to the base material 11.

[0073] 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 in deformation 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 the direction away from the base material 11 when the volume increases due to swelling.

[0074] 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 the direction away from the base material 11 in order to relieve the increase in internal pressure due to the volume increase. Thereby, 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.

[0075] 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.

[0076] 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 elastic modulus of the base material 11 to the elastic modulus of the gel layer 12, the thickness of the gel layer 12, and the like. The elastic 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, and the like.

[0077] Furthermore, by injecting a culture solution containing vascular endothelial cells C3 into the flow path 10x and culturing the vascular endothelial cells C3 in the flow path 10x, the vascular endothelial cells C3 can be arranged on the inner wall of the flow path 10x.

[0078] (Function and effect) FIG. 7 is an explanatory diagram for explaining a method for confirming the influence of VOCs on cells using the cell culture device 1.

[0079] The cell culture device 1 can simulate the structure of the stroma and epithelial tissue by regarding the flow path 10x as a blood vessel and regarding the first layer 20 and the second layer 30 as living organs.

[0080] 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 layer 20. As a result, the VOCs contained in the VOCs solution diffuse into the first layer 20. In FIG. 7, the VOCs are indicated by reference sign A.

[0081] Examples of the VOCs include formaldehyde, benzene, toluene, and trichloroethylene.

[0082] Formaldehyde is used in building materials such as processed wood products and is also contained in household goods such as glue and adhesives, and is associated with cancers of the nose and throat and lung cancer.

[0083] Benzene is contained in gasoline, tobacco smoke, industrial exhaust gas, etc., and is associated with blood cancer and lung cancer.

[0084] Toluene is contained in paints, thinners, and adhesives, affects growth and reproduction, and is associated with kidney and liver disorders.

[0085] Trichloroethylene is used as a solvent and is contained in some industrial and household products, and is associated with kidney cancer.

[0086] When more time elapses from the start of the liquid feeding of the VOCs solution, as shown in FIG. 7, the VOCs reach the fibroblasts C1 arranged in the first layer 20 and affect the fibroblasts C1. For example, if the VOCs are acetaldehyde, it is considered that the fibroblasts C1 will undergo changes such as changes in the activity of ALDH2 (acetaldehyde dehydrogenase 2) or death.

[0087] Furthermore, when more time elapses, the VOCs reach the epithelial cells C2 arranged in the second layer 30 and affect the epithelial cells C2.

[0088] When confirming the influence of VOCs on skin cells using a conventional cell culture container, it is conceivable to culture the skin cells in a state where the gas phase of the cell culture container is filled with VOCs. However, when the gas phase is filled with VOCs, the oxygen concentration in the gas phase of the cell culture container decreases, and the cells are cultured under hypoxic conditions. As a result, it becomes difficult to evaluate the state of the cells separately from the influence of the hypoxic conditions and the influence of the VOCs, and the influence of the VOCs cannot be accurately evaluated.

[0089] Furthermore, when attempting to evaluate the influence of blood-borne VOCs on skin cells, the experimental conditions with the gas phase filled with VOCs are very different from the in-vivo state. Therefore, the influence of blood-borne VOCs on cells could not be evaluated. Moreover, there was no method for confirming the influence of the shape of blood vessels when blood-borne VOCs are exposed to cells.

[0090] On the other hand, when the cell culture device 1 of the present embodiment is used, by flowing the VOCs solution through the channel 10x modeled after blood vessels, it becomes possible to easily confirm the influence of VOCs in the blood on living organs (fibroblasts C1, epithelial cells C2).

[0091] In the cell culture device 1, the channel 10x has a loop structure portion 102. By regarding this loop structure portion 102 as a model of the loop-shaped capillary in the epithelial papilla, a model experiment can be conducted to confirm the influence of blood-borne VOCs on the cells in the vicinity of the loop-shaped capillary in the epithelial papilla.

[0092] For example, usually, when VOCs (e.g., acetaldehyde) are exposed to epithelial cells C2 that are tightly adhered to each other, disappearance of the tight adhesion of the epithelial cells C2 exposed to VOCs and abnormal NOTCH signals between adjacent epithelial cells C2 may be observed. By verifying such a phenomenon, it becomes possible to verify the influence of VOCs exposure on normal cells.

[0093] In addition, when vascular endothelial cells C3 are arranged on the inner wall of the channel 10x, the influence of VOCs on the vascular endothelial cells C3 can also be confirmed simultaneously. In the cell culture device 1 having such a configuration, it becomes possible to confirm the relationship between the mutation of the vascular endothelial cells C3 and the mutation of the skin cells (fibroblasts C1, epithelial cells C2).

[0094] According to the cell culture device having the above-described configuration, it becomes possible to easily confirm the influence of VOCs on cells.

[0095] Note that the cell culture device of the present embodiment has one channel 10x, but is not limited thereto. Even a cell culture device having a plurality of channels can achieve the effects of the present invention.

[0096] In addition, in the cell culture device 1 shown in FIG. 3, all of the plurality of loop structure portions 102 have the same shape, but are not limited thereto. FIG. 8 is a schematic plan view showing the channel of the cell culture device according to the modified example.

[0097] The flow path 16x shown in FIG. 8 has a main flow path 161 and a plurality (four in the figure) of loop structure portions 162 (162A to 162D) with different shapes. The loop structure portion 162 has an annular path 162a and a connection path 162b.

[0098] The "different shapes" of the loop structure portions 162 means that when comparing two loop structure portions, at least one of, for example, the flow path width, the flow path height, the flow path diameter, the outer diameter of the annular path, the planar shape, and the length of the connection path is intentionally made different. In this sense, it does not include differences in shape due to manufacturing errors.

[0099] More specifically, the cell culture device may have different shapes of the loop structure portions 162 as shown in FIG. 8.

[0100] The loop structure portion 162A has an annular path 162a with an outer diameter D1 and a connection path 162b with a length L1.

[0101] The loop structure portion 162B has an annular path 162a with an outer diameter D1 and a connection path 162b with a length L2 (L2 > L1).

[0102] The loop structure portion 162C has an annular path 162a with an outer diameter D2 and a connection path 162b with a length L1 (D2 > D1).

[0103] The loop structure portion 162D has an elliptical annular path 162a and a connection path 162b with a length L3 (L2 > L3 > L1).

[0104] Such a flow path 16x can be formed by patterning and forming the layer 121 to create separate adhesive regions 10a and non - adhesive regions 10b. The cell culture device can be provided with a flow path having a shape according to the purpose of the experiment.

[0105] By changing the shape of the loop structure portion 162D, it is possible to locally increase the exposure amount of VOCs to the cells existing inside the loop structure portion 162D in a plan view. As a result, the region where the exposure amount of VOCs is increased can be controlled.

[0106] For example, in the esophagus, it is known that the loop-shaped capillaries (intrapapillary capillary loop: IPCL) in the subepithelial papillae of the esophageal mucosa are related to the progression degree of esophageal cancer. In the cell culture device of the present invention, a flow path is formed that mimics the morphological changes of blood vessels occurring in vivo in this way, and by exposing cells to VOCs through the flow path, it becomes possible to verify the relationship between the changes in the blood vessel structure and the influence of VOCs in the blood on the epithelium.

[0107] Even in a cell culture device having a flow path as described above, it becomes possible to easily confirm the influence of VOCs on cells.

[0108] [Second Embodiment] FIG. 9 is an explanatory diagram of the cell culture device 3 of the second embodiment, and is an exploded perspective view of the flow path forming portion included in the cell culture device 3. As shown in FIG. 9, the cell culture device 3 has a flow path forming portion 60 and a base 70. In the following description, the illustration of the first layer and the second layer described above is omitted.

[0109] The flow path forming portion 60 has a flow path formed partly of hydrogel. The flow path forming portion 60 and the base 70 are adhered.

[0110] [Flow Path Forming Portion] The flow path forming portion 60 has a base material 61 and a gel layer 62.

[0111] (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, it is shown that the base material 61 has 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 may be one each, or may be a plurality other than three.

[0112] 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.

[0113] (Gel layer) The gel layer 62 is made of a hydrogel as a forming material 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 hole 611 and the second through hole 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.

[0114] (Interface structure) FIG. 10 is a cross-sectional view taken along the line α-α in FIG. 9. FIG. 11 is a plan view of the gel layer 62 and the vicinity of the gel layer 62.

[0115] 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.

[0116] Further, 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 by a closed loop in 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.

[0117] Further, the first through hole 611 and the second through hole 612 each open to the non-adhesion region 60b.

[0118] Such an adhesive region 60a and a non-adhesive region 60b can be formed by polymerizing a monomer of a polymer material constituting the hydrogel after forming a layer 621 of a silane coupling agent in accordance with the shape of the adhesive region 60a and then overlapping the formed layer 621. Thereby, the monomer overlapping the layer 621 adheres 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.

[0119] As described above, the gel layer 62 (flow path forming portion 60) having the adhesive region 60a and the non-adhesive region 60b can be formed. The shape of the gel layer 62 can be controlled by arranging a spacer that suppresses the outflow of the monomer around the monomer when arranging the monomer. In FIG. 10, the periphery of the gel layer 62 coincides with the periphery of the layer 621.

[0120] [Base] The base 70 supports the flow path forming portion 60 on the support surface 70a. The base 70 includes 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.

[0121] 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 the contours of both overlap when the flow path forming portion 60 is overlapped.

[0122] 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 and 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 and 73a of the pipe connection portions 72 and 73 are higher than the support surface 70a.

[0123] (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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] The base 70 can be manufactured using a known 3D printing technology.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] As a result, in the gel layer 65, the portion that overlaps the non-adhesive region 60b in a planar manner greatly swells 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.

[0133] 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.

[0134] 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 at 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 at the pipe connection portion 73 via a connector 47 and used.

[0135] 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.

[0136] 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 can be variously changed based on design, specifications, etc. without departing from the gist of the present invention.

Explanation of Reference Numerals

[0137] 1, 3... cell culture device, 10, 10A, 60... flow path forming part, 10a, 60a... adhesion region, 10b, 60b... non - adhesion region, 10x, 16x... flow path, 11, 61... base material, 11a, 61a... upper surface, 12, 15, 62, 65... gel layer, 20... first layer, 21... extracellular matrix (ECM) layer, 30... second layer, 50... container, 101, 161... main flow path, 102, 162, 162A, 162B, 162C, 162D... loop structure part, C1... fibroblast, C2... epithelial cell, C3... vascular endothelial cell

Claims

1. A cell culture device capable of culturing cells while exposing them to a chemical substance, comprising a flow channel forming part, a first layer laminated above the flow channel forming part, a second layer laminated above the first layer, and a container for accommodating the flow channel forming part, the first layer, and the second layer. The first layer includes an extracellular matrix layer and a plurality of fibroblasts dispersed inside the extracellular matrix layer. The second layer includes a plurality of epithelial cells. The flow channel 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, 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, which are formed. In the non - adhesion region, the gel layer is separated from the base material, and a flow channel surrounded by the gel layer and the base material is formed. The flow channel has a main flow channel, and a loop structure part branched from the main flow channel, and is a cell culture device.

2. The cell culture device according to claim 1, wherein the flow channel has a plurality of the loop structure parts.

3. The flow channel has a first loop structure part, and a second loop structure part having a different shape from the first loop structure part, and is the cell culture device according to claim 2.

4. The cell culture device according to any one of claims 1 to 3, wherein a plurality of vascular endothelial cells are arranged on the inner wall of the flow channel.

5. The cell culture device according to any one of claims 1 to 3, wherein the epithelial cells are stratified squamous epithelial cells.

6. The cell culture device according to any one of claims 1 to 3, having a plurality of said flow paths.

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