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 an exposure to a chemical substance, the device comprising a flow channel-forming part, a diffusion layer formed above the flow channel-forming part, a culture medium layer formed above the diffusion layer, a vessel to contain the flow channel-forming part, the diffusion layer and the culture medium layer, where cells are cultured in the interface between the diffusion layer and the culture medium layer, the flow channel-forming part comprises a substrate and a gel layer formed on the upper surface of the substrate using a hydrogel as a forming material, 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 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.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 not easily experimentable. In addition, VOCs spread into the body through the blood and are exposed to the living body from the blood vessels. Therefore, a concentration gradient of VOCs in the living body occurs according to the distance from the blood vessels. However, known methods cannot form such a concentration gradient of VOCs.

[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 effect of VOCs on cells and can simulate the concentration gradient of VOCs in the living body.

Means for Solving the Problems

[0008] 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, including a flow path forming portion, a diffusion layer laminated above the flow path forming portion, a culture medium layer laminated above the diffusion layer, and a container that houses the flow path forming portion, the diffusion layer, and the culture medium layer. The cells are cultured at the interface between the diffusion layer and the culture medium layer. The flow path forming portion has a base material and a gel layer provided on the upper surface of the base material using 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, and a flow path surrounded by the gel layer and the base material is formed, providing a cell culture device.

Effects of the Invention

[0009] According to the present invention, it is possible to easily confirm the effect of VOCs on cells and provide a cell culture device capable of simulating the concentration gradient of VOCs in the living body. In such a cell culture device, the influence of the concentration gradient of VOCs can be confirmed.

Brief Description of the Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] [First Embodiment] Hereinafter, with reference to FIGS. 1 to 9, 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, ratios, etc. of each component are appropriately made different.

[0012] 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.

[0013] 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.

[0014] In the following description, "plan view" refers to viewing the object from above in the vertical direction (+z side) downward (-z direction).

[0015] 《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 diffusion layer 20, a 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.

[0016] In the cell culture device 1, the flow path forming portion 10 is disposed at the bottom of the container 50, and the diffusion layer 20 and the culture medium layer 30 are sequentially laminated above the flow path forming portion 10, and cells C are disposed at the interface between the diffusion layer 20 and the culture medium layer 30.

[0017] The cell culture device 1 is an instrument used to confirm the influence of VOCs on the cells C. Hereinafter, each component will be described in order.

[0018] [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.

[0019] (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.

[0020] 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.

[0021] Examples of the organic material that is the material for forming 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.

[0022] 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.

[0023] (Gel layer) The gel layer 15 is formed of a hydrogel as a forming material and is 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.

[0024] 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 by containing a solvent in most of their volume. The representative solvent in which the polymer material constituting the hydrogel swells is water.

[0025] In addition, as the material for forming the gel layer 15, a stimulus-responsive gel capable of adjusting the degree of swelling in response to external stimuli may be used.

[0026] For example, as gels that respond to heat (thermo-responsive gels), gels composed of poly(N-isopropylacrylamide) or poly(methyl vinyl ether) can be mentioned. A photothermal conversion material (such as a metal nanomaterial, a carbon nanomaterial, a conductive polymer, etc.) may be dispersed in the thermo-responsive gel, and the stimulus response may be triggered by heat generation due to light irradiation.

[0027] As gels that respond to pH (pH-responsive gels), gels composed of polyelectrolytes synthesized from anionic or cationic monomers can be mentioned.

[0028] As gels that respond to light (photo-responsive gels), gels composed of polymers having spiropyran or azobenzene in the backbone can be mentioned. For the photo-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 cross-linking point.

[0029] In addition, examples of the material for forming the gel layer 15 include molecular imprint gels in which specific molecules bound to a hydrogel backbone are used as cross-linking points of the gel network. For example, as protein-responsive gels, biomolecule cross-linked gels having a biomolecule complex as a cross-linking point of the gel network and antigen-responsive gels in which an antigen-antibody complex is introduced into the network as a cross-linking point of the gel can be mentioned.

[0030] In addition, 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. Furthermore, 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.

[0031] Regarding the method for synthesizing 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, when polymerizing the acrylic monomer, the acrylic group may be crosslinked to form a three-dimensional network structure.

[0032] The type of polymerization reaction when polymerizing the acrylic monomer is not particularly limited, but 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.

[0033] During radical polymerization, in order to prevent polymerization inhibition by oxygen, an oxygen scavenger may be added to the reaction system. Examples of the oxygen scavenger include a combination of glucose and glucose oxidase. Also, the radical polymerization may be carried out under an inert gas atmosphere such as nitrogen or argon.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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%.

[0039] 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.

[0040] (Interface structure) In the flow path forming portion 10, an adhesion region 10a where the base material 11 and the gel layer 15 adhere and a non - adhesion region 10b where the base material 11 and the gel layer 15 do not adhere are formed at the interface between the base material 11 and the gel layer 15.

[0041] The non - adhesion regions 10b are formed in three strip - shaped 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.

[0042] The portion of the gel layer 15 that overlaps 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.

[0043] [Diffusion layer] The diffusion layer 20 is in contact with the flow path forming portion 10 and is laminated above the flow path forming portion 10.

[0044] The material for forming the diffusion layer 20 is, for example, a gel after a polymer material constituting a hydrogel swells. That is, the material for forming the diffusion layer 20 is a material in which a liquid such as water flows into the network structure of the polymer material and swells. Therefore, it can be said that the diffusion layer 20 is a swollen product of the polymer material.

[0045] The material for forming the diffusion layer 20 may be a biological material such as gelatin, collagen, or extracellular matrix (ECM), or may be a synthetic resin (polymer material).

[0046] The polymer material constituting the diffusion layer 20 has a lower swelling degree than the polymer material constituting the gel layer 15. The swelling degree of the polymer material constituting the diffusion layer 20 is not particularly limited as long as it is lower than the swelling degree of the polymer material constituting the gel layer 15. The swelling degree of the polymer material constituting the diffusion layer 20 is preferably, for example, about 0.8 to 1.2 times the size before swelling in one - direction swelling.

[0047] Here, the "swelling degree" is measured using the polymer material constituting the gel layer 15 (i.e., the gel layer 12) before swelling and the polymer material constituting the diffusion layer 20 before swelling. Each polymer material is cut out as a disk - shaped sample with an appropriate diameter and left standing in pure water until the size no longer changes. From the size of the sample before and after standing in pure water, it is calculated by the following formula (1). (Swelling degree)=D / D0 (1) (In formula (1), D is the diameter of the largest part of the sample after standing in pure water, and D0 is the diameter of the sample before standing in pure water.)

[0048] The polymer material constituting the diffusion layer 20 is a hydrogel. The polymer material constituting the diffusion layer 20 may be the same as or different from the polymer material constituting the gel layer 15.

[0049] Examples of the polymer material constituting the diffusion layer 20 include chemically crosslinked gels crosslinked by covalent bonds through radical polymerization reactions of monomers. Examples of the chemically crosslinked gels include polyacrylamide and its derivatives (polydimethylacrylamide, poly N-isopropylacrylamide, etc.). In this case, by using methylenebisacrylamide as a crosslinking agent, the crosslinking density can be increased, and the swelling degree of the polymer material constituting the diffusion layer 20 may be within the above numerical range.

[0050] Examples of the polymer material constituting the diffusion layer 20 also include physically crosslinked gels formed by combining a polymer having a positive or negative charge and an ion having a polyvalent charge opposite thereto.

[0051] Examples of the physically crosslinked gels include · A physically crosslinked gel formed by gelling a combination of a sodium alginate solution, which is a polymer having a negative charge, and a calcium solution such as calcium chloride or calcium sulfate · A physically crosslinked gel formed by combining poly(2,2’-disulfo-4,4’-bensidineterephthalamide (PBDT), which is a water-soluble polyaramide, with various metal polyvalent cations (Ca 2+ , Fe 2+ , Al 3+ , Zr 4+ , Ti 4+ , etc.) can be mentioned.

[0052] Instead of PBDT, similarly negatively charged TEMPO-oxidized cellulose nanofibers (NIPPON PAPER INDUSTRIES CO., LTD.) or cellulose nanofibers defibrated by a phosphoric acid esterification method (Oji Holdings Corporation) may also be used.

[0053] Here, "TEMPO" is an abbreviation for 2,2,6,6-tetramethylpiperidine-1-oxyl (2,2,6,6-tetramethylpiperidine-1-oxyl).

[0054] The diffusion layer 20 covers the outer surface 15a of the gel layer 15. Therefore, when an aqueous liquid is poured into the flow path 10x, the aqueous liquid permeates through the gel layer 15 and reaches the diffusion layer 20 located outside the flow path 10x. When the diffusion layer 20 is composed of a hydrogel, the aqueous liquid that reaches the diffusion layer 20 diffuses inside the diffusion layer 20.

[0055] By changing the polymer material constituting the diffusion layer 20, the diffusion layer 20 exhibits various functions. For example, when a hydrogel having a positive charge or a negative charge is used as the polymer material constituting the diffusion layer 20, the diffusion layer 20 has a function of preventing the diffusion of low-molecular substances having a specific charge into the flow path 10x. Such a diffusion layer 20 has a function of shielding to prevent the diffusion of low-molecular substances having a specific charge from the inside to the outside of the flow path 10x.

[0056] Also, as the polymer material constituting the diffusion layer 20, a hydrogel whose hydrophilic and hydrophobic properties switch in response to an external stimulus can also be used. When the hydrogel constituting the diffusion layer 20 is hydrophilic due to an external stimulus, the diffusion layer 20 can limit the analytes diffusing inside the diffusion layer 20 to hydrophilic ones. On the other hand, when the hydrogel constituting the diffusion layer 20 is hydrophobic due to an external stimulus, the diffusion layer 20 can limit the analytes diffusing inside the diffusion layer 20 to hydrophobic ones.

[0057] In addition, as the polymer material constituting the diffusion layer 20, a hydrogel capable of changing its swelling degree in response to an external stimulus can also be used. In the diffusion layer 20 with a relatively high swelling degree of the hydrogel, the diffusion rate of the analyte diffusing inside is relatively slower compared to the diffusion layer 20 with a relatively low swelling degree.

[0058] The mechanical strength of the diffusion layer 20 is not particularly limited. For example, when the diffusion layer 20 is required to have an elastic modulus (~1.3 MPa) comparable to that of Polydimethylsiloxane (PDMS), as the polymer material constituting the diffusion layer 20, a double-network gel in which a physically cross-linked gel and a chemically cross-linked gel are combined is preferable. Since the double-network gel has a tough double-network structure, its mechanical strength is further improved.

[0059] The shape of the diffusion layer 20 is not particularly limited. The thickness of the diffusion layer 20 is thicker than the height of the gel layer 15 and can be appropriately set to any thickness.

[0060] The polymer material constituting the diffusion layer 20 may contain various additives as long as the change in the swelling degree is not extreme. By using any additive, any function can be imparted to the diffusion layer 20.

[0061] The additive in the diffusion layer 20 is not particularly limited as long as it does not inhibit gel formation. Examples include biomolecules and surfactants that improve biocompatibility.

[0062] In addition, metal nanoparticles and carbon nanomaterials can also be mentioned as additives in the diffusion layer 20. By adding these materials to the diffusion layer 20, various functions according to the additives, such as a sensor function, conductivity, and photothermal conversion function, can be imparted to the diffusion layer 20.

[0063] In addition, when the material of the diffusion layer 20 is an acrylamide gel and the diffusion layer 20 has no cell adhesiveness, a cell adhesion layer may be provided on the upper surface of the diffusion layer 20. Examples of the material of the cell adhesion layer include proteins such as collagen, fibronectin, and laminin, and anionic or cationic electrolyte polymers.

[0064] The cell adhesion layer may be formed over the entire upper surface of the diffusion layer 20, or may be formed by patterning on the upper surface of the diffusion layer 20. Examples of methods for patterning the cell adhesion layer include known methods such as microcontact printing, inkjet printing, and photolithography.

[0065] [Culture medium layer] The culture medium layer 30 is in contact with the diffusion layer 20 and is laminated above the diffusion layer 20. The culture medium layer 30 is a solid culture medium used for culturing cells, and a known culture medium can be used.

[0066] Examples of the culture medium layer 30 include, for example, general cell culture media (DMEM, RPMI), and the culture medium KSFM for esophageal squamous epithelial cells.

[0067] [Cells] Cells C are cultured at the interface between the diffusion layer 20 and the culture medium layer 30 (the upper surface 20a of the diffusion layer 20). The cells C are preferably cells obtained by two-dimensional cell culture. In FIG. 2, the cells C are arranged over the entire upper surface 20a. The cell culture device 1 is an instrument used to confirm the influence of VOCs on the cells C, and various cells for which the influence of VOCs is to be confirmed can be employed as the cells C.

[0068] Specifically, as the cells C, epithelial cells (esophageal epithelial cells, airway epithelial cells, intestinal epithelial cells), hepatocytes, mucosal cells, nerve cells, etc. can be used. These cells are useful as cells for confirming the influence of VOCs because they may be exposed to VOCs taken up in the living body or are involved in the metabolism of VOCs taken up in the living body.

[0069] [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.

[0070] 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.

[0071] The adhesive 49 fixes the pipes 41 and 42 between the base material 11 and the diffusion 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.

[0072] The adhesive 49 preferably has water resistance and adhesiveness to the base material 11 and the diffusion layer 20. Examples of the adhesive 49 include cyanoacrylate adhesives, silicone adhesives, epoxy adhesives, etc.

[0073] [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 diffusion layer 20, and the culture medium layer 30 in the internal space S.

[0074] 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.

[0075] 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, the material of the container 50 can include glass, polystyrene, etc.

[0076] The cell culture device 1 can be formed by forming the flow path forming section 10, housing the flow path forming section 10 in the container body 51, connecting the pipes 41 and 42 as appropriate, and then laminating the diffusion layers 20 and the culture medium layer 30 in the container.

[0077] FIG. 5 is a schematic perspective view of the flow path forming section 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 section 10 is obtained by forming the gel layer 12 shown in FIGS. 5 and 6 and then swelling the gel layer 12.

[0078] The adhesion region 10a and the non-adhesion region 10b of the flow path forming section 10A can be manufactured by patterning and forming a hydrogel using a known photolithography technique.

[0079] Specifically, first, using a known photolithography technique, a layer 121 of a silane coupling agent having an adhesive functional group is formed on the base material 11 in accordance with the shape of the adhesion 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.

[0080] 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.

[0081] 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 base material 11 by the surface treatment layer.

[0082] 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.

[0083] In the above manner, a gel layer 12 can be formed which has 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.

[0084] 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 a direction away from the base material 11 when the volume increases due to swelling.

[0085] As a result, in the flow path forming portion 10, 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. 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.

[0086] 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.

[0087] Also, 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.

[0088] (Function and Effect) Figures 7 and 8 are explanatory diagrams for explaining a method for confirming the influence of VOCs on cells using the cell culture device 1.

[0089] 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 diffusion layer 20 as interstitial tissue.

[0090] 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 contained in the VOCs solution diffuse into the side wall (gel layer 15) of the flow path 10x made of a hydrogel forming material and the diffusion layer 20 made of a hydrogel forming material. In Figure 7, the VOCs are indicated by reference numeral A.

[0091] Examples of VOCs include formaldehyde, benzene, toluene, and trichloroethylene.

[0092] Benzene is contained in gasoline, tobacco smoke, industrial exhaust gas, etc., and is associated with blood cancer and lung cancer.

[0093] Formaldehyde is used in building materials such as processed wood products, and is also contained in household items such as glue and adhesives, and is associated with nasal and throat cancer and lung cancer.

[0094] Trichloroethylene is used as a solvent and is contained in some industrial and household products, and is associated with kidney cancer.

[0095] Toluene is contained in paint, thinner, and adhesive, affects growth and reproduction, and is associated with disorders of the kidneys and liver.

[0096] When more time elapses after the start of feeding the VOCs solution, as shown in Fig. 7, the VOCs reach the cells C disposed on the upper surface 20a and affect the cells C. For example, if the VOCs are acetaldehyde, it is considered that the cells C will undergo changes such as a change in the activity of ALDH2 (acetaldehyde dehydrogenase 2) or death.

[0097] The amount of VOCs reaching the cells C (the exposure amount of VOCs to the cells C) 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 cells C varies depending on the configuration of the cell culture device 1, such as the material of the diffusion layer 20, the thickness of the diffusion 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, by experimentally confirming the amount of VOCs reaching the upper surface 20a either before or after, the exposure amount of VOCs to the cells C can be grasped.

[0098] Also, the exposure amount of VOCs to the cells C corresponds to the distance between the cells C and the flow path 10x, and it is considered that cells farther from the flow path 10x have a relatively lower exposure amount compared to cells near the flow path 10x. Therefore, by respectively confirming the effects of VOCs exposure on cells with different distances from the flow path 10x, the relationship between the exposure amount of VOCs and the effects received by the cells C can be confirmed.

[0099] When confirming the influence of VOCs exposure on cells using a conventional cell culture container, it is conceivable to culture the cells with the gas phase of the cell culture container filled with VOCs. However, in that case, it is difficult to control and grasp the exposure amount of VOCs to the cells. Also, when filling the gas phase with VOCs as described above, the oxygen concentration in the gas phase of the cell culture container decreases, and the cells will be 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.

[0100] Furthermore, in the method of culturing cells after mixing a medium and VOCs, there is a possibility that the organic substances contained in the medium and the VOCs react with each other before the medium and the VOCs are mixed and cell culture is started. In that case, it is conceivable that the chemical properties of the VOCs are attenuated or do not show chemical properties, and the desired results cannot be obtained.

[0101] On the other hand, when using the cell culture device 1 of the present embodiment, the exposure amount of VOCs to cells can be more easily controlled than before, and the influence of VOCs on cells can be easily confirmed. In addition, cells can be cultured without making them hypoxic, and it is easy to evaluate the influence of VOCs on cells.

[0102] Furthermore, in the cell culture device 1, after starting cell culture, VOCs can be diffused from the flow path to expose the cells to the VOCs. Therefore, the time during which there is a risk of reaction between the VOCs and the medium can be minimized, and the cells can be exposed to the VOCs.

[0103] 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, flowing a VOCs solution through the flow path 10xb, and flowing a VOCs solution with a higher concentration than that in the flow path 10xb through the flow path 10xc, the exposure amount of VOCs to the cells C arranged above the flow path 10x can be made different.

[0104] Therefore, by comparing the cells C (C1, C2, C3) overlapping the three flow paths 10x respectively, the influence of the exposure amount of VOCs on the cells C can be easily confirmed.

[0105] FIG. 8 is a schematic plan view showing an example of cells arranged on the upper surface 20a. For example, it is assumed that abnormal cells C1 and C2 are included among the plurality of arranged cells C. Examples of abnormal cells include TP53 mutant cells and cells exposed to acetaldehyde for a long time. It is assumed that a flow path 10x is formed below the abnormal cell C2 among such abnormal cells C1 and C2.

[0106] In this case, when the VOCs solution is made to flow through the flow path 10x, the cells C and abnormal cells C2 overlapping with the flow path 10x are exposed to VOCs, and the cells C and abnormal cells C1 at other positions are not exposed to VOCs, or the exposure amount is relatively small.

[0107] When culturing the cells C cultured in such a state, it is possible to confirm the influence of VOCs exposure on the expansion of abnormal cells C1 and C2 and the expansion of normal cells C. For example, when the expansion of abnormal cell C2 is faster than the expansion of abnormal cell C1, an influence due to VOCs exposure is assumed.

[0108] Also, when the expansion of abnormal cell C1 is isotropic and the expansion of abnormal cell C2 is along the flow path 10x, it can be interpreted that the cells C overlapping with the flow path 10x are weakened by the exposure to VOCs, and the abnormal cells C2 expand along the weakened cells.

[0109] When epithelial cells are used as the cells C, the cells C are tightly bound to each other. When such cells C are exposed to VOCs (for example, acetaldehyde), disappearance of tight junctions of the cells C exposed to VOCs or abnormal NOTCH signals between adjacent cells C may be observed. By verifying such a phenomenon together with the state of expansion of abnormal cells, it becomes possible to verify the influence of VOCs exposure on the spatial competition between abnormal cells and normal cells.

[0110] FIG. 9 is a schematic plan view showing the cell culture device 2 according to the modified example. FIG. 9 omits the illustration of the culture medium layer and shows the diffusion layer 20 and the flow path on the lower surface of the diffusion layer 20.

[0111] The flow path 16x included in the cell culture device 2 has four main flow paths 161x extending in the x-axis direction and connection paths 162x connecting the main flow paths 161x in the y-axis direction. Due to the connection paths 162x, the flow path 16x branches within the path and forms an annular path within the path.

[0112] On the upper surface 20a of the diffusion layer 20, cells form a plurality of colonies CL. The plurality of colonies CL includes a first colony CL1 and a second colony CL2 whose distance from the flow path 16x is different from that of the first colony CL1. Specifically, the first colony CL1 overlaps the flow path 16x in a plane, and the second colony CL2 is spaced apart from the flow path 16x in a plan view (separation distance L).

[0113] The plurality of colonies CL can be formed by patterning and forming a cell adhesion layer on the upper surface of the diffusion layer 20 and seeding and culturing cells for observation purposes (for example, epithelial cells) on the formed cell adhesion layer.

[0114] When a VOCs solution is flowed through the flow path 16x of such a cell culture device 2, a plurality of colonies CL with different VOCs exposure states are formed due to the difference in the distance between the flow path 16x and the colonies CL, for example, the difference between the first colony CL1 and the second colony CL2. Therefore, by analyzing the cells for each colony, accurate analysis can be performed without mixing cells with different VOCs exposure amounts compared to the case where cells C are cultured on the entire upper surface 20a and the same operation is performed.

[0115] In this embodiment, cells are cultured on the upper surface 20a of the diffusion layer 20, but the cultured cells may form organoids.

[0116] According to the cell culture device having the above configuration, the influence of VOCs on cells can be easily confirmed.

[0117] Note that the cell culture device of this embodiment has a plurality of flow paths, but is not limited to this. A cell culture device having only one flow path can also achieve the effects of the present invention.

[0118] [Second Embodiment] Figure 10 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 of the cell culture device 3. As shown in Figure 10, the cell culture device 3 has a flow path forming portion 60 and a base 70. In the following description, the illustration of the diffusion layer and the culture medium layer described above is omitted.

[0119] 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.

[0120] [Flow path forming portion] The flow path forming portion 60 has a base material 61 and a gel layer 62.

[0121] (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 Figure 10, 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.

[0122] In Figure 10, 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.

[0123] (Gel layer) The gel layer 62 is provided on the upper surface 61a of the base material 61 with hydrogel as a forming material. The gel layer 62 is provided so as to overlap 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 are formed.

[0124] (Interface structure) Figure 11 is a cross-sectional view taken along the line α-α in Figure 10 as viewed in the arrow direction. Figure 12 is a plan view of the gel layer 62 and the vicinity of the gel layer 62.

[0125] As shown in FIG. 11, in the flow path forming portion 60, an adhesion region 60a where the base material 61 and the gel layer 62 adhere to each other and a non-adhesion region 60b where the base material 61 and the gel layer 62 do not adhere to each other are formed at the interface between the base material 61 and the gel layer 62.

[0126] Further, as shown in FIG. 12, 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 a plan view. The shape of the non-adhesion region 60b is an example, and it can be various shapes according to the design.

[0127] Also, the first through hole 611 and the second through hole 612 each open to the non-adhesion region 60b.

[0128] Such an adhesion region 60a and 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 and then polymerizing the monomers of the polymer material constituting the hydrogel by overlapping with the formed layer 621. Thereby, the monomers overlapping with 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.

[0129] As described above, 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 a spacer for suppressing the outflow of the monomers around when arranging the monomers. In FIG. 11, the periphery of the gel layer 62 coincides with the periphery of the layer 621.

[0130] [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.

[0131] The base body 71 has a substantially rectangular parallelepiped shape. The shape of the base body 71 in plan view is the same as the shape of the flow path forming portion 60 (base material 61) in plan view, and when the flow path forming portion 60 is superposed, the contours of both overlap.

[0132] The pipe connection portion 72 has a substantially rectangular parallelepiped shape, and the corner on the +x side has a rounded shape. Similarly, the pipe connection portion 73 has a substantially rectangular parallelepiped shape, and the corner on the -x side 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.

[0133] (First flow path, second flow path) The base 70 has three first flow paths 701, which are the same number as the non-adhesive 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-adhesive regions 60b.

[0134] One end of the first flow path 701, the first inner end portion 701x, opens to the support surface 70a, and the other end of the first flow path 701, 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.

[0135] The first inner end portion 701x opens in a region X that overlaps the non-adhesive 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.

[0136] Similarly, one end of the second flow path 702, the second inner end portion 702x, opens to the support surface 70a, and the other end of the second flow path 702, the second outer end portion 702y, opens to the upper surface 23a 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.

[0137] The second inner end portion 702x opens into a region X that overlaps with the non-adhesive region 60b in a plan view. In the 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.

[0138] The base 70 can be manufactured using known 3D printing technology.

[0139] In FIGS. 13 to 15, the state in which the gel layer 62 shown in FIG. 10 is swollen is shown. Hereinafter, the swollen gel layer is denoted by reference numeral 65.

[0140] FIG. 13 is a perspective view of the flow path forming portion 60 and the base 70 having the swollen gel layer 65. FIG. 14 is a cross-sectional view taken along the line β-β of FIG. 13 and corresponds to FIG. 11. FIG. 15 is a cross-sectional view taken along the line γ-γ of FIG. 13.

[0141] As shown in FIGS. 13 to 15, 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, among 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 and 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.

[0142] As a result, the portion of the gel layer 65 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.

[0143] 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.

[0144] As shown in FIG. 13, in the cell culture device 3, a pipe 43 can be connected to a first outer end portion 701y that opens to a pipe connection portion 72 via a connector 45, and a pipe 44 can be connected to a second outer end portion 702y that opens to a pipe connection portion 73 via a connector 47 and used.

[0145] Even in a cell culture device having the above-described configuration, according to the cell culture device, the influence of VOCs on cells can be easily confirmed.

[0146] As described above, the preferred embodiments of the present invention have been described with reference to the accompanying drawings, but the present invention is not limited to such examples. The various shapes and combinations of the constituent members shown in the above-described examples are merely examples, and various modifications can be made based on the design, specifications, etc. without departing from the gist of the present invention.

Description of Reference Numerals

[0147] 1, 2, 3... cell culture device, 10, 10A, 60... flow path forming portion, 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... diffusion layer, 30... culture medium layer, 50... container, C... cell, CL... colony, CL1... first colony, CL2... second colony

Claims

1. A cell culture device capable of culturing cells while exposing them to a chemical substance, comprising a flow path forming part, a diffusion layer laminated above the flow path forming part, a culture medium layer laminated above the diffusion layer, and a container for accommodating the flow path forming part, the diffusion layer, and the culture medium layer, wherein the cells are cultured at the interface between the diffusion layer and the culture medium layer, the flow path forming part includes 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 is 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, and a flow path surrounded by the gel layer and the base material is formed. 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 cell culture device according to claim 1 or 2, having a plurality of the flow paths.

5. The cell culture device according to claim 1 or 2, wherein the cells are arranged on the entire surface of the interface.

6. At the interface, the cells form a plurality of colonies, the plurality of colonies include a first colony, and a second colony having a different distance from the flow path from the first colony. The cell culture device according to claim 1 or 2.

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